August 2003 Volume 205 • Number 1 BIOLOGICAL BULLETIN 41°00'N 40° 00' N ..;*,,-! *-SuBu IRT www.biolbull.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. 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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 Sp0t — with 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 CHARLES D. DERBY 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 GREGORY HINKLE 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 Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center, Woods Hole Hunter College, City University of New York University of California, Davis Center of Aquaculture-IRTA, Spain Bodega Marine Lab., University of California, Davis Louisiana State University Oregon Institute of Marine Biology, Univ. of Oregon 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 Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS http://www.biolbull.org Cooljast. Reliable. ••* \ I Launch a Microway® Linux cluster and solve your problems in life sciences, structure design, weather, astronomy . . . Run cool. Microway's CoolFlow™ 1U chassis is designed to provide the best cooling in the industry. Low temperatures result in higher CPU and memory reliability, plus longer cluster life. Take control. Monitor and control your cluster remotely via our secure web-based GUI. Microway's NodeWatch™ hardware and MCMS™ cluster management software report on temperatures, voltages, and chassis fans, and let you set thresholds for notification and/or failsafe shutdown if problems occur. Today's HPC market is wilder than ever. Microway's expert design team will guide you through the Whitewater of hardware, software, and storage solutions. Our clusters are competitively priced and delivered on time— and their proven reliability yields low total lifetime cost. What's more, our tech support is legendary. Call us to test drive NodeWatch™ online, arrange a benchmark, and discuss your next Linux cluster with people who speak your language. Visit our website for HPC Times technical news. CoolRak™ Cabinet Features XeonXBIade™ Technology •1.056 Teraflops per 44U cabinet running 3.06 GHz Xeons • NodeWatch "' remote control and monitoring •CoolFlow '" proprietary 1U ant1 411 chassis • Myrinet, GigE, or InfiniBand '" connectivity GSA#GS-35F-0431N Qpteron Scientists have been counting on us for over 20 years.™ 508.746.7341 • www.microway.com 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 THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to Subscription Administrator, THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, 7 MBL Street. Woods Hole. Massachusetts 02543. Subscription includes both print and online journals. 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By the time a paper is sent to the press, all nucleotide or amino acid sequences and associated alignments should have been deposited in a generally accessible database (e.g.. GenBank, EMBL, SwissProt), and the sequence accession number should be provided. 7. Reprints, page proofs, and charges. Authors may pur- chase reprints in lots of 100. Forms for placing reprint orders are sent with page proofs. Reprints normally will be delivered about 2 to 3 months after the issue date. Authors will receive page proofs of articles shortly before publication. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). Other than these charges for authors' alterations. The Biological Bulletin does not have page charges. Reference: 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 126°00'W 125°00'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 Qw 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. 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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. ZIGLER1-2* AND H. A. LESSIOS1 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: zi»lerk@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 0 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 ~^\/ uyf /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 Asn8 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° ± 2°C); 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° ± 2°C. 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% CH3CN 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% CH3CN containing 0.1% HFBA. The first step was 0%-10% CH3CN in 5 min, followed by a shallower gradient from 10% to 34% CH3CN 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 Asn8 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: X2(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 [X2(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, X2(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 0 100 80 60 40 20 0 Native Attractin Native Attractin - i 0 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 0 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 0 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 0 100 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. 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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. 45°33' N. long. 84°57' W) and Burt Lake (lat. 45°28' N, long. 84°40' 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'Ple 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) 0 09 08 07 06 05 04 0.3 0.2 0 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 (1.8±0.1s;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 r2=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 resoiiR1 ' 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 0 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 0 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 m4) and the polar moment of area (J in m4), 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 0 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 0 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 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 * • •* 0 A **• A** * A A A A o.OO A D • A 4.00 6.00 8.00 10.00 12.00 log GJ / r4 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 (rmax = 0.004 m, rmn = 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. 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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? 0 J 1 6.5 + 0 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 Mh nd 9.7 + 9 J 1 nd 9 F 1 nd 10 Fa 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_ji ,.;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 (mm3 x 10~2)§ -1 h 0 0 1 dg 2 0.0 Oh 0 0 1 dg 2 0.0 1 h 0 0 1 dg 2 0.0 2h 0 0 1 Idg n 0.0 3h 2 nd 1 Idg 4 > 0.0 4h 2 nd 0 Idg 3 >0.0 5h 2 0 2. 0.3 (I Idg 3 1.8 oh 4 0 2. 0.25. 0.35. 0.45 0 0 cecum 4 8.3 7h 4 0 1,0.1.0.2.0.55 0 0 cecum 4 9.2 2d 2 0.1.0.5 1) 0 cecum ~> 6.6 3d 0 0 6 0 Idg 6 0.0 4 d a.m. 3 nd 1 dg 5 >0.0 4 d p.m. 1 0 1 0 Idg 2 0.05 5 d a.m. 0 0 0 Idg 1 0.0 5 d p.m. 1 0.05 0 Idg T 0.007 7d 0 0 0 0 — 0 0.0 * 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. 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(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 CHRISTENSEN1'*, JAMES M. COLACINO2, AND CELIA BONAVENTURA3 ^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 (P5U = 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: christenubls1 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 (Pw = 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 0 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-cm2 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% N2, and a deoxygenated spec- trum was taken. Then 100 ju,l of 10 mM Na2S 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 O2 "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 (Na2S2O4) (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 mm3 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 107 cells. The total cell volume is equal to 4.3 mm3; and for an animal with a total WVS volume of 18.1 mm3 (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 (P5I, = 11.4 mmHg at pH 8.0, 20 °C) (Table 1 ). The P50 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 P50 values measured on the FPLC fractions are lower than those measured on the crude hemolysates and in cel- lulo. P50 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 P5{) 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 (PS() = 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 (P50 =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 P50 for measurements using intact animals may be explained by oxygen consumption of the animal. The Po-, within the tissues is lower than the external Po2 due to oxygen consumption by the tissues. The difference in Po2 can lead to an overestimation of the P50. 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 PS(, 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 P5n values were measured I'M vitro. overestimation of the juvenile P50, 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 O2 "off reaction and 5.2 x If)4 A/~'s~' and 2.8 X 104 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 0 - -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 Na2S (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 P50 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 Pw of 9 mmHg (pH 7.2) should have a Pw of about 3 mmHg. The in cellulo and in vitro analysis in the present study demonstrated a P50 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 Pso calculated for this run was 3.6 mmHg. This corresponds to the P5(j value estimated from the data of Hajduk and Cos- grove (1975). The formation of methemoglobin may explain the low P50 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 P50 of the mixture usually lies between those of the oarate fractions, not above them. During oxygen-bim1 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 P50 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. ~103 M 's ' [Colacino, 1973]; T. gemmata, 103 to 104 M~'s~' [Steinmeier and Parkhurst, 1979]) and human hemoglobins (30 X 105A/~'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 Po2 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 Po2 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. 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(August 2003) © 2003 Marine Biological Laboratory Early Development and Acquisition of Zooxanthellae in the Temperate Symbiotic Sea Anemone Anthopleura ballii (Cocks) SIMON K. DAVY1'* AND JOHN R. TURNER2 ' 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 0 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 • 6H2O 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. 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(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 sputtered (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. C«rbicula, Pisidium). The segregation may resemble the first steps of somitic segmen- tation in the early embryology of segmented animals (Wol- pert et 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 yjd = 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 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. 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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 PENNEC2, AND MARCEL LE PENNEC2 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 ;»)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. 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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^-- . 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 C02 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 2O 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 ISN 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 experiments7 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. 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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 ISN 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 Ca2' 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 f1 esota Harvey, Sarah, Medi< Virginia Hong, Eun-Jin (Eric " | 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 19th 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'icwitz Mr David Space ar ' in/is Dr and Mrs Alan Dr. and Mrs W I1'. phenson Mr and Mrs !" : ewart Dr Elijah Ste~ Ms Jasmin Bihler Drs. Alber , urd 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 STAFF1 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, Gavin7 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 L2 Grossman, William M Klimm III, Henry W Sexton, Andrew W Sterling, Andrew2 Sterling, Christian2 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, Jessica2 Reuter, Laura Westburg, Joanne for Information Technology Division Inzina, Barbara, Network Manager Callahan, Michael- Cohen, Alex2 Dematos, Christopher Fournier, Pamela Jones, Patricia L Leary, Patrick2 Lowell, Gregory Mountford, Rebecca J Remsen, David P, Renna, Denis J Space, David B. Wagner, Paul2 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, Kristopher2 Research Space Administration Kaufmann, Sandra J Satellite/Periwinkle Children's Programs Robinson, Paulina H 2 Butler, Meredith2 Camire, Aaron2 Guiffrida, Beth2 Halter, Sarah2 Karalekas, Nina2 Langill, Susan2 Leveque, Rachel2 Noonan, Brendan2 Noonan, Patrick2 O'Connor, Caitlin2 Orfila, Cecelia2 Plourde, Anna2 Sturbaum, Sonja2 Swetish, Margaret2 Swiniarski, Kathryn2 Thamm, Jennifer Urciuoli, Karen2 Vachon, Julia2 | Services, Projects and Facilities Cutler, Richard D , Director Enos, Joyce B Stackhouse, Aaron2 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, Matthew2 Duane, James2 Illgen, Robert F Malchow, Robert2 McHugh, Clare2 Mendoza, Guy2 Rogers, William2 Santoro, John2 Plant Operations and Maintenance Abbott, Thomas E., Supervisor Anderson, Lewis B Atwood, Paul R Auclair, Donald2 Bailey, Jeffrey B Barnes, John S Bryant, Horace2 Cadose, James W. Callahan, John J Cormier, Garrett2 Elias, Michael Ficher, Jason Goehl, George Gonsalves, Jr, Walter W. Henderson, Jon R House, James Howell, Robert Langill, Richard Laurmo, Frank Mancevice, Connne2 McAdams III, Herbert M McHugh, Michael O McQuillan, Jeffrey2 Mendoza, Duke Mills, Stephen A Pratt, Barry Rattacasa, Frank2 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://www.biolbull.om 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 (23°C), 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 Editor Associate Editors Section Editor Online Editors Editorial Board Editorial Office MICHAEL J. GREENBERG Louis E. BURNETT R. ANDREW CAMERON CHARLES D. DERBY MICHAEL LABARBERA SHINYA INOUE, Imaging and Microscopy JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG JOAN CERDA ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL KENNETH M. HALANYCH GREGORY HINKLE 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 Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center. Woods Hole Hunter College. City University of New York University of California. Davis Center of Aquaculture-IRTA, Spain Bodega Marine Lab., University of California, Davis Louisiana State University Oregon Institute of Marine Biology. Univ. of Oregon 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. 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Easily scales to address rapid expanding storage requirements. Consulting Services: Microway offers expertise designing custom software for financial analys genomics, proteomics, plus data warehousin project management tools, network/data centi design, and lab integration/management systems AMDJ1} Opteron Technology you can count on 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 tin1 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.\ nil transposed as in some BIOLOGICAL ABSTRACTS listin' H. Alv i.i>own international journals in their preferred forms rath" _. . WORLD LIST or USASI usage (e.g.. Nature. Science :<>n NOT Nature, Land.. Science, N.Y.; Evolution, Lancaster, Pa.) 6. Sequences. By the time a paper is sent to the press, all nucleotide or amino acid sequences and associated alignments should have been deposited in a generally accessible database (e.g.. GenBank. EMBL, SwissProt). and the sequence accession number should be provided. 7. Reprints, page proofs, and charges. Authors may pur- chase reprints in lots of 100. Forms for placing reprint orders are sent with page proofs. Reprints normally will be delivered about 2 to 3 months after the issue date. Authors will receive page proofs of articles shortly before publication. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). Other than these charges for authors' alterations. The Biological Bulletin does not have page charges. Reference: 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 )O2g~' h~' (Table 1 ), an oxy-calorific conversion of 4.7 kcal 1~' O2, 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 O2 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 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. 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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 70 - R — 7/20exptj I, •' . bU - = sn X n — 8/2expt movement 1 stopped .5 3U " I/I v- Q> oU - / X y ••^^ TT- o> ^0 - "D 1 n -/ ^ 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 pCO2) 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. 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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. HUNT1, AND STEVEN H. D. HADDOCK Monterey Bay Aquarium Research Institute, 7700 SamihoUlt /?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 H2 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 4°C 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 . 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. ZIMMER1 2'§ 1 Department of Biology, 2Neurosciences 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 (34°24'16" N, 119°31'3()" 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 cm3) 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 1°C 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 21°C (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 21°C (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 (21°C) and light (40 /j,mol/m2/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 1 r 20 If |T t S3 EI & j. 15 n 34.0 330 j ft F i i a P i j d : [3 i f i f * 320 11 0 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-0^00'0'^ 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 0 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 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 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 I1* 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. 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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 2nd cleavage 9.45 h 3rd cleavage 22 h Morula 24—48 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 nd°e; 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 _ f V 5 2 • ill f 1 1 f 1 o I Id OF H ^ CQ CD CH -; O D-; i 1 1 i ii i CO (3 H 5 a.H CO t»_ O ^s c -s s -5 1 o I ^o ^ I "° 73 "° OJj ^ O r i en — i sO r , J E & Si) 1 E E I E ? E p ^ ,_ .S B^ E 4 44444 0 ^ ^ ^ "§ .3 1 1 ^ 1 1 £ ^ S S ° 5 OC *^J O >/"j — u-i OO r*-, X i o ^i* —. 0 o u u u u U 0 2lP E a _,. rl oo 00 r- O 01 0 1 - , - ri - r, "O "3* "O ' 13* *O T3 "O 1 1 SIS •a' TS -a' -d l_ Ha:- g ^ 2 °° 2 ^ ^ O OJ — . -H- 1 c, !___„__ - CQ > ^ H •a ~ 0 "5= dj a ^ *,l. 3 w g. • « RR 1 "-I" - - "3 - | r- | -r - - "~" "rt |j c: c •£ -S ">> ^ > T3 -^ "58 §g c I s (U ^ i*% W) c/- 2 j i 8 ^j O iff (8 S'8 IS III 1 D o' 1 GO' w : 8 1 2 o ri i^, .5 O -T " ._ C >_! ° 2> £ J ^ rt ^ lit « < « < < « ca < « < | ca co D3< < -C -3 n-J 5 £• 3 rt -C £ E E E E E E 'o c c aS JH re ^ •£ 3. 3. 3. i i i i § IE oo tJ PJ c/5 O GO OO r*~, O OO 'GO ON ON O O -T 1 • 1 ' si ' ^r 1 a -c _ 2 c CL s> > '- C £ > c i: « « w O ffi ed §> c S "O ^ C 3 __ Q ^ -X Cu tlj t •!s i s c a pulchra, with emphasis on the sensory receptor cells. Cell Tissue Res. 225: 235-248. Chia. F.-S.. and R. Koss. 1984. 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(October 2003) © 2003 Marine Biological Laboratory Effects of Allogeneic Contact on Life-History Traits of the Colonial Ascidian Botryllus schlosseri in Monterey Bay NANETTE E. CHADWICK-FURMAN1 * AND IRVING L. WEISSMAN2 ^ Life Science Department, Bar-llan University; Ramat Can, Israel, and Interuniversity Institute for Marine Science, P.O. Box 469, Eilat, Israel; and 'Department of Pathology, Stanford Medical School, Stanford University. Stanford, California 94305-5323 Abstract. The formation of chimeric colonies following allogeneic contact between benthic invertebrates may strongly affect colony fitness. Here we show that, in a field population of the colonial ascidian Botryllus schlosseri in Monterey Bay, California, more than 20% of all colonies occur in allogeneic contact with conspecifics. We experi- mentally assessed the effects of allogeneic contact on the following life-history traits under natural field conditions: growth, age and size at first reproduction, and egg produc- tion (fecundity). When compared with isolated colonies, and in some cohorts also with colonies that rejected alloge- neic neighbors, colonies that fused with neighbors incurred reduced fitness in terms of most life-history traits measured. We propose that one of the benefits of precise allorecogni- tion is that, in fused colonies, it limits the unit of selection to chimeric individuals composed of closely related kin. Introduction Tissue fusion and chimera formation between allogeneic individuals occurs in sessile invertebrates such as sponges (Ilan and Loya, 1990), corals (Chadwick-Furman and Rinkevich. 1994; Hidaka et al.. 1997; Frank et til., 1997), and protochordate ascidians (Chadwick-Furman and Weiss- man. 1995a: Rinkevich. 1996). The formation of chimeras between kin may confer benefits on colonial invertebrates due to increased body size, early onset of sexual reproduc- tion, and increased survival following partial predation (Buss, 1982; Grosberg and Quinn, 1986; Sabbadin. 1994). Received 3 July 2002; accepted 8 July 2003. *To whom correspondence should be addressed. E-mail: furmants1 mail.biu.ac.il However, fusion of different genotypes also may come at a cost to individuals, in terms of germ and somatic cell parasitism (Stoner and Weissman, 1996; Rinkevich, 1996; Stoner et al., 1999). Studies of fused allogeneic colonies of the protochordate Botryllus schlosseri have shown that the eggs of one partner may be retained and brooded by the other partner over several reproductive cycles (Sabbadin and Zaniolo, 1979). In laboratory studies, fusion between allogeneic colonies of B. schlosseri leads to costs rather than benefits in terms of several fitness parameters (Rinkevich and Weissman, 1987, 1992a, b); indeed an inevitable result of such fusion is the death and resorption of all zooids (colonial units) of one colony, and the survival of the zooids of the other colony for up to many weeks after fusion (Rinkevich and Weissman, 1992a, b). In nature, however, resorption is not the inevita- ble conclusion of fusion prior to the onset of reproductive competence (Chadwick-Furman and Weissman, 1995a). Further, the resorbed partner also may parasitize the germ cell line of the resorbing partner in chimeras under both laboratory and field conditions (Pancer et al., 1995; Stoner and Weissman, 1996; Stoner et al., 1999). Thus, the genetic composition of chimeric colonies in nature may be more complex than previously observed in the laboratory. Previously we reported on seasonal variation in life his- tory traits of B. schlosseri colonies in a field population in Monterey Bay, California (Chadwick-Furman and Wiess- man, 1995b). Here we determine natural frequencies of allogeneic contact in the same field population, and assess the resulting impacts on life-history traits in this colonial ascidian. We also describe the morphology and stability of chimeric colonies under natural field conditions. 133 134 N. E. CHADWICK-FURMAN AND I. L. WEISSMAN Figure 1. ( i-.onies of the ascidian Btitryllux xchloxxcri that were grown under three Upe.s oi allogeneic contact conditions in Monterey Bay. California. Scale .bar-. 5 mm. (A) An isolated colony at 49 days old, consisting of 70 clonal units, teimed zooids, that are arranged into six circular groups (systems) of 10-14 zooids each. The zooids are embedded in a clear gelatinous tunic and connected by a closed circulatory system. Materials and Methods Under field conditions, individuals of the cosmopolitan ascidian Botiyllits schlosseri pallas form compact, disc- shaped colonies (Fig. la), which occur in protected shallow marine environments, such as bays, harbors, and marinas, in temperate areas of both the northern and southern hemi- spheres (Chadwick-Furman and Weissman, 1995b, and ref- erences therein). Our experimental studies were conducted in the Monterey Municipal Marina, Monterey Country, Cal- ifornia (36°37.4'N;121°54'W), where colonies of B. schlos- seri are a dominant component of the fouling community on hard submerged surfaces (Chadwick-Furman and Weiss- man, 1995b). We observed colonies of B. schlosseri on submerged columns and docks in the Monterey Marina at depths from the surface to 1 m and determined the frequencies of natural contacts between these colonies and other encrusting macroorganisms. This survey was conducted during No- vember 1990, in the season of low abundance of fouling organisms in the marina (Boyd et til.. 1986; Carwile, 1989); thus our estimates represent minimal contact rates. Three hundred and nine colonies of B. schlosseri were observed in the marina for determination of their contact status. To test the effects of allogeneic contact on life-history traits in B. schlosseri, we set up three treatments using each of four cohorts of newly settled offspring from field-col- lected colonies. The four cohorts settled on 19 May 1990, 3 July 1990, 15 October 1990, and 25 January 1991 (after Chadwick-Furman and Weissman. 1995b). In each cohort, newly settled, one-system colonies of 1-7 zooids each ( = one circular system of zooids. Fig. la), were either (1) isolated on plates, (2) placed in incompatible pairs that rejected each other, or (3) placed in compatible pairs that fused. We distinguished between fusible and incompatible pairs by placing the small, one-system colonies into contact and observing the outcome. We used only colony pairs that established contact during the one-system stage of develop- Bulbous ampullae, or sacs of the circulatory system, are visible around the perimeter of the colony. This colony settled in May 1990. began sexually producing eggs in August, and produced a total of 683 eggs in four clutches before it died in September. (B) Fused chimeric colony at 142 days old. The left genotype, according to developmental characters (see text), con- sisis of 151 zooids; the right genotype, which is slightly darker, consists of 215 zooids. The line of fusion of their tunics is visible at center. Both members of this chimera settled in October 1990. came into contact and fused in January 1991, and died simultaneously in March 1991 at 149 days old, without having produced any eggs. (C) Pair of rejecting colonies at 68 days old. Both colonies settled in May 1990, and came into contact and rejected during June 1990. Along their interacting borders at center. 25 pairs of blood-system ampullae are in allogeneic tissue contact. The right colony, which consists of 129 decaying zooids. is in the process of senescing and dying. The left colony, which consists of 89 zooids, died one week later at 75 days old. Neither colony produced eggs. ALLOCONTACT AND ASCIDIAN FITNESS 135 merit. In each cohort, offspring from 10 field-collected colonies were assigned randomly to each of the three treat- ments (n = 8-36 newly settled colonies per treatment). A total of 274 colonies from all cohorts were monitored. Each experimental colony or pair of colonies was placed on LI glass plate measuring 5.0 X 7.5 cm and allowed to attach firmly for 1 week in the laboratory (after Rinkevich and Weissman, 1992a; Chadwick-Furrnan and Weissman. 1995b). Colonies that did not firmly attach to the plates, or that appeared damaged, were removed from the study at this point. All well-attached colonies were then transferred to the marina field site, in an area where abundant colonies of B. schlosseri grow naturally on fouling surfaces. About every 7 days, depending on the time of year, all the zooids in each colony passed through an asexual growth cycle (hereafter termed "cycle"). During each cycle, the zooids produced buds, then shrank and were replaced by their buds; thus a new asexual generation of zooids was formed in each colony. To examine cycle-related life-history traits, every 4-7 days we collected all the experimental colonies, ob- served them under a dissecting microscope in the labora- tory, and returned them to the field within a few hours (after Chadwick-Furman and Weissman. 1995a. b). We examined the following life-history parameters for each colony: ( 1 ) growth rate of somatic tissues, as measured by the number of clonal units (zooids. Fig. 1) produced per cycle; (2) age and size at sexual maturity, defined as the beginning of egg production; and (3) sexual reproductive output, as measured by the number of eggs produced by each zooid during each cycle, the number of cycles in which eggs were produced (# clutches), and the total number of eggs produced by each colony throughout its lifespan (fe- cundity) (after Sabbadin and Zaniolo, 1979; Sabbadin and Astorri. 1988; Chadwick-Furman and Weissman. 1995b). We assigned zooids in chimeric colonies to genotype on the basis of morphological and developmental characters, such as their relative positions in the chimera, the number of buds produced, and in some cases, color patterns (after Chad- wick-Furman and Weissman, 1995a; Yund et til., 1997). Since colonies were observed every 4-7 days, we counted directly the number of buds produced by each zooid at each cycle, and thus accurately assigned each new budded zooid to original colony genotypes in chimeras. All statistical analyses were performed using STATA, version 7.0 (Statacorp. 2001). Effects of allogeneic contact treatment on life-history traits were examined only within each cohort, since between-cohort comparison of life-his- tory traits were made previously (Chadwick-Furman and Weissman, 1995b). For life-history traits that were exam- ined on a per-cycle basis (i.e., number of zooids produced per cycle and number of eggs per zooid per cycle, see above), we measured the value for each cycle within a colony, but we present only the mean of these values for each colony. Thus, at one-way model was used in analyzing these traits. Log-transformed values of all life-history traits had approximately equal variances between treatment groups within each cohort, so ANOVA tests were applied to the data. Results Frequencies of natural contacts We observed high frequencies of natural contact between colonies of Botiyllus schlosseri and other encrusting macro- organisms. About one-third of all colonies (28.2%, n = 309) contacted encrusting bryozoans. Many colonies of B. schlosseri contacted the other colonial ascidians Botryl- loides violaceous (23.6%) or Diplosoma macdonaldi (4.8%), or individuals of solitary ascidians (5.5%). In addi- tion, 21.4% of Botrylliis schlosseri colonies occurred in allogeneic contact with conspecifics. Only one colony was observed to contact macroalgae (0.3%), and some colonies were isolated from contact with other sessile macroorgan- isms (16.2%). Morphology and growth Colony morphology was similar in all cohorts and exper- imental treatments. All colonies were flat and disc-shaped when small, with closely spaced groups of zooids (Fig. la). As they grew, some of the colonies developed irregular outlines, but the zooid systems remained compact and close together (Fig. Ib. c). In fused chimeric colonies, the area of fusion became barely visible over time, and some zooid systems straddled the area of initial fusion (Fig. Ib). The zooids of all genotypes in fused colonies appeared to grow constantly and to coexist in chimeras during their entire lifespan (Fig. Ib). We did not observe any shrinkage or somatic resorption of one genotype by another in chimeras. Until the time of chimeric colony senescence and death, robust blastozooids from all partners appeared to coexist within a single fused colony (Fig. Ib). Colonies that contacted noncompatible partners under- went rejection reactions that persisted along an extensive border of contacting tissues (Fig. Ic). As colonies grew, the contact area expanded along this border, and the number of points of rejection increased. Up to 15 points of rejection were observed during each sampling period throughout the lifespan of rejecting colonies. All rejecting colonies main- tained a long, continuous border throughout their lifespans, until one of them senesced and died (Fig. Ic). Pairs of rejecting colonies were compact, grew actively, and neither retreated nor grew away from each other. Colonies grew until they reached the edges of the glass culture plate, then grew around the plate edges, and contin- ued to spread over the back sides of the plate. None of the colonies filled all of the space available on both sides of the plate (Fig. 1). 136 N. E. CHADWICK-FURMAN AND I. L. WEISSMAN Juvenile colonies givw • ponentially, regardless of treat- ment (Fig. 2). Durir January and October, exponential growth began a .:g time of 3-5 cycles (= 32 to 64 days. Fig. 2). cohorts, colonies in the isolated treat- ment reachf :;ie largest maximum size (Fig. 2). This pat- tern persis... ,.; even in the October cohort, in which some isolated colonies experienced partial predation during cycle 9 that reduced their size to almost zero, after which they recovered and became the largest colonies in the cohort (Fig. 2). Growth rate slowed upon commencement of sexual reproduction in all cohorts (Fig. 2). In most cohorts, there was a significant effect of treatment on colony growth rate (Tables 1 and 2, Fig. 3a). Isolated colonies grew faster than did both rejected and fused colo- nies in the cohorts born during January and May (Table 2). In two of the cohorts, rejected colonies also grew faster than did colonies that fused to become chimeras (Table 2). In the October cohort, none of the fused colonies grew past the juvenile stage, and so were not included in statistical anal- yses of life-history differences among colonies that reached sexual maturity (Fig. 3, Tables 1 and 2). Sexual reproduction There was no effect of allogeneic contact treatment on the age at which colonies reached first reproduction, except in the May cohort, where rejected colonies reached sexual maturity at a significantly later age than did both isolated and fused colonies (Tables 1 and 2, Fig. 3b). The age at which colonies began to reproduce sexually appeared to be I oooo 1000 January •Isolated -Rejected -Fused pairs 10000 1000 May 0 5 10 15 20 25 30 Isolated Rejected Fused pairs 10 15 20 25 30 July Isolated Rejected Fused pairs 10000 1000 October isolated Rejected Fused pairs Age (cycles) Figure 2. Typical growth curves of colonies of the ascidian Botrylhis schlosseri for three allogeneic contact treatments and four cohorts in Monterey Bay. California. The shape of growth curves varied widely within each treatment, so mean and error values cannot be shown clearly here. Thus, only the largest colony in each treatment is shown for each cohort (for mean growth rates, see Fig. 3a). Note that colony size is plotted on a logarithmic • :ile. Arrows mark the commencement of sexual reproduction (egg production) for the first colonies to reach maturity in each cohort. Data on isolated colonies were published previously as Figure 1 in Chadwick-Furman and VvVissman (I995b). ALLOCONTACT AND ASCIDIAN FITNESS Table 1 137 One-wa\ ANOVAs of life-history traits hetu'cen allogeneic contact treatments within each of four cohorts of the colonial ascidian Botryllus schlosseri grown in Monterey Bay, California Life-history trait Cohort Source of variation DF Mean square F P Growth rate January Treatment i 0.105 10.44 *** IMI en 33 0.010 May Treatment 2 0.521 41.20 : Error 53 0,013 July Treatment 2 0.200 4.71 * Error 47 0.049 October Treatment 1 0.030 1.62 ns Error 8 0.019 Age at first reproduction January Treatment 2 0.356 3.02 ns Error 33 0.118 May Treatment 2 0.073 9.90 **# Error 53 0.007 July Treatment 2 0.016 1.03 ns Error 47 0.016 October Treatment 1 0.107 4.61 ns Error 8 0.023 Size at first reproduction January Treatment 2 1.953 3.72 * Error 33 0.525 May Treatment 2 14.400 41.66 *** Error 53 0.346 July Treatment 2 3.036 3.35 * Error 47 0.907 October Treatment 1 2.142 4.56 ns Error 8 0.470 Number of eggs/zooid/cycle January Treatment 2 0.641 3.25 ns Error 33 0.147 May Treatment 2 1.008 5.00 * Error 53 0.202 July Treatment 5 0.343 2.31 ns Error 47 0.148 October Treatment 1 0.305 4.10 ns Error 8 0.074 Clutch number January Treatment 2 2.696 9.83 *** Error 33 0.274 May Treatment 2 0.179 1.34 ns Error 53 0. 1 33 July Treatment i 0.795 2.35 ns Error 47 0.339 October Treatment 1 1.692 12.04 ** Error 8 0.140 Fecundity January Treatment 2 12.S44 15.79 *#* Error 33 0.813 May Treatment 2 19.587 25.71 *** Error 53 0.762 July Treatment 2 4.695 3.17 * Error 47 1.480 October Treatment 1 16.656 23.43 ** Error 8 0.711 ' P < 0.05; ** P < 0.01: P < 0.001: ns = not significant. controlled mainly by environmental factors, such as tem- perature, that varied with season of birth (see Chadwick- Furman and Weissman. 1995b). Variation in the size of colonies at first reproduction followed the same pattern as did colony growth rate (Table 2). but the differences between groups were magnified (compare Figs. 3a and c). In both the January and May cohorts, isolated colonies, which grew relatively rapidly as juveniles (Fig. 3a), were significantly larger at maturity than were fused and rejected colonies (Fig. 3c, Table 2). Where 138 N. E. CHADWICK-FURMAN AND 1. L. WEISSMAN Table 2 Tnk.e\-Kramer mii!i :• !i TO h wv for differences in life-history traits beftveen all "'act treatments within each of 4 cohorts of the colonial a- m) llus schlosseri grown in Monterey Bay, California Life-history trait Cohort 1 "reatment* Growth rate (# buds/zooid/cycle) January >RF May >R~SF July R>F October ~R Age at first reproduction (# cycles) January ~RF May F> R July ~RF October R Size at first reproduction (# zooids) January > RF May >R~SF July R > F October ~R Number of eggs/zooid/cycle January "RF May >RF July RF~ October R Clutch number January >RF May RF~ July RF October > R Fecundity (total # eggs/colony) January >RF May > RF July _RF~ October > R * Symbols for treatments: I = isolated, R = rejected, F = fused. Treatments that did not differ significantly (P > 0.05) are conjointly underlined. > signs indicate which treatments had significantly larger values of each life-history trait within each cohort. In the October cohort, none of the fused colonies survived to reproduce, and so they were not included in analysis of variation in life-history traits among colonies that survived to maturity. there were significant differences, isolated colonies were, on average, 1.5-2.5 times larger at sexual maturity than re- jected colonies, and 2-3 times larger than colonies that fused to form chimeras (Fig. 3c). The number of eggs produced per zooid per cycle ( = reproductive effort) varied widely between colonies within each treatment, and did not vary between treatments, except in the May cohort (Table 1, Fig. 3d). For colonies born during May, isolated individuals produced significantly more eggs per zooid per cycle than did colonies in either of the allogeneic contact treatments (Table 2). The total number of egg clutches produced by each colony was affected by treatment in only the two cohorts that overwintered, those born during October and January (Table 1, Fig. 3d). In both cases, colonies that were isolated from contact produced significantly more egg clutches than did those in either of the two allogeneic contact treatments (on average, 2-3 times more clutches. Fig. 3e, Table 2). The lifetime fecundity of colonies varied significantly with treatment in all cohorts (Table 1 ). The combined ef- fects of relatively rapid somatic growth, large size at matu- rity, and a large number of egg clutches in the isolated colony treatment (Fig. 3a-e) resulted in much higher life- time fecundity in isolated than in either the fused or rejected treatments (Fig. 3f. Table 2). The mean fecundity of isolated colonies ranged from 1.8 to 2.5 times that of fused or rejected colonies in summer cohorts (May and July). In the winter cohorts (January and October), the mean fecundity of isolated colonies was more than 5-10 times that of fused or rejected colonies. Fused colonies that were born in October did not produce eggs at all (Fig. 30. Colonv longevity and survivorship Colonies in all treatments and cohorts had short, suban- nual lifespans (Fig. 4). Within each cohort, colonies in all treatments reached sexual maturity at about the same age (Fig. 3b). reproduced sexually for a few cycles, and then all died within a few cycles of each other (Fig. 4). The per- centage of colonies that survived to reproduce was high and did not vary significantly among treatments in the January and May cohorts (chi-square tests, ;t o.05(2) = 5.99, G - 0.68 and 3.78 respectively. Fig. 5). In the July cohort, survivorship also was high, but did vary with treatment; rejected colonies had the lowest survivorship to maturity (G = 13.16). Colonies born in October had low survivor- ship that did not vary significantly with treatment, even though all colonies in the fused treatment died as juveniles (G = 5.04. Fig. 5). Colony longevity in all treatments and cohorts was con- trolled mainly by the timing of colony senescence (Fig. Ic). Senescence occurred in four distinct stages that began 1-2 weeks before death (details in Chadwick-Furman and Weissman, 1995b). The stages of senescence did not vary with treatment or cohort. When senescence began in the zooids of one genotype, it spread to the zooids of fused, but not rejected, partners (Fig. Ib, c). After one of the partners in a rejecting pair died, the other colony continued to live for a few cycles (Fig. Ic). Discussion We document here that allogeneic contacts, whether they lead to fusion or rejection, result in significantly reduced fitness in field-grown colonies of the colonial ascidian Bot- ryllus schlosseri. This is the first demonstration in a proto- chordate that, under natural field conditions, allogeneic con- tacts leading to both fusion and rejection come at a cost to life-history processes such as growth and reproduction. We also show that colonies of Botryllus schlosseri in the wild frequently contact those of conspecifics and of other species of sessile invertebrates, so associated fitness costs may be a ubiquitous and important phenomenon in nature. Contact rates with the colonial ascidian Botr\lloiikjs viola- AU.OCONTACT AND ASCIDIAN FITNESS 139 D Isolated H Rejected • Fused A B (18X6X12) C5K6IC5) Cl 1(131(16) (/X3)(0) E z 6000 i ??4000 - = 2000 - January July October January October Cohort Figure 3. Variation in life-history traits among three allogeneic contact treatments and four cohorts in the colonial ascidian Botryllus .schlnsseri, grown in Monterey Bay, California. Note that for fused colonies, traits are presented for each genotype within the colony. Means plus one standard deviation are shown. Sample sizes for all life-history traits are given in parentheses in graph A. Sample sizes are low in some groups due to mortality of some colonies before reaching sexual maturity (compare sample sizes with those in Fig. 5). Data on isolated colonies were published previously as Figure 2 in Chadwick-Furman and Weissman (1995b). ceous were especially high at our site (see Results). Yet, because our survey of contact frequencies was based on a one-time observation, which inherently underestimates con- tacts throughout the life of a colony, lifetime contact rates between colonies of Botrvllus xchlosseri and other sessile organisms at Monterey are even higher than those presented here (see Results). Our limited manipulation of colonies in one of the cohorts of Botnilux schloxseri (born on 15 October 1990) indicates that xenogeneic interaction with Botrylloides violaceous results in a level of fecundity inter- 140 N. E. CHADWICK-FURMAN AND I. L. WEISSMAN 100 T 10 : .£• 1 o 7 u January 100 i - Isolated (N=28) - Rejected (1M=8) - Fused (N= 16) May - Isolated (N=35) - Rejected (N=14) - Fused (N=36) 100 q 10 : 25 30 July 100 - Isolated (N=26) - Rejected (N=28) - Fused (N=24) 10 15 20 25 30 October - Isolated (N=33) - Rejected (N=14) - Fused (N=12) 10 15 20 25 30 10 15 20 25 30 Age (cycles) Figure 4. Survivorship curves for colonies of the ascidian Bittnllits schlosscri grown in Monterey Bay, California, in four cohorts and three allogeneic contact treatments. Arrows indicate the commencement of sexual reproduction in each cohort. The last point in each line represents the last surviving colony of each group. Note that survivorship is plotted on a logarithmic scale. Data on isolated colonies were published previously as Figure 3 in Chadwick-Furman and Weissman (I995b). mediate between those of isolated and allocontacted colo- nies [total number of eggs produced = 1383 + 769 (x + SD), n = 9 xenocontacted colonies of Botryllus schlosseri that survived to maturity, N. E. Chadwick-Furman, pers. obs.; compare with October cohort in Fig. 3f], Thus, xeno- geneic contact appears to affect colony fecundity, but not as severely as allogeneic contact. The reduced fitness of colonies following fusion or re- jection may result from energetic or physiological costs associated •• ith recognizing and reacting to non-self tissue. The process oi interaction along the borders of rejecting colonies involves extensive tissue damage and resource demand on both colonies (Scofield and Nagashima, 1983: reviewed in Rinkevich, 1992). In addition, competition be- tween somatic and germ cell lines within fused chimeras also may draw heavily on the physiological resources of the partners involved (Buss, 1982). Colonies that are isolated from allogeneic contact do not face these costs. The lack of resorption observed here in field-raised chi- meras of Botiyllus schlosseri is in striking contrast to pre- vious results from laboratory studies (Rinkevich and Weiss- man, 1987, 1992a, b; Pancer et al., 1995). The reduced chimeric stability of laboratory colonies has been demon- strated by growing genetically identical replicates of chime- ALLOCONTACT AND ASCIDIAN FITNESS 141 D Isolated CD Rejected • Fused (281(81(161 January (351(141(361 ' (261(28)124) May July Cohort (33H14M12) October Figure 5. Variation in percent survivorship to first reproduction among four cohorts and three allogeneic contact treatments of colonies of the ascidian Bottyllus schlosseri grown in Monterey Bay, California. Sample sizes for each treatment are given in parentheses. ras under field versus laboratory conditions (Chadwick- Furman and Weissman, 1995a). The present results show that, under field conditions in Monterey, the partners of a chimera appear to grow in a stable manner and do not undergo somatic resorption. Previous field studies indicate a high level of environmentally dependent plasticity in fit- ness-related life-history traits of B. schlosseri (Chadwick- Furman and Weissman, 1995a; Yund et al., 1997). The reduced reproductive success of interacting versus isolated colonies (Fig. 3 and Table 1 ) reveals effects of allogeneic contacts on sexual reproduction as well as on somatic tissue production. Recruitment of larvae at Monterey in the springtime may be derived from a small number of parent colonies that overwinter (Carwile, 1989; Chad wick- Furman and Weissman, 1995b). Thus, the costs of interactions in this experiment would have resulted in reduced representation of the offspring of winter allo-con- tacting colonies in the summer bloom. Allogeneic interactions do not alter the survivorship of colonies in most cohorts (Fig. 5). The longer lifespans (Figs. 3b and 4) and lower survivorship (Fig. 5) of colonies during winter, as compared to summer, appear to be due to a slowing of colony growth and development during low temperatures in the winter in Monterey Bay (Boyd et al., 1986; Chadwick-Furman and Weissman. 1995b). As found in past studies, whole-colony senescence causes the death of most colonies (Chadwick-Furman and Weissman. 1995a, b) and is genetically controlled (Rinkevich et ui. 1992). At the time of our experiments, we did not have markers to identify the genotypes of blood cells, bud cells, or ga- metes in the fused colonies, and so we did not test for somatic or germ cell parasitism as a result of colony fusion. However, germ cell parasitism has been reported in this species (Rinkevich and Weissman, 1987; Sabbadin and Astorri. 1988; Pancer et al.. 1995), and recent work has verified that it occurs in both male and female gametes capable of fertilization (Stoner and Weissman, 1996; Stoner et al., 1999). The process of germ cell parasitism, in which one partner in a chimera uses the somatic resources of the other to produce its own germ cells, may alter the relative fitness of fused genotypes in chimeras (Buss, 1982; Stoner and Weissman, 1996; Stoner et al., 1999; Weissman, 2000). However, because the fitness of fused pairs of genotypes was less than half that of isolated colonies in all cohorts (Fig. 3f), the reproductive output of all the genotypes com- bined in chimeric colonies was less than that of genotypes in isolated colonies. Thus, germ cell parasitism may alter the relative amount of fitness lost due to fusion in chimeric colonies, but cannot prevent an overall reduction in fitness due to fusion. Even if germ cell parasitism were extensive in the chimeras tested here, chimera formation causes reduced fecundity, regardless of which genotype dominates (Fig. 3f). In 30% of the field chimeras examined by Stoner and Weissman ( 1996) at the same Monterey marina site, little or no germ or somatic cell parasitism was found. Thus, the values presented here for genotype-specific measures of fitness (Fig. 3) may represent realistic estimates for at least some chimeras that retain a stable genetic composition in the wild. A drawback of the present study is that we could not set up, as controls, undissected pairs of isogeneic colonies, to determine whether isogeneic contact affects fitness. Thus, an evaluation of the actual costs of allogeneic contact per se is problematic. However, set-up of this control group would have required dissecting apart and re-uniting systems from multi-system colonies, thus introducing further manipula- tion of all colonies in this experiment. As the colonies grew, they produced lobes of tissue that contacted along their edges and fused along the undulating margins of the colony in all treatments (Fig. Ib. c). Thus, if isogeneic contact affected fitness, it did so equally in all treatments here. We show here that egg production in fused colonies is greatly reduced (Fig. 30. possibly due to competition be- tween the genetically different individuals that fused to make up that colony. Thus, one benefit of precise allorecog- nition in this species may be that it limits the unit of selection to chimeras composed of closely related kin (Grosberg and Quinn. 1986; Rinkevich and Weissman. 1987; Stoner and Weissman, 1996; Stoner et al.. 1999). Because of the high polymorphism of the Fii/HC gene locus (that permits fusion rather than rejection to occur; Scofield et al., 1982), fused individuals in the wild most likely represent kin rather than a random assortment of genotypes (Grosberg and Quinn. 1986). In Botryllus schlosseri. the proportion of fusions occurring between siblings is higher 142 N. E. CHADWICK-FURMAN AND I. L. WEISSMAN than between nonsiblinus (Scofield et ai, 1982; Magor et til., 1999). Thus. Mr to genetic inheritance for chimeric colonies of fusee i would be the outcome of germline competition K:i rfti the mother colony and the diverse sperm thai i r^ed her. In addition to kin fusion, regulated by Fu/HC matching, kin cosettlement is encouraged by the limited dispersal of tadpole larvae from the maternal colony and nonrandom cosettlement according to shared Fu/HC genotype (Grosberg and Quinn. 1986). A common selected trait in these chimeras is allele-sharing at the Fu/HC locus (Weissman et til., 1990). Kin selection would act also on shared genes other than the selected Fu/HC types that are common to these siblings. Reproductive outcomes in these chimeras could be as simple as the direct gametic represen- tation of the diverse blastozooid units in the chimera: or could be as complex as the outcomes of selective resorption or germ cell parasitism that generate skewing from that simple representation (Pancer et til.. 1995; Stoner and Weissman, 1996; Stoner et til., 1999; Weissman, 2000). No matter whether allogeneic colony contact results in fusion or rejection, if it leads to reduced fitness, as measured by growth and fecundity, with no increase in survivorship, why have these organisms developed and maintained an elaborate system of allorecognition? Perhaps, in this spe- cies, genetically based allorecognition is nonadaptive. It may be linked to other processes that are adaptive, and thus have evolved as a by-product of processes such as disease recognition (Buss and Green. 1985; Magor ct til., 1999) or gametic compatibility (Scofield et til.. 1982). However, the ability to recognize and reject nonrelated colonies, and to fuse only with closely related kin that share alleles at the Fii/HC locus, may be directly beneficial in that it reduces the costs of germ cell parasitism in colonies (Stoner et til., 1999). The phenomena of cosettlement. fusion, and development of reproductive competence in chimeras are not limited to protochordates, and may be important selective factors in other sessile organisms as diverse as fungi, sponges, and cnidarians (reviewed in Buss. 1982; Rinkevich and Weiss- man, 1987; Pancer etui, 1995). Our findings that allogeneic contact, and especially chimera formation, reduce individ- ual fitness under natural field conditions may have broad implications for the evolution of allorecognition systems. Acknowledgments We thank Buki Rinkevich for discussions and collabora- tions that led to and resulted from these studies. We also thank Kathi Ishizuki and Kurlu Palmeri for assistance in data collection. Simona Bart! and Robert Lauzon for dis- cussions, and Uri Frank and Buki Rinkevich for comments that improved the manuscript. Karen Tarnaruder assisted with the graphics. Prof. Maureen Lahiff of the School of Public Health at the University of California at Berkeley is gratefully acknowledged for advice on statistical analysis. Funding was provided by a Frederick B. Bang Scholarship from the American Association of Immunologists, and PHS Grant CA09302 awarded by the National Cancer Institute. DHHS. to N.E.C.-F.. and by PHS Grant CA42551 to I.L.W. Literature Cited Boyd H. C., S. K. 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Marcum, and J. Stewart-Savage. 1997. Life-history variation in a colonial ascidian: broad-sense heritabilities and tradeoffs in allocation to asexual growth and male and female reproduction. Biol. Bull. 192: 290-249. Reference: Bio/. Bull. 205: 144-159. (October 2003) © 2003 Marine Biological Laboratory Effect of Disturbance on Assemblages: an Example Using Porifera J. J. BELL1'* AND D. K. A. BARNES2 Department of Zoology ami Animal Ecology, University College Cork, Lee Mailings, Co. Cork, Ireland; and 2British Antarctic Sun>ey, N.E.R.C., High Cross, Madingley Road, Cambridge CB3 GET, United Kingdom Abstract. Extensive sponge assemblages are found in a number of habitats at Lough Hyne Marine Nature Reserve. These habitats are unusual in experiencing a range of envi- ronmental conditions, even though they are only separated by small geographic distances (1-500 m), reducing the possibility of confounding effects between study sites (e.g., silica concentrations and temperature). Sponge assemblages were examined on ephemeral (rocks), stable (cliffs), and artificial (slate panels) hard substrata from high- and low- energy environments that were used to represent two mea- sures of disturbance (flow rate and habitat stability). Sponge assemblages varied considerably between habitat types such that only 26% (25 species) of species reported were com- mon to both rock and cliff habitats. Seven species (of a total of 96 species) were found in the least-developed assem- blages (slate panels) and were common to all habitats. Sponge assemblages on rocks and panels varied little be- tween high- and low-energy environments, whereas assem- blages inhabiting cliffs varied considerably. Assemblage composition was visualized using Bray-Curtis similarity analysis and Multi-Dimensional Scaling, which enabled dif- ferences and similarities between sponge assemblages to be visualized. Cliffs from high- and low-energy sites had dif- ferent assemblage compositions compared to large rocks, small rocks, and panels, all of which had similar assem- blages irrespective of environmental conditions. Differ- ences in assemblages were partially attributed to sponge morphology (shape), as certain morphologies (e.g., arbores- cent species) were excluded from 2-D rock habitats. Other mechanises were also considered responsible for the sponge assen, vuges associated with different habitats. Received 27 August 2001: accepted 23 May 2003. * To whom correspondence should be addressed. Current address: In- stitute of Biological Sciences. Edward Llwyd Building. The University of Wales. Aberystwyth, Ceredigion SY23 3DA. Introduction The development of any community (e.g., mature or young) is controlled by a suite of biological and physical factors that may be closely related and interlinked (Buss and Jackson, 1979). In some marine environments, such as caves, communities (all species) are well developed (ma- ture); in others, such as ice-scoured polar shores, all com- munities tend to be poorly developed (young). Yet there are other localities that contain communities in various stages of development in proximity to one another. Lough Hyne is such an example, where adjacent communities may experi- ence some similar conditions (e.g., flow rate, temperature, food concentration) but are at different stages of develop- ment. For example, rocks in areas with fast currents, where disturbance is high, may have less well-developed commu- nities than rocks in areas experiencing slight current flow, where disturbance is reduced (Maughan and Barnes, 2000a), even though they are separated by less than 300 m. The same may be true for sublittoral cliff habitats where climax community states may be achieved only under cer- tain environmental conditions. In the case of rocks, size may also be important to community development because smaller rocks are more likely to be moved than larger rocks, causing differences in community (all species) and assem- blage (component phyla) composition (Barnes et al., 1996). Unstable or highly disturbed (e.g., small rocks) habitats are often colonized by fast-growing, opportunistic, and r-stra- tegic species that are able to quickly take advantage of newly created habitats or space (Lilly et al., 1953; Osman. 1977; Sousa, 1979; Barnes et al.. 1996; McCook and Chap- man. 1997). Sponges are an important component of hard-substratum communities throughout most polar (e.g., Dayton, 1978). temperate (e.g., Hiscock et al., 1983; Bell and Barnes. 2000a), and tropical regions (e.g., Alcolado, 1990). There is a wide body of literature that considers the influence of 144 SPONGB COMMUNITY COMPOSITION 145 environmental parameters on the composition ot sponge assemblages (e.g., Wilkinson and Cheshire. 1989; Alcolad, 1990; Alvarez et til., 1990; Diaz et ul.. 1990; Schmahl. 1990; Witman and Sebens, 1990). There are also examples of studies comparing sponge assemblages on large (100- 1000 km) spatial scales (Maldonado and Uriz, 1995; Hooper et al.. 2002) and on smaller scales (2-20 km) between sponges inhabiting mangrove pools (Riitzler et til.. 2000). However, comparisons between different habitat types (e.g., between loose rock and cliffs) under fixed environmental conditions or on local scales (hundreds of meters) where confounding effects are reduced are less common. Studies of sponge assemblages suggest that a number of physical factors control species distributions; these factors include water flow rate (Bell and Barnes, 2000a), sedimen- tation (Konnecker, 1973). nutrient levels (Storr, 1976), depth (Alvarez et til.. 1990; Witman and Sebens, 1990). light (Sara et al., 1978; Cheshire and Wilkinson, 1991 ), and habitat availability (Konnecker, 1973; Barthel and Tendal. 1993). Habitats classified within these physical parameters (e.g., fast water flow at a depth of 20 m) often contain many smaller or cryptic habitats with their own predefined phys- ical characteristics. For example, at a site of slight water flow, where sedimentation is high, the amount of sediment falling on inclined, vertical, or overhanging cliff surfaces varies considerably, resulting in different assemblages on each surface type (see Bell and Barnes, 2000b. c). Although not as well studied, biological factors are also known to influence the composition of sponge assemblages (see Paine. 1974; Wulff, 1995, 2000), and predation may also limit the local distribution of sponge species (Dunlap and Pawlik, 1996; Wulff. 2000; Bell, 2001). Rich sponge as- semblages on sublittoral cliffs at Lough Hyne have been the focus of recent studies, but abundant and diverse sponge populations also occur on the undersides of loose rocks (Lilly et til.. 1953: van Soest and Weinberg, 1980; van Soest et al., 1981). At many sites within Lough Hyne, these two habitats can be separated by less than 1 m, providing the opportunity to investigate differences between sponge as- semblages occurring in habitats experiencing different lev- els of stability, without the confounding effects created by larger spatial scales. The high overall diversity and richness found on hard substrata within Lough Hyne, coupled with the large range of local habitat stability, results in assemblages at various stages of development existing in proximity. Such environ- mental characteristics provide opportunities to examine the contribution of certain taxa (in this case sponges) to the overall community. This study investigates assemblages inhabiting three substratum types — artificial panels (the most ephemeral), rocks, and cliffs (the most stable) — which represent a qualitative series in terms of habitat stability (i.e.. amount of disturbance). The degree of development of sponge assemblages at different levels of habitat stability can be investigated within a particular environmental re- gime (e.g., fast flow rates). Also, the use of a common substratum (i.e.. panel, rock, and cliff) between sites with different environmental conditions (flow regime) gives a second environmental gradient based on flow-rate-gener- ated disturbance (rather than habitat stability). This study attempts to answer four questions: ( 1 ) How do sponge assemblages vary with environmental conditions and habitat stability? (2) How does the composition of sponge assem- blages vary with water flow rate compared with different rock sizes; both are surrogate measures of disturbance, so do they have similar consequences? (3) Are there discriminat- ing species for (local) habitats of differing stability and flow rates? (4) Are there consistent similarities or differences in assemblage composition in extremes of flow rate and habitat stability? Materials and Methods Stutl\ site Lough Hyne Marine Nature Reserve (Fig. 1 ) is a small (0.5 km2) temperate sea lough on the southwest coast of Ireland (51°29'N, 9°18'W). It is characterized by a large number of habitats within a small area (Kitching, 1987). Habitats range from current-swept cliff faces to soft-sedi- ment basins where water currents are slight. The lough is connected to the adjacent Atlantic coast by a shallow and narrow channel (The Rapids). This constriction results in an unusual tidal regime whereby water flows into Lough Hyne for about 4 h and out for 8 h. The squeeze causes fast current velocities (>250 cm s~') in the eastern parts of the lough during inflow, but only slight surface currents in the vicinity of the rapids during outflow (Bassindale et al.. 1957). As water moves from east to west across the lough, there is a quick reduction in current flow rate with a corresponding increase in sedimentation. The small size of Lough Hyne means that communities are only separated on small spatial scales (hundreds of meters). Nevertheless, these communities are discrete be- cause cliff and rock habitats in different parts of the lough are separated by soft sediments. Sponge assemblages are thus isolated and predictable, rather than occurring along a continuous gradient. Different substratum types (i.e., rocks, cliffs, and panels) within a specific environment were sep- arated by less than several meters and therefore not discrete. Panels were sited at Labhra Cliff and Whirlpool Cliff, and rock habitats were sampled at West Cliff and Whirlpool Cliff (Fig. 1). West Cliff and Labhra Cliff have similar sedimentation and rates of water flow, and the sponge assemblages are also similar. Both sites are characterized by slight current flow rates (<5 cm s~") and heavy sediment accumulation, which increases with depth. Hard substratum extends more than 30 m at both of these sites. Rocks were not sufficiently abundant at Labhra Cliff to sample. Whirl- pool Cliff experiences a very fast (>200 cm s~'), unidirec- tional flow regime, resulting in high disturbance. This site 146 J .1 BELL AND D. K. A. BARNES Figure 1. Sites where sponge rock and cliff assemblages were sampled in Lough Hyne Marine Nature Reserve. extends to about 18 m, and current flow rates decrease with depth, falling to 100 cm s~' at 18 m (Fig. 2). Some data were taken from the literature (Bell and Barnes, 2000a) to enable greater comparison between sponge assemblages from different habitats within Lough Hyne. These data, which were included in the analysis, concerned sponge assemblages inhabiting vertical and inclined surfaces on sublittoral cliffs at Labhra Cliff, West Cliff, Whirlpool Cliff. and Bullock Island (an adjacent Atlantic coastal site with a turbulent flow regime). The tidal range within Lough Hyne is about 1.5 m. Sampling and observation methods The artificial substrata (panels) used were square ma- chined slate panels (15 x 15 cm), prepared and assembled as for Turner and Todd ( 1994). They were used to investi- gate an ear!> pioneer stage in community succession. A blue square (10 cm K 10 cm) was drawn in the center of each panel with a permanent blue marker pen. Panels were placed in running water iVr 24 h and then dried; this process was repeated twice more prior to deployment. The blue back- ground makes it easier to identify recruits that are small or translucent. Three panels (forming one panel array) were attached by cable ties to welded steel bars. The panel array was positioned with the blue surfaces facing down (to simulate the undersides of a boulder). Bolts at the corner of each steel frame allowed the panels to be adjusted so they were 20 mm above the substratum. Panel arrays were de- ployed at depths of 0 m. 6 m, and 12 m at Labhra Cliff and Whirlpool Cliff. The first panels were deployed at the start of October 1997 and were replaced bimonthly until March 2001. Before panels were replaced, they were cleaned using a razor blade. Each panel was examined under a binocular microscope, and the number of sponge recruits was re- corded. Spicule preparations of sponge recruits were made to confirm identification. Additional panel arrays were placed at the same depths, but were not replaced bimonthly. Each panel was photographed 1, 2, 6, and 12 months after the date of deployment. Photographs were then projected onto a grid composed of 400 random dots, and the percent- age cover of sponges on each panel was estimated. The mean percent cover was calculated for panels submerged at each time interval. Twenty-five rocks were randomly selected within three size classes — small (10-150 cm2), medium ( 151-500 cm2), large (501-1000 cm s~'), and very large (1001-2000 cm s ' ) such that equal numbers of boulders of each size were examined (i.e.. this does not represent the true boulder size distribution). The surface areas of both upper and lower rock surfaces were measured using a transparent cloth marked in square centimeters. The number of sponges (number of patches) for each species on each rock was recorded. Each species was photographed, and samples were taken for spicule analysis to confirm identification. Sponge samples were dissolved in concentrated nitric acid, or bleach as required, and were examined through a com- pound microscope at high power (X400). All observations were made between October 1999 and February 2000, which eliminated fuunistic inter-site differences caused by seasonal growth or tissue retraction over the study period. The morphologies of all sponges observed on rocks were classified within the following groups (after Boury-Esnault and Ruizler, 1997; Bell and Barnes, 2000d): encrusting (EN), flabellate (FL), clathrate (CL), massive (MA), arbo- rescent ( AR). repent (RE), tubular (TU), ficiform (FI), mas- sive globulose (MG), and papillate (PA). Data visualization and statistical procedures Data were subjected to Bray-Curtis similarity analysis using hierarchical agglomerative group-average clustering for all habitats and depths (including data from Bell and Barnes, 2000a). This analysis was performed using the unweighted pair group method using arithmetic averages (UPGMA) with the PRIMER program (ver. 5.2.8. Plymouth Marine Laboratory, Plymouth. England). Data were log (.v + 1 ) transformed to reduce the importance of extreme SPONGK COMMUNITY COMPOSITION 147 T3 U OT 60 40 30 20 10 0 60 50 - 40 • c £ a 30 C "-^ •3 OJ 00 20 10 60 50 - 40 30 ] 20 10 60 c 2? 3 on - 50 • 40 • 30 • 20 10 0 Glannafeen APR 99 MAY 99 JUN 99 JUL 99 AUG 99 SEP 99 OCT 99 NOV 99 DEC 99 JAN 00 FEBOO MAR 00 APR 00 MAY 00 Goleen APR 99 MAY 99 JUN 99 JUL 99 AUG 99 SEP 99 OCT 99 NOV 99 DEC 99 JAN 00 FEB 00 MAR 00 APR 00 MAY 00 Labhra Cliff APR 99 MA Y 99 JUN 99 JUL 99 AUG 99 SEP 99 OCT 99 NOV 99 DEC 99 JAN 00 FEB 00 MAR 00 APR 00 MAY 00 West Cliff APR 99 MAY 99 JUN 99 JUL 99 AUG 99 SEP 99 OCT 99 NOV 99 DEC 99 JAN 00 FEBOO MAR 00 APR 00 MAY 00 Month Figure 2. Annual variation in sedimentation rates (g sediment m :d ') at 6-m depth intervals at tour sublittoral cliff sites within Lough Hyne. 6 m (•) 12 m (D) 18 m (T) 24 m (O) 30 m (•). Standard error bars are shown. [After Bell (2001).] values (rare species). Ordination by non-metric Multi-Di- mensional Scaling (MDS in PRIMER) was undertaken on a dissimilarity matrix created from Bray-Curtis similarity analysis. SIMPER analysis (in PRIMER) was used to de- termine the contribution of each species to the average Bray-Curtis dissimilarity between habitats and sites. This method of analysis determines which species are responsi- ble for any differences that occur. Data were transformed as for Bray-Curtis analysis. Because sampling areas differed between habitat types, the area of each habitat and the abundance of each species were scaled up or down to standardize sampling areas between sites, depths, and hab- itats (sponges m~~). Patterns of species diversity for sponge assemblages on 148 J. J. BELL AND D. K. A. BARNES rocks were described v-i;!i the Shannon and Weiner (Krebs, 1989) informal!'"- n tion H' -S/?,/» /',. Paired Stu- dent's t tests we- . d to examine differences between rock surface typ • . -neral linear model analysis of variance (GLM • . \) was used to compare logarithmically (LogH •. .--.lornied sponge species diversity, richness, and rock surface area relationships between West Cliff and Whirlpool Cliff. The same method was used to compare the untransformed relationship between sponge density and rock surface area. This method compares the average linear relationship to an average linear relationship fitted to all the data. Kruskall-Wallis tests were used to compare the differ- ences between percentage cover on submerged panels after 1, 2, 6, and 12 months. Results Assemblage composition A total of 96 species were reported during this study, 44 of which were found on rocks and 76 from cliff habitats (Table 1 ). Twenty-five sponge species were shared between rock and cliff habitats. All but two of the species (Hvmeni- acidon perlevis and Halichondria bowerhanki) of sponge found on rocks occurred on the undersides. These two species were often found growing on the fronds of algae as well as on rock surfaces. Although sponge assemblages varied considerably between habitat types, there was little variation between rock sponge assemblages inhabiting the different sites (extremes of water flow rate) (Table 2). Of the 44 species inhabiting rocks, only 7 (16%) differed between West Cliff and Whirlpool Cliff, with only 1 of these species (Mycale rotalis) being found exclusively at Whirlpool Cliff. The remaining 6 species were only found at West Cliff. Nineteen of the 44 species found on rocks were exclusive to rock habitats, illustrating the high degree of species exclu- sivity within this habitat type. The panels at Labhra Cliff and Whirlpool Cliff showed no exclusive species, and only 7 species of sponge were reported (Table 1 ). Therefore, no juvenile settlement was recorded for the majority of sponge species reported during this study despite the nearly 4-year duration of panel deployment. All species found on panels occurred in both rock and cliff habitats. Because panels can be considered a similar habitat to rocks (but younger in terms of community development), community age was the most likely determinant of the composition of the sponge assemblage. Of the total 76 species of sponge reported from cliff habitats, 31 species (data included from Bell and Barnes, 2()00a) were found at Bullock Island, compared to 40 species at Whirlpool Cliff and 52 from both Labhra Cliff and West Cliff (Table 3). Although many of these species were shared between cliff habitats, only 10 of the total 76 species were shared between all cliff habitats. Environmen- tal parameters, therefore, proved important in determining the sponge assemblage in cliff habitats. A much higher proportion of species were shared within the same habitat than between different habitat types (Table 3). However, the proportion of species shared between sites was higher for rock and panel habitats (>85%) than for cliff habitats Sponge morphology On rocks, encrusting morphologies were the most abun- dant form (20 of 44 species), followed by massive (15 of 44 species) and repent forms (7 of 44 species) (all pooled rock data). The remaining species exhibited cylindrical (Scypha ciliatum) and clathrate forms (Clathrina coriacea). On cliff surfaces, 30 species were encrusting, 19 were massive, and 6 exhibited repent forms (pooling cliff data). Sponge species on panels had a wide range of morphologies (given the small number of species), including tubular, repent, encrust- ing, massive, and clathrate forms. Differences were ob- served between the proportions of sponge morphologies between certain habitats and sites (Table 4). There was a considerable difference between the proportion of morphol- ogies between cliff sites (Table 3). Encrusting forms were more abundant at the high-energy cliff sites (Bullock Island and Whirlpool Cliff) than at low-energy sites (Labhra Cliff and West Cliff), where they were replaced by arborescent and papillate forms. In rock habitats, high proportions of encrusting and massive forms were found, with no differ- ences between sites. Panel assemblages were also very similar in the proportions of morphologies between sites. Identification of different sponge assemblages Small sponges were difficult to identify, (particularly on panels) and potentially represent an error in the analysis. However, this problem was reduced by the transformation of the data prior to analysis, which decreased the importance of extreme values or apparent rare species. Bray-Curtis similarity analysis and Multi-Dimensional Scaling (MDS) (Fig. 4) were used to distinguish between sponge assemblages within different habitats and sites. The Bray-Curtis analysis identified five major assemblage groups that showed increasing similarity, as follows: rocks (all sites and sizes), panels (all sites), Whirlpool Cliff, Bullock Island, and Labhra Cliff/West Cliff. However, each major assemblage group had a very low similarity (<25%) with the others. MDS distinguished two additional, smaller assemblages, which were not obvious from the Bray-Curtis analysis. The first of these assemblages was at 30 m at Labhra Cliff; it showed the greatest affinity with the other cluster at Labhra Cliff and West Cliff (cliff habitat). The second assemblage was composed of 0-m sponges at Labhra Cliff and West Cliff (cliff habitat). These assemblages were most similar to those at Whirlpool Cliff. The subtidal (6 and 12 m) panels had high levels of correspondence (=65%) between sites, but differed considerably from those in the intertidal zone (0 m) (=30% similarity). To account for morphological differences created by the nature of the dif- SPONGE COMMUNITY COMPOSITION 149 Table 1 Sponge species found within Lough Hyne on sublittoral cliff and rocks and on the adjacent Atlantic coast B W L U H A W c B U W H L W A C Antho ini'oh-ens (EN) * * * * * * Hymeraphia stellifera (EN) lophon hyndnumi (EN) lophon ingalli (EN) * * * * * * * Antho inconstant # (EN) Aplvsilla rosea (EN) Aplysilla sulfurea (EN) lophonopsis nigricans (EN) Axinella damicornis (FL) Axinella dissimilis (AR) Biernnti variantia (MA) Clathrina coriacea (CL) Cliona celala (MA) Dercitus bucklandi # (MA) Dysidea fragilis (MA) D\sidea pa/lcscens (MA) Esperiopsis fucorum ( RE ) Etirypon sp. 1 (EN) Eurypon sp. 2 (EN) Euiypon sp. 3 (EN) Eurypon sp. 4 (EN) Eurypon sp. 5 (EN) Eurypon sp. 6 (EN) Eurypon sp. 7 (EN) Eurypon sp. 8 (EN) Halichondria bowerbanki (RE) Halicliondria panicea (EN) Haliclona cinerea (MA) Haliclona fistulosa (RE) Haliclona simulans (RE) Haliclona sp. 1 (MA) Haliclona sp. 2 (MA) Haliclona sp. 3 (RE) Haliclona sp. 4 (MA) Haliclona sp. 5 (MA) Haliclona sp. 6 # (MA) Haliclona sp. 7 # (RE) Haliclona sp. 8 # (RE) Haliclona sp. 9 # (RE) Haliclona sp. 10 # (MA) Haliclona sp. 1 1 if (MA) Haliclona sp. 12 #{RE) Haliclona urceolus (TU) Haliclona viscosa (MA) Halicnemia patera (EN) Halisarca sp. (MA) Hemimycale columella (EN) H\medrsmia hrondstedi # (EN) Hymedesmia jecusculum # ( EN ) Hymedesmia pansa # (EN) Hymedesmia paupertus (EN) Hymeniacidon per/e\'is (MA) Laxosuberites incrustans (EN) Leucosolenia complicata (RE) Lettconia nivea (EN) Microciona fallax # ( EN ) Microciona streps itoxa # (EN) Mvcale contarenii (MA) Mvcale macilenta (MA) Mycale rotalis (MA) Myxilla rosacea (MA) Myxilla incrustans (EN) Mvxilla fimbriata (MA) Pachymatisma johnstonia (MA) Paratimea constellata (EN) Phakellia sp. (PA) Phorbas fie titius (EN) Plakortis simplex (EN) Plocamilla coriacea # (EN) Polymastia sp. 1 (PA) Polymastia sp. 2 (PA) Pohmastia sp. 3 (PA) Polvmastia sp. 4 (PA) Pol\inastia sp. 5 (PA) Polymastia sp. 6 (PA) Polymastia sp. 7 (PA) Polymastia sp. 8 (PA) Psetidosuberites sulphureus (EN) Rhaphidostyla kitchingi (AR) Raspailia hispida (AR) Raspailia ramosa (AR) Scypha citatum (TU) Spanioplon annatiinun # (EN) Stelligera rigida (FL) Stelligera stuposa (AR)* Stylostichon dives (EN) Stvlostichon pliimoswn # (MA) Suberites camosus (FI) Suberites ficus (FI) Suberites sp. 1 (EN) Suberites sp. 2 (EN) Suberites sp. 3 (EN) Suberites sp. 4 (EN) Terpias fitgax # ( EN ) Teth\a auranaum (MG) Unidentified sponges An asterisk indicates species found at a particular site (BU. Bullock Island; WH. Whirlpool Cliff: LA. Labhra Cliff; WC, West Cliff). Underlining denotes species found on rocks, and the symbol # indicates that the species was exclusive to rocks; boldface type denotes species found on panels (none were exclusive). Uppercase letters in parentheses after the species name are macro-morphological descriptions: AR. arborescent; CL. clathrate; EN. encrusting; FI. ficiform; FL. flabellate; MA, massive; MG. massive globulose; PA. papillate; RE. repent; TU. tubular. ferent habitats, the same MDS and Bray-Curtis analysis was repeated using only species that exhibited encrusting or massive morphologies. However, no differences were ob- served between the original dendrogram and the 2-D scaling plot produced (output not shown). SIMPER analysis was used to determine which species 150 J. J. BELL AND D. K. A. BARNES Table 2 Sponge species tin:: wei • 'I iroiti the under (*) and upper ( + ) sides of rocks found at rim sites at Lough Hyne West Cliftt Whirlpool Clifft Species ! Morphology ># S M L VL S M L VL Anthn UK . v;sM».v (EN) * * * * * * A/H/io inmlvens (EN) * * * * A/I/VM/IH \iilfurca (EN) * * * * * * * * Aplysilla rii\cii (EN) * * * * * * * * Cliitlirimi ciiriaccit (CL) * * * * * * * * Clioiui celala (MA) * * * * Dercitus buckltimli (MA) * * * * * * D\Mth'ti tnivilis (MA) * * * * * * * * Pvwc/ci/ pullescenx (MA) Esperiopsis fiiconiin (RE) Haliclomi sp. 1 (MA) Haliclomi sp. 2 (RE) Haliclomi sp. 3 (RE) Haliclomi sp. 4 (RE) Haliclona sp. 5 (MA) Haliclomi sp. 6 (MA) Haliclomi sp. 7 (MA) Haliclomi sp. 8 (MA) Halichondria bowerbanki (RE) + Hymedesmia hroiulstetli (EN) Hymedesmia jecit.\ci<; 20 - /K /\ a) Goleen b) Goleen 15 - 1. 10 • 5 0 20 • a) West Cliff b) West Cliff 15 • 1- 10 • • 5 • 0 • 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 1 | | Time(h) | | HW LW HW LW Figure J. Spring (a) and neap (b) tide flow rates over one tidal period at four sites within Lough Hyne. Note that graphs have different v axes. [After Bell (2001).] Assemblages on rocks Bray-Curtis analysis and MDS showed that sponge as- semblages on rock had a very high level of similarity (65%) between sites and different rock sizes (Fig. 4). But, when the cluster composed of assemblages from various sized rocks was considered alone, assemblages inhabiting medium, large, and very large rocks had a greater similarity between different sites than the assemblages on small rocks had with other rock sizes within the same site. At both sites, assem- blages on medium, large, and very large boulders gave rise to clusters that then formed a larger cluster with the small rock assemblages. Rock size had important influences on sponge assemblages (Table 2). A higher number of species was reported from larger rather than smaller rocks at both sites (as expected given the area effect). Yet few differences were observed between the sponge assemblages on the small and medium-sized rocks at West Cliff and Labhra Cliff. There was a logarithmic relationship between substratum area and sponge species diversity on rocks from both West Cliff and Whirlpool Cliff. Data were LoglO transformed, resulting in linear relationships (Fig. 6). The relationship between the number of sponges (per rock) and the area of rock surface at both sites was linear (Fig. 7). General linear model (GLM) ANOVA was used to compare the linear relationships between substratum area, number of sponges, and diversity. In the relationships between species diversity and area, no significant difference was observed in slope or SPONGE COMMUNITY COMPOSITION 153 1 - WHO (V) 2 - WH6 (V) 3-WHI2IV) 4-WH18(V) 5 - WHO (I) 6-WH6(I) 7-WH12(l) S-WHIS(I) 9-BUO(V) IO-BU6(V) 11-BU12(V) 12-BU18(V) 13-BUO(I) 14-BU6(I) 15-BU12(I) 16-BU18(I) 17- LAO (V) 18-LA6(V) 19-LA12(V) 20 -LA 18 (V) 21 -LA 24 (V) 22 - LA30 (V) 23 - LAO (I) 24 - LA6 (I) 25-LA12(I) 26 -LAI 8 (I) 27-LA24(I) Stress = .17 28-LA30(I) 29-WCO(V) 30-WC6(V) 31-WCI2(V) 32-WC18(V) 33 - WC24 (V) 34-WCOd) 35-WC6(I) 36 - WC12 (I) 37- WCI8(I) 38 -L12 panels 39 - L6 panels 40 - LO panels 41 -WH 12 panels 42 - WH6 panels 43 - WHO panels 44 - WC Sm boulder 45 - WC Med. boulder 46 - WC Lar boulder 47 - WC V.Lar boulder 48 - WH Sra. boulder 49 - WH Med. boulder 50 - WH Lar Boulder 51 - WH V Ur boulder Figure 4. Bray-Curtis similarity and Multi-Dimensional Scaling (MDS) analysis to compare the sponge assemblages inhabiting panels, rocks, and cliff at a number of sites, depths, and surface inclinations from Lough Hyne. The dashed circle indicates the grouping that MDS produced for loose rock assemblages. intercept between West Cliff and Whirlpool Cliff (GLM ANOVA; F ratio = 0.07, P = 0.78. denominator df = 1, numerator df = 278). This was also true for the transformed relationships between species richness and rock surface area at West Cliff and Whirlpool Cliff (graph not shown) (GLM ANOVA; F-ratio = 1.1. P = 0.25, denominator df = 1, 154 J. J. BELL AND D. K. A. BARNES 2 JS E •? c \ _^ * •~ S itl 3 ii 1 *E S •§ t w— 2 ^ S> .Sc ci. u ?3 t~ 1 1 t £ i ^ -| -| .Jf ^ "-^ ^ ^' ~5j "c 3; £ Q' & 3: -^_ * * i I Jc ^ ^ Zt. * c 5 ° « O — s ^ ^ t ^ 2 i> 5 "S: 5" c. '5-. ^ B '^ 5- 2 a c ^5 5 -2 2 5 2 ^ J3 ~ If 111 2 5 5, ^ ^ £.^ El ? &' Q' t^ £ £ ^ S kl Q -J -> \ - a -2 *3 -2 o ~ s 5 5 •2 ^2 ^~ 5 \- -2 a *o -3 * ~- "~ ? "2 S ^ ^i ? ^ ^S £ "•" * S. S ^ ; J 3 1 '= "1 -^ 1 •2 -^4; g- 5 .2 § '=••. •" "^ — • "C =: ^ r- ^ "2 a g g "g "S S .§• o "5 "S ^2 '= § 15 .SP -5 [C 2 TO r"' Q f*-i ^ ^ Illlc 3 & 2. s §• — ^ Q ^ U. * c * Q ta s = s :- _ u 1 § a o c ^ £ * 1 £.5 ~ — ^- ~ ~5 ~ -1 -^ -£ ~~* S S -r *-. -2 £, ^ o -3 *•• II •> ^ -.— t f = 1 •3; »2 5 ~s f 1 1 "1 s- 1 'S 3^2 !j -2 %• '=' '£' 1 1 •? II S " 5- G o -i ^ ^ _5 -£ •t" 3 - S S S .1 5 | S "5 6* ^-* 3 ^ ^~ ^. ^JC -i "1 § -=' "^ ill % «§ 5 kj co f-^ Q' ^t t $ 3^1 c^ •2 O ^>" W .iT' ^ LO Uj h*. Q ^ a a a > 7: §> w ^ 3" '? & i? s 'in ^ U 7 t 3 ^ 5 - T > i; ll I 1 1 d, y: 1 S ex "5 ^ I | d^ I 1 d.1 S ^ •s B i S r£ -2 "S .s z 3 2 ~i -C' "S '- | g i 1 1 § 1 5 •> 5 '£ _^ C "^ -i; S: •i 1 2 2 -3 £ ^s 3: 5 5; •£• C ^ 3: S -r 3^ 3; C S -r 5 £ i £ -»; C £ 3: * C ^ 11 - 1 1 * * •- ? - ~ i C ||l ^ "S- 'C a; •"- ^ ^J r 'i s ^ s ^ i 1 1- = "1 2 S 1 - l| eniacii C ,G 2 5 •2 I ^ 5 £ ? || •2 § •§ X o 1 1 1| | •3 2 a "S — ^- ^ ^S 5 5" ^ '£ 5 g s II 3? "5. S $5 -i — ^ -2 -2 "s; a, O t: a- J S C 3: j to £ C -? 3; .j 5; C -5" 3; -j ^. ^j ^ ^ «^j * -X- * # g 2 -y.- * * ^ * ~ 0 1/3 £ •5 = 1, 2 I 1 = 1 J 1 1 1 11. 1 1 I 1 5; C •? 3: ^j s; -t C g 2 C C Haliclona sp. 7* H. brondstedi* Esperiopsis fucon Scvpliu ciliiitnni' Si/heritex carnoxu Eurypon orange Stelligera rigida Halichondiiii pun, Suberites carnoxu Stelligera rigida g c V. | I He/niinyciilc coliimella* Scypha ciliatum* Stelligera rigida Suberitex carnoxu. Raspailia ramosa Eurypon orange Suberites carnoxu: Haliclona sp. 4* Eurypon orange Raxpailia ramosa* Piirutimea cunxtcl Hiilichoiulria pant Scypha ciliatum* Esperiopsis fiicnru Stelligera rigida Suberites carnosm ta & •a •^ o 0 c fe •j- U U c/5 _^" '-J ^ O .'— P *- O .c — £> ^ le 1 - | £ U XI ~£ ^ C CO a 1 H '£ c a. ^ ^ ^ m 3 j ^ SPONGE COMMUNITY COMPOSITION 155 12 5? 10 1 6 "3 4 2 0 12 10 -3 6 a, I = -- o Labhra Cliff Whirlpool Cliff 140 0 2 4 6 8 10 Number of months since panels deployed 12 Figure 5. The proportion of artificial settlement panels covered in sponges after 1. 2. 6, and 12 months of deployment at high-energy (Whirlpool Cliff) and low-energy (West Cliff) sites at Lough Hyne. numerator df = 278). However, when GLM ANOVA was used to compare the linear relationship between sponge numbers and rock surface area, a significant difference was found between the slopes of the relationships at West Cliff c Whirlpool Cliff R-squared = 0.54 234 Boulder surface area (Log 10) Figure 6. The relationships between sponge species diversity and richness v.s. rock surface area at high-energy (Whirlpool Cliff) and low- energy (West Cliff) sites on the undersides of rocks at Lough Hyne. Species diversity (antilog 10) = 2.09 (± 0.15) + 2.09 <± 0.04) X surface area. 120 - 2 100 - s H. s | 60 - Numbers • West Cliff R-squared = 0 73 o Whirlpool Cliff R-squared = 0.54 0 500 1000 1500 ") Boulder surface area { cm" ) 2000 2500 Figure 7. The linear relationship between sponge densities and rock surface area at high-energy (Whirlpool Cliff) and low-energy (West Cliff) sites on the undersides of rocks at Lough Hyne. Number of sponges (West Cliff) = 60.3 (± 35.84) + 14.8 (± 0.85) x surface area. Number of sponges (Whirlpool Cliff) = 47.3 (± 46.54) + 22.6 (± 1.87) x surface area. and Whirlpool Cliff (GLM ANOVA; F ratio = 16.16. P < 0.001, denominator df = 1, numerator df = 278). Any unit increase in surface area resulted in a greater increase in sponge density at West Cliff than at Whirlpool Cliff. Also, any given rock size harbored a greater number of sponges at West Cliff than at Whirlpool Cliff. Discussion Animal communities are known to vary between large geographic areas (scale of kilometers), but the variability between localized habitats (1 to 100 m) under similar en- vironmental conditions is far less well known. This is true for sponge assemblages: broad distributions have been de- scribed by habitat at Lough Hyne (Picton, 1991; van Soest and Weinberg, 1980; van Soest et «/., 1981). and recently more detailed studies have added environmental variables to such distributions (Bell and Barnes, 2000b, c). As with most, if not all organisms, environmental factors are critical to the distribution of sponge species. To date, little quanti- tative information has been provided on localized (where confounding factors are minimized) habitat-associated dif- ferences between sponge assemblages in temperate locali- ties. Morphological variability with habitat stability and environmental characteristics The studied sponge assemblages (within Lough Hyne) varied considerably between habitats of differing stability (i.e., cliff and boulders) and environmental conditions (i.e., high and low flow), even when habitats were separated by only several meters or less. Sponges inhabiting hard sub- 156 J. J. BELL AND D. K. A. BARNES stratum have been shown in exhibit considerable morpho- logical adaptation in ; iio;ise to a variety of factors, includ- ing flow regime, icntation, and substratum type, at both assemblage t.rli and Barnes, 2000d) and individual levels (Mancr.-v ,nd Pronzato, 1991; Bell et ai. 2002). It seems, tluivture, that morphology alone may account for some of these differences in assemblage composition be- tween sites and habitats. The two-dimensional nature of rock (under-surface) habitats undoubtedly accounts for the high proportion of encrusting forms because the overt three- dimensional nature of many other sponge species common to cliff habitats is inhibited (e.g., arborescent forms). High- energy cliff sites were dominated by encrusting and massive forms and showed greater levels of similarity (in assem- blage composition) to rock habitat assemblages than to those of low-energy cliffs. However, high proportions of encrusting and massive morphologies were found in both cliff and rock habitats, although the species compositions were very different. Morphology alone cannot account for all the differences found. Organisms living on the under- sides of rocks have a number of advantages, including reduced competition from fast-growing algae found on up- per rock surfaces, since many sponges are considered slow- growing (Ayling, 1983), and protection against potentially harmful UV radiation. Underside rock communities also experience reduced sediment settlement in areas of low current flow, reduced effects of desiccation (in the inter- tidal), and reduced predation from fish and other large invertebrates (Dunlap and Pawlik, 1996; Wulff, 2000). A combination of these factors may account for the presence or absence of any single species on the undersides of loose rocks. Therefore, the distribution and habitat of each species should be considered individually. Morphology alone has already proved valuable in sepa- rating sponge assemblages inhabiting sublittoral cliffs (Bell and Barnes, 2000a). Increased numbers of delicate morphol- ogies, exhibited by a number of species exclusive to low- energy sites, have been associated with decreasing flow rate and as a mechanism to reduce sediment settlement (Bell, 2001). However, the rate of water flow had little effect on the sponge assemblages found on rocks and apparently was not a regulatory factor of such assemblages (in the present study). One aspect of morphology that was not considered within this study is the potential importance of the surface texture of encrusting sponges in response to habitat distur- bance or stability. For example, sponges with smooth sur- faces may be suited to areas of high current flow since they experience reduced drag. Also, the actual flow (in terms of speed and direction) experienced by sponges may be sig- nificantly influenced by seabed characteristics (Hiscock, 1983), such that their morphology and species distribution may be influenced by microscale environmental character- istics, which are themselves difficult to characterize. For example, the overlying nature of loose rocks may lead to localized areas of negligible current flow, even in high- current areas. Assemblage composition variability with flow rate and rock size Negligible (between-site) differences in sponge diversity or species richness attributable to flow rate were found on the under-surfaces of rocks. This suggests that in rock habitats unlike cliff habitats (Bell and Barnes, 2000a), water flow rate is not directly important in determining the com- position of sponge assemblages. Sponge diversity and rich- ness did, as with most organisms, increase with area (rock size), which is usually termed an area effect. The density of sponges for any given rock size was greater at West Cliff than at Whirlpool Cliff, most likely due to reduced compe- tition in low-energy environments from superior spatial competitors such as colonial ascidians and anthozoans (Maughan and Barnes, 2000b; Bell, 2001a; Bell and Barnes, 2003). Increased sedimentation, to which sponges may show morphological and physiological adaptation, has been credited for the shift in competitive ability (Bell, 2001a). However, since the organisms inhabiting the undersides of rocks experience little direct sedimentation, other factors must be responsible for the lack of superior competitors, such as increased sediment loading (rather than direct set- tlement) in the water, a condition that sponges appear to tolerate (Lilly et al., 1953). Also important is the difference between upper and lower rock surfaces. The domination of sponges on lower surfaces relates to the absence of mac- roalgae, which has been considered to control both large- and small-scale sponge distributions (Witman and Sebens, 1990). Sponge proliferation on upper surfaces may also be prevented by interference competition from the sweeping action of algal fronds, as suggested for other invertebrates (Jenkins ct til., 1999). The linear increase in the number of sponges with increasing surface area of rock may appear unusual. One might expect that the dominant sponge species would occupy most of the primary substratum (Russ, 1982; Maughan and Barnes, 20()()b). However, competitive en- counters and interactions between sponges may be non- hierarchical and resemble a network (Buss and Jackson, 1979; Bell and Barnes, 2003), thereby preventing monospe- cies dominance. Even if certain sponge species inhabiting rocks are dominant over others, these species may show seasonal growth and retraction of tissue that prevents them from monopolizing rock surfaces (Sara, 1970; Stone, 1970; Elvin, 1976; Barthel. 1989). The distribution of sponges may also be limited by other biological factors, in particular predation (e.g., Wulff, 1995); but in the observations of sponge assemblages at Lough Hyne, predation of sponges was rarely observed. Fish feeding on algae sometimes broke branches from arborescent species on cliffs, but extensive predation was not seen. Sponge population heterogeneity is also affected by competitive processes (Becerro et nsen: 1 I: 851- 885. Jenkins. S. R., T. A. Norton, and S. J. Hawkins. 1999. Settlement and post-settlement interactions between Seinibalainis halanoules (L.) (Crustacea: Cirripedia) and three species of fucoid canopy algae. J. £v/>. Mar. Biol. Ecol. 236: 49-67. Kid In ML' J. A. 1987. Ecological studies at Lough Hyne. Atlv. Ecol. Res. 17: 115-186. Ko'nnecker, G. 1973. Littoral and benthic investigations on the West Coast of Ireland. I. The sponge fauna of Kilkieran Bay and adjacent SPONGH COMMUNITY COMPOSITION 159 areas. Section A: Faunistic and Ecological Studies. /'/•<«•. A' // Acn,/. 73B (26): 450-472. Krehs. C. J. 1989. Ecological Metli,20°C) temperatures, measurements from data loggers were sorted into 5°C intervals, and measurements in each class were summed together. Each data point was assumed to represent about 15 min of exposure to the recorded temperature. In nearly all cases the approximate duration of exposure to warm temperatures was longer at the high tidal height (Fig. 2) for the 2 weeks preceding the summer sampling. During the summer 2000 period, the total exposure to temperatures in excess of 20°C was about 67 h at the high tidal height compared to 44 h at the lower tidal height. The thermal regime based on mean emersion is summarized in Figure 3. The majority (>70%) of the time (not shown), oysters at both tidal heights were submerged and encountered temperatures in the 15-20°C range. Oys- ters encountered elevated (>20°C) and reduced (<15°C) temperatures at or above the point of emersion. No significant difference in mortality was recorded in animals at either site during the experiment. Total mortality at both sites was less than 1(Y7c annually. Animals at the high tidal height (1.2 m) showed a cumulative mortality of 16.5% (62 out of 375 animals): those at the low tidal height (0.3 m) experienced an 18.4% (69 out of 375 animals) mortality. Most of the mortalities occurred during the wann- est months (June-September). HSP70 protein ami Iisp70 mRNA levels The levels of HSC70 (HSC77 and HSC72) in control (non-heat-shocked) Pacific oysters were associated with the level of environmental thermal stress (Fig. 4). At both study sites the control levels of HSC77 and HSC72 were as much as 100-fold greater in summer than in winter (P < 0.001). These differences were not attributed to differences in total protein levels, as mean control protein levels were 41.5 mg/g wet weight and 46.2 mg/g wet weight in winter and summer, respectively (Bradford protein assay, BioRad). and these differences were not significantly different (P > 0.07). During the winter months there was no statistically signif- icant difference in the control levels of HSC70 between the high and low tidal height sites (P = 0.917). The threshold temperature for induction of HSP69 ap- peared to be associated with the level of environmental thermal stress (Figs. 1 — !•). In January. HSP69 accumulation was induced in oysters from both sites after heat shock at 37°C. In contrast, limited or no induction of HSP69 and HSC72 was observed following heat shock at 37°C in August oysters living at high or low tidal height (Fig. 4: see also Fig. 6A). Full induction (i.e.. HSP69 expression at levels approximating those of HSC77 or HSC72) of HSP69 was not observed in August until after heat shock at 40- 43°C. Thus, both the threshold temperature for HSP69 induction and the control levels of HSC70 exhibited signif- icant seasonal plasticity. The level of hsp70 mRNA in summertime high tidal height oysters increased dramatically relative to control after heat shock at 40 and 43°C, but not after heat shock at 33 and 37;C (Fig. 5). This result reflected the observed increases in HSP69 protein after heat shock at these tem- peratures in the same animals. A subset of oysters from each site was sampled and heat-shocked at 37°C monthly during the summer, begin- ning in April and ending in September (not shown). Based on the results of these observations, the greatest change in threshold temperature for HSP69 induction and control level of HSC77/HSC72 temperature occurred between our June and July sampling dates and winter (January) (Fig. 6A, B; Fig. 7). In June, oysters at the high tidal height com- pletely induced HSP69 after a 37°C heat shock. In contrast, by July, induction of HSP69 was much more limited in these oysters. Control levels of HSC70 also increased mark- edly during this period. Although there were slight increases 164 A. M. HAMDOUN ET AL. Totten 0.3m Totten 1.2m • CH'Sler 1 • Ovster 2 • Ch'Ster 3 Bag Temp Figure 1. Internal temperatures of three living oysters (solid lines) at two tidal heights ( 1.2 m and 0.3 m at Totten Inlet, Washington) and external temperatures from a temperature probe placed alongside the oysters (dotted lines). Internal temperatures closely track external temperatures. in mean levels of control hsp70 mRNA in summer relative to winter oysters, the differences were not significant (P = 0.13; Fig. 7). Thermal limits and induction of thermal tolerance Laboratory heat shock of oysters at both tidal heights suggested both seasonal and tidal height influences on the upper thermal limits for survival (Fig. 8). In January there was no significant difference in lethal temperatures between oysters at the two tidal heights. However, by August there were significant increases in the baseline thermal limits of oysters at both study sHes. Moreover, oysters at the high tidal height appeared to exhibit elevated thermal limits compared to their low tidal height counterparts during the summer. August survival of high tidal height oysters after 44°C heat shock was significantly higher than that of the low tidal height oysters (P = 0.03). These differences occurred in the absence of HSP69 expression, suggesting that control thermal limits in Pacific oysters are likely to be more closely associated with cognate HSC70 expression than to inducible HSP70 expression. In contrast, induced thermal tolerance appeared to be associated with inducible HSP70 expression (Fig. 9). Oysters at the high tidal height that were heat-shocked at 37°C during the winter were able to completely withstand a subsequent lethal treatment of 44°C. Heat shock at 37°C did not induce thermotolerance HSP GENE EXPRESSION IN OYSTER 165 35 o January 2000 | 30 fjU 1800 D Totten 1.2m ^ i drift rri D HSP69 EHSC72 * "5 g* 20 S Tolten 0.3m T 0HSC77 £ 4( §H , V^ Q. 100° T = 1 ^N 800 I 1 9 « > w Sx — 2. •*• 11 10 NX & 600 ^ T •3 — X\X ^^1 H^^^ .Qf. i aly 77 I ' u 0 • ^ ^ M 1 li 2°I m m m 1 % to m .m i i 20C-25C 25C-30C 30C-35C 35C-40C 40C-45C CON 25 29 " " CON 25 29 33 " Temp, range Totten 0.3m Totten 1 -2m Figure 2. The approximate hours of exposure to elevated (>20°C) air temperatures in oysters at high and low tidal heights for the 2 weeks prior to the summer sampling. The vast majority of the time, oysters experienced temperatures (15-20°C) below the range shown. (to 46°C) during the summer. However, when August oys- ters were heat-shocked at 40°C, they were able to withstand heat shock at 46°C. These results suggest that induction of thermal tolerance is more closely associated with inducible HSP70 family expression. Discussion Our results demonstrate significant phenotypic plasticity in the heat-shock response of Pacific oysters. Massive changes (10- to 100-fold increases) in the amounts of con- stitutively expressed HSP70 proteins were measured in summer as compared to winter. These changes are associ- ated with concomitant increases in thermal limits and set point for HSP69 induction. In oysters at both high and low tidal height, the threshold for complete (i.e., equivalent to levels of HSC77 and HSC72) induction of HSP69 increased 5000. DHSP69 4500 T August 0HSC72 B,HSC77 * 4000 ^^ -•- •s 350° •*" 4n S ,000, T -»- i- ,g i rr^r- | 2S00 - S' 2000 ~t- -t 3 1500 • ^ 1000 - 500 J 0 4- I No data [Vyl 1 1 i. 1 i i \ 1 CON 33 37 40 43 CON 33 37 40 43 TottenO 3m Totten 1.2m Tidal Height/Temperature Figure 4. Relative levels of HSP70 as determined from western blots for animals sampled in August and later in January. Bars represent the mean values of total HSP70 for 5-6 animals (± s.e.m. for each isoform). The relative amount of each isoform is indicated within the total amount. Three isoforms were resolved by one-dimensional electrophoresis: two cognate forms (HSC77 and HSC72) and one inducible isoform (HSP69). The .Y-axis of each graph indicates the heat-shock temperature series for the low tidal height (0.3-m) group, followed by the temperature series for the high tidal height (1.2-m) group. D Rotten 0.3m Tbffen A 2m Temperature class Figure 3. Mean level of emersion for 5°C temperature intervals en- countered during the month of August. For the vast majority of time (> 70%). temperatures were in the range of 15-20°C. At this temperature range, oysters were completely submerged. At temperatures outside of this range, oysters were at or above the point of emersion. Control 33C 37C 40C 43C 1234 6 ' 8 9 10 Figure 5. Northern blots of hsp 70 mRNA in high tidal height (1.2-m) oysters 48 h after heat shock (HS) in August. Lanes are loaded with equivalent amounts (4 p.g) of total mRNA. HSP70 was analyzed with an antisense digoxygenein-labeled UTP probe corresponding to a 660-bp portion of oyster HSC72. Each lane represents mRNA from one oyster. Four oysters were subjected to each treatment (1-4 are Controls, 5-8 are 33°C heat shock (HS), 9-12 are 37°C HS. 13-16 are 40°C HS. 17-20 are 43°C HS). 166 A. M. HAMDOUN ET AL. January August Control Control 33C 37C 37C 40C B. Control 37C June July ~i.. :HSC77 :HSC72 ; HSP69 ) Sixth cleavage (arrowhead), 9.6 hpf. (ct Blastoderm formation, 76.25 hpf. The boundary created at sixth cleavage {arrowhead} is well preserved at this stage of development, (d, e, f) Blastoderm formation to the onset of epiboly, 26-27.5 hpf. (d) A distinct blastoderm with radiating pairs or clusters (*) of outer blastomeres, 26 hpf. (e) Blastoderm, 26. 7 hpf: the inner blastomeres from each marked pair have migrated toward the developing blastoderm while their outer sister cells and the one lone cell simultaneously collapse into the cytoplasmic cortex of the yolk cell. If) Blastoderm. 27.5 hpf: the migrating inner blastomeres have reached the blastoderm while their outer sister cells are no longer visible. Scale bar = 200 jj.m. For further description, see text. Video Note. Supplementary video clips are available for viewing on The Biological Bulletin web site at [http://www.mbl.edu/BiologicalBu/letin/ VIDEO/BB.video.htmll. around the blastoderm. An asterisk (*) has been placed by each of four pairs of these radiating sister blastomeres. in addition to one lone cell (Fig. Id). By 26.7 hpf, the inner blastomeres from each marked pair have migrated toward the developing blastoderm, while their outer sister cells simultaneously collapse, a cell behav- ior first described by J. P. Trinkaus in fish (7), into the cortex of the yolk cell (Fig. le). One hour later, the migrating inner blastomeres have reached the blastoderm, while the nuclei of their outer sister cells have entered the yolk cell, contributing to the yolk syncytium (Fig. If I. In teleosts. the YSL is critical to patterning and development (7. 8), and time-lapse video analysis has played an important role in our understanding of this model system (9). As in squid, the YSL separates the yolk from the embryo and regulates the transfer of all nutrients and factors present within the yolk to the developing embryo. Formation of this important layer involves the collapse of blastomeres at the boundary between the developing blastoderm and the yolk cell cytoplasm. That cephalopods form their YSL through similar developmental mechanisms to those of teleosts exemplities the fundamental similarities that exist between em- bryos faced with similar structural constraints and highlights the importance and need for further comparative study. This work was made possible by support from a Faculty Devel- opment Grant and the Aldom-Plansoen Distinguished Endowed Professorship in Contemporary Studies to K.C. from St. Mary's College of Maryland. K.C. is most grateful to Bill Eckberg, Howard University, who graciously provided laboratory space, collaborative guidance, and digital imaging assistance. We thank Rudi Rottenfusser. MBL Zeiss representative, for allowing his summer intern (P.H.W.) to participate in this work. Finally, K.C. wishes to thank J.P. Trinkaus and his many students for reminding us to "watch." Literature Cited 1. Brooks. VV. K. 1880. Anniv. Mem. Boston Soc. N.H. 1-22. 2. Arnold. J. M. 1965. Biol. Bull. 128: 24-32. 3. Segawa, S., W. T. Yang. H.-J. Marthy, and R. T. Hanlon. 1988. Vcliser 30: 230-243. 4. Crawford, K. 2000. Biol. Bull. 199: 207-208. 5 Crawford, K. 2001. Biol. Bull. 201: 2? 1-252. f>. Crawford, K. 2002. Btol. Hull. 203: 216-217. 7 Trinkaus, J. P. 1993. J. Exp. Zoo/. 265: 258-284. X Trinkaus. J. P. 1996. Dev. Biol. 177: 356-370. 9. Concha. M. L., and R. J. Adams. 1998. Development 125: 983-994. DEVELOPMENTAL BIOLOGY 181 Reference: Biol. Bull 205: 181-182. (October 2003) £> 2003 Marine Biological Laboratory Lithium Chloride Inhibits Development Along the Animal Vegetal Axis and Anterior Midline of the Squid Embryo K. Crawford St. Mary's College of Maryland, MD Marine Biological Laboratory, Woods Hole, MA When squid embryos. Loliga pealeii, are cultured in vitro ( 1 ) they may be individually manipulated with classic and molecular techniques, providing insights into the conservation of develop- mental pathways. Dorsoventral polarity in many embryos is asso- ciated with precise gene transduction cascades involving the Wnt signaling pathway (2). Results from experiments with frog (3), fish (4). and mouse (5) embryos suggest that a component of this cascade, /3-catenin. plays a major role in axis formation. Addi- tional support for the role of j3-catenin in the early development of many embryos comes from studies using lithium chloride (LiCl). LiCl is a known vegetalizing agent for sea urchins (6), and in echinoderms generally, it enhances and expands levels and regions of nuclear /3-catenin localization coincident with increases in endoderm and mesoderm (7, 8). In contrast, its effect on amphibian embryos is species- and stage-specific. For example, when gastru- lating embryos are treated with LiCl, they develop reduced noto- chords and enhanced vegetal structures, but when treatments are given at earlier or later cleavage stages, dorsalizing and anterior- izing effects, respectively, are observed (3, 9). In this study, embryos cultured in vitro were treated with lithium chloride to determine its effect on development: this is the first step towards understanding the molecular mechanisms of patterning in squid. Embryos were fertilized in vitro (1) and cultured at 17 °C in 60-mm plastic petri dishes (Falcon) that were lined with 0.2% agarose (Type II-A. Sigma), filled with Millipore (0.22 ;u.M) fil- tered seawater (MFSW), and supplemented with bovine serum albumin (BSA) (0.5%). Each LiCl treatment dish also contained 20, 40, or 60 mM LiCl (Sigma). Three trials of 15 embryos per treatment were performed. Dishes and solutions were changed every other day. Embryos were treated with LiCl for the first 6 d of development, by which time epiboly of the outer yolk cell was complete. Development was observed until the control embryos began to hatch from their chorions. at 19-20 d after fertilization. The classical stages of J. M. Arnold ( 10) were used to describe embryonic development. Embryos cultured in the presence of LiCl exhibited a dosage-dependent inhibition of development that was evident by 6 d in culture (Fig. la. b. 6 d post-fertilization (dpf), stage 18). but it was more easily detected later, during organogen- esis (Fig. Id. e. f, 17 dpf. stage 27). Development was inhibited in many structures normally associated with ectodermal tissues, such as tentacles, eyes, mantle, fins, and funnel. Moreover, convergence and inhibition of anterior midline structures was observed in embryos treated with 40 and 60 mM LiCl. In one trial, for example. E-mail: kcrawford@snicm.edu 8 of 16 embryos cultured in the presence of 60 mM LiCl had anterior midline structures that were inhibited. As a result, the eyes were abnormally placed: either converged (5/16, Fig. If), fused (2/16), or cyclopic (1/16). In contrast, structures that normally form on the posterior body wall such as the funnel or paired statocysts, although reduced, were always present in these em- bryos. These observations suggest that various regions in the embryo are differentially sensitive to LiCl treatment. Lithium chloride treatment inhibits development along the ani- mal-vegetal axis in the squid embryo and causes convergence and fusion of anterior cephalic structures. This result is consistent with the notion that LiCl treatment induces vegetalization of the squid embryo and thereby enhances mesodermal and endodermal struc- tures at the expense of ectodermal derivatives, as it does in other invertebrate and vertebrate embryos (3, 6, 7. 8, 9). That LiCl treatment may induce convergence, fusion, and cyclopia in squid embryos strengthens this interpretation (see 1 1 for a survey of the early literature on cyclopia produced by experimental means) and provides insights into the possible conservation of developmental mechanisms in cephalopods. This work was made possible by support from a Faculty Devel- opment Grant and the Aldom-Plansoen Distinguished Endowed Professorship in Contemporary Studies to K.C. from St. Mary's College of Maryland. K.C. is most grateful to Bill Eckberg. Howard University, who graciously provided laboratory space, collaborative guidance, and digital imagine assistance. Literature Cited 4 Crawford, K. 20(12. Biol. Bull. 203: 216-217. Sokol, S., J. L. Christian, R. T. Moon, and D. A. Melton. 1991. Cell 67: 741-752. Schneider, S., H. Steinheisser, R. M. Warga, and P. Hausen. 1996. Mech. Dev. 57: 191-198. Haegel, H., L. Larue, M. Ohsugi, L. Fedorov, K. Herrenknecht, and R. Kemler. 1995. Development 121: 3529-3537. 5. Kelly, G. M., D. F. Erezylimaz, and R. T. Moon. 1995. Mech. Dev. 53: 261-273. 6. Lallier, R. 1975. Pp. 473-507 in The Sea Urchin Embryo: Biochem- istry and Morphogenesis. G. Czihak, ed. Springer. New York. 7. Logan, C. Y.. J. R. Miller, M. J. Ferdowicz, and D. R. McClay. 1999. Development 126: 345-357. 8. Kitazawa, C., and S. Amemiya. 2001. Dev. Growth Differ. 43: 73-82. 9. Kao, K. R., and R. P. Klinson. 1989. Dev. Biol. 132: 81-90. 10. Arnold, J. M. 1965. Biol. Bull. 128: 24-32. 11. Rogers, K. T. 1963. Dev. Riol. 8: 129-150. 182 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Figure 1. Lithium chloride inhibits the uninml vegetal body rlv differentiated. (/) Embryo treated with 60 mM LiCI. 1 7 dpf. Large arrowheads indicate lightl\ pigmented eves with lenses visible in their centers that have converged toward the anterior mid-line. An * indicates each of the four tentacle pnmordia on the left side of the embryo, ys = yolk sac. a, b and c, and e and fare the same magnification. Scale bars = 500 jum in each view. Reference: Biol. Bull. 205: 182-184. (October 2003) © 2003 Marine Biological Laboratory HNK-1/N-CAM Immunoreactivity Correlates with Ciliary Patterns During Development of the Polychaete Capitella sp. I Susan D. Hill1'* and Barbara C. Bayer2 ' Michigan State University, East Lansing, MI Union College, Schenectady, NY Capitt'l/i: p I is an opportunistic polychaete that is widespread and is amoni: -.he early colonizers of disturbed areas that are high ' Corresponding author: hillss@msu.edu in organic content. Larvae are lecithotrophic and are competent to settle within a lew moments of emerging from the brood tube, although they can remain viable in the water column without feeding for two or three days if environmental cues for settling are not present. Under experimental conditions larvae demonstrate a DEVELOPMENTAL BIOLOGY 183 notable ability to select the appropriate environment for settlement and metamorphosis (1. 2). Fertilized eggs develop in a brood tube, emerging after eight or nine days as segmented, motile metatrochophores. The muscular framework that will be used by the juvenile worm after metamor- phosis is already in place (3). but swimming is powered by two bands of cilia, an anterior prototroch. and a posterior telotroch. A large cluster of cilia, the neurotroch. is apparent on the ventral surface in scanning electron micrographs (4). These ciliary bands are not only important in locomotion, but may also have a che- mosensory role in settlement and metamorphosis (5). This study is part of an ongoing investigation of the interaction between nerves and muscles during development and its role in locomotion. We used a monoclonal antibody against HNK-l/N- CAM. which is expressed on neuroepithelial cells in early embryos (6), as a tool to follow neural development; we have found that it is also a marker of ciliary pattern formation in capitellid larvae. In the present study. HNK-1 antibody is used to visualize the se- quence of ciliary development in Capitella sp. 1. Capitellids were cultured in filtered seawater and mud in finger bowls (J. P. Grassle. pers. comm.). and brood tubes were collected. Larvae at different developmental stages were removed from the brood tubes and fixed in 4% formaldehyde in PBS. They were then permeabilized in acetone, treated with blocking serum, and incu- bated in anti-HNK-1/N-CAM (Molecular Probes). This was fol- lowed by incubation in a Texas red-labeled secondary antibody. Whole mount preparations were made and observed with an Olym- pus BX60 fluorescence microscope and imaged with an Olympus Magnifier digital camera (model S99860). There is no evidence of HNK-1 reactivity during cleavage or gastrulation. If larvae are removed from the brood tube immedi- ately after gastrulation, no locomotion or distinct morphological characteristics are visible. For a brief period before they begin to swim, the larvae are able to glide slowly over the substrate. Most of the changes in patterns of fluorescence occur as the larvae acquire swimming ability. A positive reaction is first observed in nonmotile, postgastrula larvae several days prior to emergence from the brood tube. The label is seen in the developing prototrochal region in the form of a row of sparse fluorescent dots. This is followed by a small amount of punctate fluorescence in the perianal region and in the episphere, the region anterior to the prototroch. which will become the prostomium (Fig. la I. The fluorescent dots then become more numerous, delineating the prototrochal region and forming a slightly thickened band around the larva. Concurrently the punctate fluorescence, which at first seems to be randomly arranged, continues to appear in the episphere. In the pygidial region, posterior to the telotroch. scat- tered fluorescence is seen. The width of the prototrochal band of fluorescence next in- creases significantly, although the posterior edge remains less defined than the anterior. The amount of label in the episphere continues to increase. Antibody labeling reveals a distinct perianal ring and beginning organization of the pygidial region. In contrast to the labeled prototroch. there is not yet a distinct band of telotrochal label (Fig. Ib). At this stage the larvae are able to glide on the substrate. After reaching its maximum width, the prototrochal fluores- cence begins to recede towards the initial band, which is now prominently labeled. Concurrently the neurotroch becomes evident as a midventral mass of punctate fluorescence, with the densest reactivity just posterior to the stomodeum. Label in the pygidium is becoming dense and organized except in the dorsal region where there is no fluorescence. This reflects the ciliary patterns seen in scanning electron micrographs in which the dorsal area of the pygidium in the hatched larva is devoid of cilia (4). The telotroch is now prominently labeled, forming a band of fluorescence several dots wide (Fig. Ic). Although they have not yet emerged from the brood tube, the larvae are now capable of swimming. Next, the anterior boundary of the prototroch remains lightly labeled, and some scattered fluorescence persists in the episphere. The neurotrochal labeling is greatly diminished, while the pygidial region and telotroch are heavily labeled (Fig. Id). By this stage, the ciliary patterning appears to be complete. The patterns of cilia in newly emerged metatrochophores of Capitella sp. I have been described by Eckelbarger and Grassle (4). These include well-developed prototrochal and telotrochal bands, and a distinct neurotroch — a midventral cluster of cilia beginning posterior to the prototroch and terminating a short distance anterior to the telotroch. There is no apical tuft, although there are scattered cilia and mucous glands in the episphere. Discrete rays of cilia radiate from the perianal ring toward the telotroch except in the dorsal area, which is free of cilia. Our labeling, which proceeds from anterior to posterior and corre- sponds to these patterns, indicates that the HNK-1 /N-CAM protein is being expressed sequentially as the ciliary organization is laid down. We suggest that anti-HNK-1/N-CAM labeling may prove to be an important tool for studying the development of cilia. This work was supported in part by Michigan State LIniversity and the Union College Faculty Research Fund. The authors grate- fully acknowledge the generous assistance of Dr. William Eckberg in creating the figure. Literature Cited 1 . Butman, C. A., J. P. Grassle. and C. M. Webb. 1988. Nature 333: 771-773. 2. Butman. C. A., and J. P. Grassle. 1992. J. Mar. Res. 50: 669-675 3 Hill. S. D., and B. C. Boxer. 2001. Biol. Bull. 201: 257-25S. 4. Eckelbarger. K. J.. and J. P. Grassle. 1987. Biol. Soc. Wash. Bull. 1: 62-67. 5. Biggers, W. J., and H. Laufer. 1999. Biol. Bull. 196: 187-198. 6. Kruse, J., R. Mailhamnier. H. \\ernecke, A. Faissner, I. Sommer, C. Goridis, and M. Schathner. 1984. Nature 311: 153-155. 184 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Figure 1. Whole mounts of developing Capitella ,s/>. / larvae showing labeling of developing ciliary hands. Anterior is to the left. Early embryos measure approximately 240 X 175 JLUH. As development proceeds they become more elongated without increasing in size, (a) The first HNK-1 expression is seen in the developing prototroch (arrow), (b) The wide prototrochal band is apparent. Note scattered fluorescence in the epispliere. In the p\gidial region a distinct perianal ring is labeled (arrow), (c) Labeling of the prototroch has narrowed and the ventral neurotroch and the telotroch are visible. Kays of fluorescence (arrow) extend from the perianal ring toward the telotroch except on the dorsal surface of the pygidinm. which remains cilia-free, (d) Labeling is most prominent in the telotroch and pygidiufn. The prototroch continues t<> be lightlv labeled. Ep. episphere: N. neurotroch: P. prototroch: Py, pygidial region: T. telotroch. CELL BIOLOGY 185 Reference: Biol. Hull. 205: 185-186. (October 2003) €> 2003 Marino Biological Laboratory1 Ryanodine-Sensitive Calcium Flux Regulates Motility of Arbacia punctulata Sperm D. E. Heck1 * anil J. D. Laskiir 1 Rutgers University, Piscataway, NJ 2 UMDNJ -Robert Wood Johnson Medical School, Piscutawav, NJ The motility of sea urchin spermatozoa and the direction of their movement are due to the beat of the flagellum, which is controlled by an ion flux across the plasma membrane. When sea urchin sperm contact egg jelly, channels are activated that rapidly (<5 s) and transiently elevate intracellular calcium, initiating a cascade of events that lead to the sperm acrosome reaction (1-3). This process, in turn, induces a complex se- quence of events that include protein phosphorylation and ele- vation of calcium levels thought to be dependent on intracellu- lar calcium mobilization (4). Recent reports have identified ryanodine-gated intracellular calcium stores as critical for controlling the motility of mammalian sperm (5). In the present studies, we determined whether increases in intracellu- lar calcium and motility in the sperm of the sea urchin Arba- cia punctulata that are mediated by egg-derived products are also mediated by ryanodine-gated intracellular calcium stores. In initial studies, we found that Arbacia punctulata egg- derived products (egg water) that stimulate sperm motility also increase sperm intracellular free calcium (Fig. 1, panel A). The increase was about 10-fold, occurred within 2 min, and per- sisted for at least 10 min. These experiments and all further experiments were repeated three times with similar results. The increase in intracellular calcium did not require calcium in the seawater. indicating that the ion was likely released from in- ternal stores (data not shown). In further experiments, therefore, we used calcium-free seawater. Sperm motility and increases in intracellular free calcium were also found to be stimulated 2- to 3-fold by ryanodine (0.3-1 ^M) or caffeine (1-3 p,M). both agonists of ryanodine-gated ion channels (for reviews see refs. 5 and 6) (data not shown). We next examined the effects on sea urchin sperm of ruthenium red. a potent inhibitor of ryanodine- sensitive channel activity (6-8). We found that 1 H.M ruthe- nium red would inhibit the calcium mobilization and sperm motility initiated by egg water, or by ryanodine and caffeine (Fig. 1, panel C and not shown). Taken together, these data indicate that a ryanodine-sensitive calcium flux regulates the motility of Arbacia piinctiilata sperm. Recent studies indicate that ryanodine receptors are regulated by nitric oxide-induced oxidation and reduction of critical sulfhydryl residues (9, 10). In previous studies, we and others have demon- strated that nitric oxide is also important in regulating motility in sea urchin sperm, and we have found further that this process is likely to be dependent on the activity of GTP-binding proteins (11-13). In these reports, sperm motility and nitric oxide produc- tion were inhibited by cholera toxin, and motility was recovered by * Corresponding author: heck@eohsi.rutgers.edu the addition of the nitric oxide/peroxynitrite-releasing drug 3-mor- pholinosydnonimine (SIN-1). We found that SIN-1 (1 /u,Af) also stimulated sperm calcium mobilization (Fig. 1, panel B). Increases in intracellular calcium were approximately 3- to 4-fold, persisted for at least 10 min. and were also independent of extracellular calcium. Motility and calcium mobilization induced by SIN-1 in Arbacia punctulata sperm were also found to be inhibited by ruthenium red (Fig. 1, panel D. and not shown). These data indicate that nitric oxide, like egg water, is effective at mobilizing intracellular calcium. Moreover, nitric oxide appears to function in sea urchin sperm upstream of ryanodine-sensitive calcium chan- nels. The lower panel in Figure 1 shows a schematic representation of events leading to the activation of Arbacia punctulata sperm. In this model, the interaction of the egg-derived mediator with sperm receptors activates the guanylate cyclase activity of the receptor, resulting in the formation of cGMP. The resulting cascade of events, including an intermediate calcium-induced release of sequestered intracellular calcium ("gray box") that provokes the activity of high capacity sodium channels in the plasmalemma. ultimately culminates in motility. We hypothe- size that nitric oxide, produced in response to enzymatic bind- ing of calcium/calmodulin made available by the initial brief rise in intracellular calcium, interacts with ryanodine-gated ion channels to mediate the release of calcium from internal stores. We speculate that, in a manner similar to that of ryanodine- gated release of calcium from stores in mammalian skeletal muscles (7, 14), nitric oxide-mediated alterations to channel proteins are central to the prolonged and hyperactive motility of egg mediator-activated sperm. Literature Cited 1. Ward, G. E., D. L. Garbers, and V. D. Vacquier. 1985. Scie?ice 227: 768-770. 2. Schackmann, R. \V., and P. B. Chock. 1986. J. Biol. Client. 261: 8719-8728. 3. Su, Y. H., and V. D. Vacquier. 2002. Proc. Natl. Acad. Sci. USA 99: 6743-6748. 4. Schackmann, R. 1986. Methods Cell Bio/. 27: 57-71. 5. VValensky, L. D., T. M. Daw son, J. P. Steiner, D. M. Sabatini, J. D. Suarez, G. R. Klinefelter. and S. H. Snyder. 1998. Mol. Med. 4: 502-514. 6. Ehrlich, B. E., E. Kaftan, S. Bezprozvannaya, and I. Bezproz- vanny. 1994. Trends Phunmicol. Set. 15: 145-149. 7. Salama, G., E. V. Menshikova, and J. J. Abramson. 2000. Anti- oxid. Redox Signal. 2: 5-16. 8. Ozawa, T. 2001. Int. J. Mol. Med. 7: 21-25. 9. Galione, A., and G. C. Churchill. 2002. Cell Calcium 32: 343-354. 10. Heck, D. E. 2001. Antioxid. Redox Signal. 3: 249-260. 186 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS I 1. Heck, D. E., W. Troll, anri J. D. La.skin. 1996. Biol. Bull. 191: 275-276. 12. Heck, D. E., W a i-i J. D. Laskin. 1996. Pp. 16-17 in Tin- Biology of Ni i . S. Moncada, S. Gross, J. Stamler, and A. Higgs. ed- u Press. Portland. ME. 13. Herrem, B., E. de Lamirande, and C. Gannon. 20(13. Ciin: Plwiii, I •>: 419-425. 14. Sun. J.. L. Xu, J. P. Eu, J. S. Stamler, and G. Meissner. 2(103. ./. Biol. Cliem. 278: 8184-8189. 100 1000 01 1.0 10 100 1000 Log Fluorescence (free calcium) . "^ MOTILITY Egg-derived Mediators f Cytosol Ca Figure 1. Effects of egg water and SIN-1 on calcium mobilization in sperm from the sea urchin Arhacia punctulata. Freshly isolated sea urchin spenn were incubated with ihe cell permeant calcium-sensitive fluorescent indicator fluo-4 AM (Molecular Prohes. Eugene. OR. 5 jLtM. 10 mill} and [hen stimulated with (%'t; water (panels A and C) or SIN- 1 (Molecular Probes, I /xM, panels B and D}. After 3 mill, mlracellular levels of free calcium lure analysed using a Coulter EPICS flaw cytitmeter (excitation wavelength 488 nm. emission wavelength 525 mn). Data are presented on a 4-deende log scale. US, iinstimulated spenn. In experi- ments where cells were treated with ruthenium red, the spenn were first transiently penneahili:ed using a Live Cell Penneahili:ation Kit (Gihco. Grand Island, NY) to allow uptake of the compound (panels C and D). Note that spenn treated with ruthenium red tailed to mobilise calcium in response to egg water or SIN-1 (panels C and D). (Lower panel): Schematic representation of events leading to activation o/"Arbacia punctulata spenn. Events originate with the binding of egg-dcriml mediators to their receptors on the surface of the spenn and progress through signaling to stimulate the release of calcium from internal stores. Tliis release potentially involves protein interaction with intracelhilarly produced nitric oxide and presumably occurs within the endoplasmic reliculuin. which mediates the opening of high-capacity sodium channels in the phisnialetnina and ultimately leads to motility. PM, plasma membrane: RYR. minodinc receptor: ER. endo- plasmic reticulum: NO. nitiic oxide. CELL BIOLOGY 187 Reference: «/«-/. Hull. 2(15: IS7-IS8. (October 2003) ;l ••§ 7$ kDa «•• °\ C -r 2 3 pH 3 "tlT ~2sin-\' rail fGST-GTD) was expressed in E. coli. The 84 kDa fusion protein was bound ro n GST-affinity column, then clarified squid brain extract was applied to the column, washed, and elated with gliitathionc ro identity binding partners of the myosin-V tail. (A) SDS-PAGE gel of the fraction containing GST-GTD is shown in lane I. A band at 84 kDa indicates the presence of GST-GTD. The lower molecular weight proteins are putative m\-osin-\' tail-binding partners. An SDS-PAGE gel of the fraction eliitcd with glntathione from the control column (no GST-GTD) is shown in lane 2. There were no proteins in this fraction. A blot of the GST-GTD fraction probed with GST antibody (lane 3) confirmed the presence of the m\nsin-V tail fragment (84 kDa). The lower molecular weight bands represent breakdown products of the GST-GTD fusion protein. A blot of the GST-GTD fraction probed with H2 (lane 4i -.how. the presence of kinesin (120 kDa I. indicating interaction between myosin-V and kinesin. (B) A 2-D gel of the GST-GTD fraction shows approximately 35 spots. Two identified spots, a and b. are a-tiibii/m and ft-tubulin. respectively. (C) A plasmid containing GST-Rab-GDI (GST-GDII was expressed in E. coli. applied to a GST affinity column, and eluted with glutathione. An SDS-PAGE gel of the fraction containing GST-GDI (75 kDa) is shown in lane I. A blot of the GST-GDI fraction probed with GST antibody indicates the presence of GST-GDI at 75 kDa (lane 2). A blot of GST-GDI probed with QLLQ indicates interaction between native myosin-V 1 196 kDa) and Rab-GDI (lane 3). (D) Motility assavs in extruded \unid axop/asm were used to determine the effect of Rab-GDI on actin-based vesicle transport. The number of vesicles moving per field per min (V/F/M) was measured at concentrations of 0-20 /^M GST-GDI. Vesicle transport was inhibited by 99<7c at 20 fiM GST-GDI. lane 3). Therefore, these data show that myosin-V interacts with a Rab-G protein. Motility assays were performed with GST-Rab-GDI to deter- mine whether Rab GTPases are involved in vesicle transport in the squid giant axon. Rab-GDI blocks the exchange of GTP for GDP and thereby inactivates Rab proteins. Motile activity at 20 juM GST-Rab-GDI decreased from 54 ± 4 vesicles/field/min (V/F/M) in the control assay to 0.5 ± 0.2 V/F/M (Fig. ID). Therefore, the GST-Rab-GDI inhibited vesicle transport by 997r at this concen- tration. At 10 p.M GST-Rab-GDI. motile activity decreased by 86rf . These data show that Rab activity is required for myosin-V- mediated vesicle transport in the axon. These data are consistent with published results showing that myosin-V is recruited to melanosomes and endosomes by Rabs 27a and 1 la respectively. Our studies support the hypothesis that Rab GTPases are required for the recruitment of myosin-V to vesicles for transport. This work was supported by NSF Grant IBN-0131470 and MBL-Shifman Award to CJD. Literature Cited 1 . Huang. J. D., S. T. Brady, B. W. Richards, D. Stenoien, J. H. Resau. N. G. Copeland, and N. A. Jenkins. 1999. Nature 397: 267-270. 2. Kuznetsov, S. A., G. M. Langford. and D. M. Weiss. 1992. Nature 356: 722-725. 3. Langford, G. M. 2002. Traffic 3: 859-865. 4. Hammer. J. A.. Ill, and X. S. \Vu. 2002. Curr. Opin. Cell Biol. 14: 69-75. 5. Wu, X., F. Wang. R. Kang, J. R. Sellers, and J. A. Hammer. III. 2002. A/o/. Cell. Biol. 13: 1735-1749. 6 Bahadoran. P., E. Aberdam, F. Mantoux. R. Busca. K. Bille. N. Valman. G. de Saint Basile. R. Casaroli-Marano. J-P. Ortonne, and R. Ballotti. 2001. ./. Cell Biol. 152: 843-849. 7 Wu, X.. K. Rao, H. /hang. F. Wang. J. Seller, L. Matesic, N. Copeland. N. Jenkins, and J. Hammer, III. 2002. Nat. Cell Biol. 4: 271-278. 8. Chin, G., and S. Goldman. 1992. Brain Res. 571: 89-96. 9. Brown, J. R., E. M. Peacock-Villada, and G. M. Langford. 2002. Biol. Bull. 203: 210-21 I. 10. Brown, J. R.. P. Stafford, and G. M. Langford. 2003. J. Neuro- biol. (In Press). 192 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull 2(15: 19 -193. (October 2003) © 2003 Marine Bioloci ; atorv skeletal Events Preceding Polar Body Formation in Activated Spisula Eggs R. M. Pielak, V. A. Gaysinskaya, and W. D. Cohen* Hunter College, New York. NY Marine Biological Laboratory, Woods Hole, MA Polar body formation is of interest both as a fundamental pro- cess in sexual reproduction and as an extreme example of unequal cytokinesis in cell biology. Eggs of the surf clam Spisula solidis- sima, released in the germinal vesicle stage, are readily induced to form polar bodies by activation with KC1 or by fertilization. However, although Spisula eggs are utilized in current studies of centrosomes and the cell cycle (e.g., 1), polar body formation in this model system is described only in older literature (2, 3). We have examined changes occurring in major cytoskeletal ele- ments— microtubules and F-actin — in the stages immediately pre- ceding formation of the first polar body. These stages are thought to be critical for docking of the meiotic spindle with the cell cortex, and for meiotic apparatus-cortex signaling that mediates the posi- tioning and generation of the contractile ring. These processes occur by as yet unknown mechanisms. In this study, eonfocal fluorescence microscopy was used to localize actin and tubulin relative to meiotic chromosomal stage. Ripe Spisula were obtained from the Aquatic Resources Division of the Marine Biological Laboratory (MBL) and maintained at 1 1-13 "C until used. Clams were opened by dissection, the gonad was removed, and the eggs were filtered through cheesecloth. Before use, the eggs were washed 3 times in 0.2-^m filtered seawater (FSW), and resuspended to a concentration of 1:10 (^eggs/^Hsw)- The eggs were activated, either by adding excess KC1 to the seawater or by fertilization with freshly obtained sperm, according to standard methods for this species (4. 5). Time of KC1 or sperm addition was recorded as t = 0. After germinal vesicle breakdown (GVBD). the eggs were resuspended to a concentration of 1:100 ( Veags/VFSW). Time-course samples, taken approximately every 2 min after GVBD. were prepared by simultaneous lysis and fixation of eggs in a medium consisting of 0.6% Brij-58, 4% formaldehyde in PEM (100 mM PIPES, 5 mM EGTA, 1 niM MgCl,. pH 6.8 using NaOH). After incubation for 1 h. the samples were washed in PEM and then stained. For F-actin, the eggs were stained with rhodamine or Alexa Fluor 568-phalloidin. Microtu- bules were stained with a 1:1 mass mixture of mouse monoclonal anti-a and anti-0 tubulins (Sigma T9026, T-4026) pre-labeled with Alexa fluor 488-iabeled anti-mouse Fab fragments (Zenon™. Mo- lecular Probes), and chromosomes and nuclei were stained with DAPI. These procedures followed protocols developed in previous work on microtubule localization in a variety of cell types (6). and our measurements showed that normal Spixula egg diameters ( — 50-55 jam) were retained after such treatment. Confocal fluo- rescence microscopy of stained samples was performed using the : Corresponding author: cohen(s'genectr.hunter.cuny.edu. Zeiss Laser Scanning System LSM 5 PASCAL, and images were processed with Zeiss LSM Image Examiner software. Using the KCl-activation method at 23 °C, GVBD occurred in about 7 min. and was followed at approximately 13 min post- activation by the appearance of the first metaphase meiotic spindle. At first, the metaphase spindle was slightly eccentric (Fig. la). Subsequently, it moved toward the cell surface and approached the cortex while remaining in metaphase (Fig. Ib; —18 mini. Micro- tubules of the peripheral aster, initially straight, were now ob- served to curve outward along the cell cortex, symmetrically away from a central microtubule-poor region (Fig. Ib, b' arrow). At —20 min the spindle entered anaphase (Fig. Ic), and a ring of thickened F-actin. —17-20 p,m in diameter, then appeared in the cortex (Fig. Id, d'). This ring surrounded a small circular cortical area, —7-9 fim in diameter, in which the F-actin was considerably thinner, creating a "bulls-eye" appearance in 3-D computer-generated ro- tated images (Fig. Id', and online supplemental animation at www.mbl.edu/BiologicalBulletinA/IDEO/BB.video.html). The peripheral aster was now greatly diminished in size, and no longer visible along the cortex. Subsequently, it became apparent that the bulls-eye center of the F-actin ring (Fig. Id. d') was the region through which the polar body nucleus and associated remaining centrosomal material passed (—22-24 min post-activation; Fig. le). The first polar body appeared fully formed at about 26 min; it was enclosed by an actin-containing cortex that followed an out- wardly and inwardly bulging contour (Fig. If). Results similar to those shown in Figure 1 were obtained with KCl-activated eggs from several different clams, and also with sperm-activated eggs. These observations are consistent with, and extend, older electron microscopic work on polar body formation in eggs of Spisula and Tubifex (2,7) and are of value for comparison with current findings on unequal cell division in Saccharomyces cerevisiae and Caeno- rhtihilitis elegans (8. 9). The sequence of cytoskeletal events observed in Spisula eggs prior to polar body formation, as illustrated in the Figure 1 dia- gram, is suggestive of mechanisms. The initially eccentric meta- phase meiotic apparatus moves toward the cortex, and the periph- eral aster contacts the surface (diagram b). Curvature of the astral microtubules and spreading outward along the cortex at the meta- phase-to-anaphase transition follows (diagram c. d), rather than shortening of straight astral microtubules on contact. Such behav- ior is consistent with models in which spindle movement toward the surface is brought about by the capture of plus ends of micro- tubules and microtubule transport, either by cortical dynein. or by a formin-based mechanism (10. 11 ). The thickened ring of cortical F-actin — the inner layer of which (at least) is presumed to repre- sent formation of the contractile ring — is not established until late CELL BIOLOGY 193 Figure 1. Singes in KCI-activated eggs at 23 °C, with triple staining for F-actin, microtubiiles, ami chromosomes (white letters) and a diagrammatic summon' (black letters), (a) First metaphase meiotic spindle, eccentrically positioned; t = 16 min post-activation, (b) Metaphase spindle at cell cortex with astral microtubiiles curving outward from central microtubitle-poor region; ~!8 min post-activation, (b1 1 Higher magnification view of stage (b); arrow: microtubule-poor central region, (c) Early anaphase with microtubiiles spread along cortex, (d) Edge view of thickened cortical F-actin ring, only chromosomes and actin stained; —20 min post-activation. Inset: same stage, with microtubiiles also stained, (d' ) Computer-generated, rotated image of the F-actin ring shown in (d). (e) Te/ophase chromosome set within F-actin ring; only chromosomes and actin stained; —24 min post-activation. Inset: same cell, microtubiiles also stained, (f) First polar body, enclosed by an actin-containing cortex; —26 min post-activation. Magnification bars = 10 \im; bar for nil tii>urcs I other than b' I as shown in it. anaphase (diagram d). This suggests the involvement of anaphase signaling following a metaphase checkpoint. It is evident that the sequence involves a mechanism for periph- eral aster disassembly (Fig. Id, inset), but such disassembly is delayed until after astral spreading along the cortex. In addition, the earlier pattern of contact between peripheral aster microtubules and the cortex, with its central microtubule-poor area (diagram b). is a close match to the "bulls-eye" pattern of thickened F-actin, with its central thinner area, which appears later (diagram d). The dimensions of the later pattern also correspond to those of the earlier aster (~- 16-20-jxm outer diameter, ~7-9-|xm inner diam- eter). Taken together, these observations strongly suggest that the signaling mechanism for contractile ring generation involves the astral microtubules. We thank Kyeng-Gea Lee, Dr. David Burgess, Dr. Robert Palazzo, Dr. Shirley Raps, Ruben Pinkhasov. and Alex Braun, for assistance and helpful discussion. Support by the Howard Hughes Medical Institute Undergraduate Science Education Program in Biology (grant 52002679), and by PSC-CUNY64249 and NSF9726771. is gratefully acknowledged. Literature Cited 1 . Palazzo, R. E., E. A. Veisberg, D. G. Weiss, S. A. Kuznetsov, and W. Steffen. 1999. J. Cell Sci. 112: 1291-1302. 2 Longo, F. J., and E. Anderson. 1970. J. Ultrastmct. Res. 33: 495-514. 3. Kuriyama, R., G. G. Borisy, and Y. Masui. 1986. Dev. Biol. 114: 151-160. 4. Allen, R. D. 1953. Biol. Bull. 105: 213-239. 5. Costello, D. P., and C. Henley. 1971. Methods for Obtaining and Handling Marine Eggs and Embryos. 2nd ed. Marine Biological Lab- oratory, Woods Hole. MA. 6. Lee, K-G., A. Braun, I. Chaikhoutdinov, J. DeNobile, M. Conrad, and W. Cohen. 2002. Kiol. Bull. 203: 204-206. 7. Shimizu, T. 1983. Eur. J. Cell Biol. 30: 74-82. 8. Knoblich, J. A. 2001. Nut. Kev. Mol. Cell Biol. 2: 11-20. 9. Guertin, D. A., S. Trautmann, and D. McCollum. 2002. Micro- biol. Mol. Biol. Rev. 66: 155-178. 10 Busson, S., D. Dujardin, A. Moreau, J. Dompierre, and J. R. De Mey. 1998. Cnrr. Biol. 8: 541-544. 1 1 Gundersen, G. G., and A. Bretscher. 2003. Science 300: 2040- 2041. 194 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Bio/. Bull. 205 i" .; -195. (October 2003) © 2003 Marine BioloL itwatory Three-Di «al Birefringence Distribution in Reconstituted Asters of Spisula Oocytes Revealed by Scanned Aperture Polarized Light Microscopy Michael Shribak and Rudolf Oldenbourg Marine Biological Laboratory, Woods Hole, MA 02543 Ten years ago we reported the first measurement of the distri- bution of birefringence in asters dispersed in lysates of Spisulu oocytes: the observations were made with a new type of polarized light microscope, the Pol-Scope ( 1 ). Since then, the Pol-Scope and its commercial version, the LC-PolScope (CRI, Inc., Woburn, MA (www.cri-inc.com)), have become a mature microscopical tech- nique used in many laboratories around the world for studying living cells and other specimens (2). Asters consist of microtubules radiating in all directions from centrosomes — microtubule-organizing centers found in almost all animal cells. Nevertheless, both the structure of centrosomes and the mechanism by which they nucleate microtubules are still poorly understood. The Pol-Scope reveals the birefringence retar- dation, also called retardance, of astral microtubules irrespective of their orientation within the plane of focus. But when microtubules are inclined away from the plane of focus, the measured retardance is reduced, and the angle of inclination cannot be discerned. Now we are introducing a new technique called scanned aper- ture polarized light microscopy (Fig. 1). The Scanned Aperture Pol-Scope not only measures the microtubule orientation within the plane of focus, but it also measures the angle of inclination away from the focal plane. In addition, the Scanned Aperture Pol-Scope measures the retardance of all microtubules equally, irrespective of their orientation and inclination angle. Figure 2 shows images of an aster reconstituted by polymerizing tubulin off a centrosome purified from Spisulu oocytes (3). The images were recorded with the LC-PolScope (panel B) and with the Scanned Aperture Pol-Scope (panel C and D). As illustrated in the schematic of panel A. the focal plane was offset by 5 jum relative to the center of the aster (focal depth approximately 0.3 juiii). While the LC-PolScope image lacks retardance in the center of the structure, the image recorded with the Scanned Aperture Pol-Scope reveals the retardance of the dense microtubule arrays that are located near the center and are oriented almost parallel to the microscope axis. In addition, panels C and D also indicate the measured inclination and orientation angle of the birefringence axis, which also indicates the orientation of the astral microtu- bules. We also recorded images after adjusting the focal plane to include the centrosome in the center of the aster (images not shown). In this configuration, the retardance values in images recorded with the Scanned Aperture Pol-Scope were below 0.3 nm near the center of the aster, increased rapidly at a radius of 3 ju.m. and peaked at a radius of 7.4 /xm (peak retardance ~ 3 nm). In addition, the inclination angle was close to zero for all retardance values measured in the focal plane that included the center of the aster. This latter result conforms to the expectation that all astral microtubules are oriented parallel to the focal plane at this focal CCD camera circular analyzer objective lens specimen on slide condenser lens CD LC-B § g , LC-A ™ > linear polarizer: LC-M a. linear polarizer] y interference filter lamp Figure 1. Schematic of optical I left I and electronic components of the Scanned Aperture Pol-Scope. The optical set-up is based on a traditional light microsi ope enhanced by an aperture .scanning device and an analyzer for circular polarized light. The scanning device includes the liquid crystal devices LC-A, LC-B. and LC-M. LC-M is sandwiched between two linear polarizers. All liquid crv.stal devices are linear retarder plates with .S or l> individuull\ controlled sectors. LC-M and the two linear polarizers are used to block or transmit light passing through 8 pie-shaped sectors. A pair of sectors in LC-A and LC-B. arranged in series, function as a universal compensator, controlling the polarization state of the passing light /-//. All components of the scanning device are bonded together and form a 7 mm-tlnck 171/11 (// flat with electrical connections and an approximate diameter of 25 mm. The computer-controlled scanning device is placed in the front aperture plane of the condenser lens and is used to sequentially illuminate the specimen with a polarized, converging light beam whose central ia\ is tspically tilled to the microscope a.\i.s. Tilt angles are controlled bv blocking or passing the light in the K pie sectors. By passing the light in all fs sectors, the tilt angle is zero, and recorded images are equivalent to LC-PolScope images. Currently, images are recorded at ? Jil/ereni till angles and are combined using specially developed algorithms for measuring the .^-dimensional birefringence distribution in every re- solved specimen point {see t-'ig. 2: for a more detailed description of the instrument see /5/J. position. All results reported here are typical of our observations of at least 3 asters and repeated measurements at different focus positions. Our findings indicate that the centrosome essentially lacks aligned microtubule arrays. However, the density of astral micro- tubules increases sharply at the surface of the centrosome. We propose to interpret the radius at which the measured retardance has the steepest gradient as the location of the centrosome surface (radius = 3.1 ju.m). In the future we plan to measure the density of microtubule arrays as a function of radius from the center of asters that are exposed to various physiological conditions to determine their potential for microtubule nucleation. In conclusion, we have demonstrated that the Scanned Aperture CELL BIOLOGY 195 A Side View microscope axis focal plane Figure 2. An aster reconstituted from purified centrosomc ami tuhiilin was imaged with the LC-PolScope and the Scanned Aperture Pol-Scope using mi nil immersion condenser and objective lens \Zeiss Neoftuar 100 X /1. 3 NA Poll. IAI Schematic side view of the aster indicating the position of the plum' i'/ tui if. in panels B, C. and D and the definition of the inclination angle. IB) LC-PolScope image of microtubule retardance in the focal plane. The measured retardance in the center of the image is close to ~ero because the microtubule arrays are oriented nearly perpendicular to the focal plane I parallel to the microscope a.\is>. (Cl Microtubule retar- dance measured with the Scanned Aperture Pol-Scope. Gray scale image shows retardance of all microtubule arra\s, regardless of inclination angle. Over/av indicates lines of equal inclination angle in steps of 20°. (D) Same microtubule relardance image as in panel C: the image is overlaid with lines indicating the orientation of measured birefringence axis in the plane of focus. For clarity, both overlays show only a subset of orientation and inclination angles, which tire measured in every resolved image point. In images of panels B. C. and D. black is ~ero retardance. and white is 3 run retardance. Pol-Scope can be used to measure the orientation and inclination angle of the birefringence axis of objects exhibiting low birefrin- gence, such as asters. In addition, the technique measures the retardance of all birefringent objects equally, regardless of their orientation and inclination angle. The measurements are performed at all resolved specimen points simultaneously, at high spatial resolution (~ 0.3 /urn), and high sensitivity (0.1 nm retardance). This seems to be the first reported technique that can measure the 3-dimensional distribution of birefringence in microscopic speci- mens. We expect this technique to impact many application areas of the traditional polarized light microscope, including the imaging of living cells, tissues, and functional model systems in biology and medicine. Purified components for reconstituting asters in-vitro were kindly provided by Robert E. Palazzo of Rensselaer Polytechnic Institute and the Marine Biological Laboratory. The research is supported by NIH grants GM49210 and EB002045. Literature Cited I Oldenbourg, R., G. Mei, and R. E. Palazzo. 1993. Biul. Bull. 185: 288. 2. Oldenbourg, R. In Lire Cell Imaging: A Laboratory Manual. D. L. Spector and R. D. Goldman, eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY. (In press). 3. Schnackenberg. B. J., and R. E. Palazzo. 2001. Methods Cell Bioi 67: 149-165. 4. Shribak, M., and R. Oldenbourg. 2003. Appl. Opt. 42: 3009-3017 5. Shribak, M. I., and R. Oldenbourg. 2002. Pp. 104-109 In Three - Dimensional and Multidimensional Microscop\: Image Acquisition and Processing IX. Proceedings of SPIE 4621, San Jose. CA. Reference: Biol. Bull. 205: 195-197. (October 2003) © 2003 Marine Biological Laboratory Rho-kinase Is Required for Myosin-II-Mediated Vesicle Transport During M-Phase in Extracts of Clam Oocytes Torsten Wollert', Ana S. DePiiur. Carl J. DeSelnr, and George M. Langford' Rostock University, Rostock, Germany ~ Dartmouth College, Hanover, NH In mammalian cells. Rho proteins Rho/Rac/Cdc42 regulate the formation of the actin cytoskeleton in stress fibers, lamellipodia, and filopodiu ( I ). One of the ways in which Rho proteins mediate effects on the actin cytoskeleton is via the Rho-ROK/Rho kinase- myostn phosphatase pathway. In this pathway, myosin light chain phosphatase (MyoP) is phosphorylated by ROK/Rho kinase and is thereby inhibited (2). The net result is the activation of myosin- 11-mediated activities. In addition, Rho-family proteins have also been shown to regulate the actin cytoskeleton during cell division (3). To study the role of Rho proteins in myosin-II mediated vesicle transport during the M-phase of the cell cycle, we used Y27632 to inhibit Rho kinase activity in extracts of clam oocytes. We have shown previously that actin filaments assemble spontaneously in 196 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS such extracts and organize into a three-dimensional network of interconnected til < This self-organized network of actin filaments resent'" ytokinetic ring of dividing cells in the following v a it exhibits myosin-II-mediated, anti-parallel sliding of li .nents (4, 5), and (ii) it assembles during the M-ph:; , cell cycle. Fortuitously, actin filaments in the extracts co-align to form bundles (10-20 parallel filaments) that are visible by AVEC-DIC microscopy. We have also shown that vesicles are moved along the actin filaments by a class II myosin motor (4). In this report, we show that a specific inhibitor of Rho kinase. Y27632, blocks vesicle transport in these extracts, thereby providing additional evidence that vesicle movement on actin in these extracts is mediated by myosin-II. Extracts prepared from mature oocytes arrested at the G2/M phase of the cell cycle were snap frozen and stored at —80 °C. To begin an experiment, the pH of the cytoplasmic extracts was shifted from 6.8 to 7.2 by diluting 2-fold with pH-8 buffer to initiate progression through M-phase. Nocodazole (30 JJ.M) was added to the extracts to block microtubule assembly; an ATP regenerating system was added to maintain ATP levels; and the preparation was incubated at 1 8 °C to assemble actin filaments and reconstitute motor activity. Rhodamine-phalloidin (0.5 ^.M) was 45' J_ .S 40- T JL_ E -JL_ T 1 fl 35' ~ 1 !5 30- 09 — JL 1 > 20' 0* I | IS- 0 S 10- 1 0-15 15-30 30-45 45-60 60-75 75-90 90-105 105-120 120-135 135-150 Time (min) -35 30 — [3 25 ' ~l '> O 20 Control 2 jiM Staurosporine 300 nM Y-27632 Figure 1. Vesicle transport was measured at regular intervals. Motile activity is defined as the number of vesicles moving/video field/min. Motile activity is /j/'v/i /, ft, :\ hut declines at 15-30 min. Motile activity returns to high levels benveen 45 and 120 min. (B) Xenopus sperm nuclei were added to the extract at time era to determine the phase of the cell cycle. The initial elongated shape changed to a round shape at 30 min, and the chromosomes condensed ai 45 iiu/i . .,.:/, wi, of M -phase. The nucleus remained condensed at 90 min but then began to expand at 120 min. (O Inhibition of motile activity in the present i n/ )'27f>32 and Manrosporine. A reduction of the motile activity was observed. CELL BIOLOGY 197 added to stain the net in filaments, and the myosin-Il motor activity was monitored by AVEC-D1C and fluorescence microscopy. In control extracts, vesicle transport was measured at regular time points to determine whether motile activity varied upon entry into M-phase of the cell cycle. Vesicle transport was measured by counting the number of vesicles moving per video field per min (v/f/m: motile activity) at 15-min time intervals. We found that the motile activity was high during the first 15 min of incubation at 18 °C, then declined during the 15-30-min interval but rose again and remained stably high between 45 and 60 min (Fig. 1A). Motile activity declined again after 120 min at 18 °C. The actin network, as revealed by rhodamine-phalloidin staining, did not change dur- ing the 2-h period of incubation. To determine when the extracts were in the M-phase of the cell cycle, a Xenopus sperm-nucleus shape-change assay was performed. Xenopus sperm nuclei were added to the extract, stained with DAPI, and observed by fluores- cence microscopy at regular intervals during incubation. In the initial period, the Xenopus sperm nuclei remained elongated, but they began to expand and appear uniformly bright at 30 min (Fig. IB). At 45 min. the nuclei assumed an irregular shape as the chromosomes condensed. Chromosome condensation is diagnostic of M-phase. The nuclei remained irregular in shape with con- densed chromosomes from 45-120 min, the period when motile activity was high (Fig. 1A). Based on this assay, extracts incubated for 45 to 60 min were judged to be in M-phase. To determine whether Rho kinase is required for vesicle trans- port, extracts were incubated at 18 °C for 45 min, and then the inhibitor Y27632 was added at a concentration of 300 nM. We found that Y27632 inhibited vesicle transport by 65% compared to the control (Fig. 1C). The inhibitor had no effect on the actin cytoskeleton. The fact that vesicle transport was strongly inhibited at low concentrations of the inhibitor suggested that the specific target of the inhibitor was Rho kinase, the most sensitive target of this inhibitor. Inhibition was also achieved with 2 mM staurospor- ine (Fig. 1C), a general kinase inhibitor. Based on the results with the Y27632 inhibitor, we conclude that Rho kinase is required for movement of vesicles on actin filaments. In summary, these data show that Rho kinase is directly in- volved in vesicle transport. Because there was no effect on the actin cytoskeleton in this case, the downstream target of Rho kinase is mostly likely myosin II. This is the only myosin-mediated pathway known to be regulated by Rho GTPases. The downstream effector of the Rho proteins is most likely myosin light chain phosphatase ( 1 ). Therefore, we can conclude that the Rho-ROK/ Rho kinase-myosin phosphatase pathway regulates vesicle trans- port during M-phase in clam oocytes. This work was supported by NSF Grant IBN-0131470 and MBL Shifman award to CJD. Literature Cited 1 Bishop, A. L., and A. Hall. 2(100. Biochem. J. 348: 241-255. 2. Kimura, K., M. Ito. M. Amano, K. Chihara, Y. Fukata, M. Naka- fuku, B. Yamamori, J. Feng. T. Nakano, K. Okawa, A. Iwamatsu, and K. Kaibuchi. 1996. Science 273: 245-248. 3. Yoshizaki, H., Y. Ohba, K. Kurokawa, R. E. Itoh, T. Nakamura, N. Mochizuki. K. Nagashima, and M. Matsuda. 2003. J. Cell Bi»l. 162: 223-232. 4. Wollert, T., A. S. DePina, R. F. Reid, and G. M. Langford. 20(12. Biol. Bull. 203: 20S-210. 5. Wollert, T., A. S. DePina, L. A. Sandberg, and G. M. Langford. 2001. Biol. Bull. 201: 241-243. Reference: Biol. Bull. 205: 197-199. (October 2003) © 2003 Marine Biological Laboratory An Experimental Approach to the Study of Gap-Junction-Mediated Cell Death A'. Cusato' ', J. Zakevicius2, and H. Ripps"* 'Albert Einstein College of Medicine, Bronx, NY ~ University of Illinois College of Medicine, Chicago, IL The vertebrate retina is a highly specialized sheet of neural tissue derived from an undifferentiated population of neural pro- genitor cells, which, upon completion of their final division, mi- grate to their laminar positions and differentiate ( I ). In the adult retina, almost every class of neuron and glial cell is linked to its neighbors by gap junctions — aqueous channels that allow the intercellular exchange of ions, second messengers, and other small molecules (< 1 kDa). Thus, this communication pathway aids in the synchronization of cellular activity, and plays a significant role in maintaining cellular homeostasis (2). During development, however, many retinal cells undergo programmed cell death, or apoptosis, and both intracellular and intercellular communication are known to regulate the process (3). Indeed, we recently reported * Corresponding author: harrripp@uic.edu that gap junctions mediate a form of "bystander" cell death in the developing retina (4). Although this process is largely arrested in the mature retina, differentiated neurons and glia undergo apopto- sis in neurodegenerative diseases (5), as well as in ischemia and trauma. In all of these cases, the spread of cell death from one dying cell to its otherwise unaffected neighbors (bystanders) may increase the total number of cells that enter the apoptotic pathway. Because the retina is a complex tissue, some studies of by- stander cell death are technically unfeasible at this time. We have developed a model system for the study of gap junction-mediated cell death using Xenopus oocytes which express an endogenous gap-junctional protein (connexin38, [Cx38]). Oocytes are paired at their vegetal poles following removal of their vitelline membranes and become electrically coupled via gap junctions. Because the Xenopus oocyte can serve to express many different connexins, the system should enable us to identify which gap junctional channels 198 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 0 CcCell Bystander Cell 05- < o- n -0.5- 05' < o- -05 5s -*— Cc injected Cell o Bystander Cell -50 20 40 60 Time(min) 80 100 120 Figure 1. (a.b) Photographic images of two pair.', of Xenopus oocytes tluit have been juxtaposed at their vegetal poles. The oocytes express an endogenous connexin (C\38) and one member of each pair (asterisks) was injected with cytochrome c. Images were collected at 20 min (a} and 3 h (b) following cylochrotne-c injection. Both cytochrome c-injected cells have undergone cell death (evidenced h\ /v.v/.s or cell swelling). Note that the noninjected cell in h (arrowhead in right pair) a/so showed clear signs of cell death, whereas the noninjected cell paired to an injected cell that had deteriorated completely in less than 41) min did not. (c) Currents recorded from paired oocyles under dual electrode voltage clamp, shortly after cytochrome c injection, illustrate that the cells are eleclricallv coupled. The upper trace shows currents from a cell in which hyperpolarizing voltage steps ( — 100 to —20 mV) were applied, vhile the bottom trace shows currents from the coupled cell, (d) Same cell pair and same protocol 311 min later. Traces indicate that cells remain foii/i/fil , los\ of membrane integrity: note the greater currents reauired to maintain the cells in voltage clamp, (e) The time course of membrane potential chang recorded from a pair of oocvtcs c\prc\sing C.\3K following cytochrome c injection into one cell. Note the rapid loss of membrane potential in llic injf: h '• ,:>id the slower changes associated with death of the bvstander cell. See text for details. CELL BIOLOGY 199 remain open under apoptotic conditions, to study the dynamics of gap junctional coupling during cell death, and to determine which molecules may pass from a dying cell to its neighbors to trigger the apoptotic process. Conversely, it is important to recognize that gap-junction-mediated cell death may result from the depletion of essential molecules (e.g.. ATP) passing from the healthy cell to its dying neighbor. Previous studies of single Xenopus oocytes have shown that microinjection of cytochrome c induces apoptotic cell death, ac- companied by a progressive loss of membrane potential, activation of caspase 3. and DNA fragmentation (6). In the present study we have shown that injection of cytochrome c into one oocyte of a Cx38-coupled pair causes death in both the injected and nonin- jected cells over a period of 2-3 h (Fig. la,b). Pairs of oocytes preinjected with an antisense oligonucleotide to Cx38 did not exhibit bystander cell killing following cytochrome c injection; although the cell into which cytochrome c had been injected did die, its paired neighbor did not. Similarly, in cases where the cells were electrically coupled, but where the cytochrome c injected cell lysed in less than 40 min. the noninjected cell did not die (Fig. la.b. left pair). This result suggests that the intercellular channels joining the cytoplasm of the coupled cells must remain intact for longer than 40 min for bystander cell death to occur. It also indicates that bystander killing is not mediated by contact or by extracellular toxins, since the surviving cell continued to be in close apposition to the dying cell but remained intact. Vehicle injections failed to induce cell death in either injected or nonin- jected cells of electrically coupled pairs. Dual electrode voltage-clamp using GeneClamp 500 amplifiers controlled by pClamp 8 software (Axon Instruments, Foster City, CA) was used to monitor gap junctional coupling between cell pairs after injecting one of the cells with cytochrome c. The two cells were voltage-clamped to the same potential (-40 mV). voltage steps were applied to the noninjected cell, and the current responses of both cells were recorded (7). As shown in Figure 1 (c.d), cells remained electrically coupled despite the gradual death of the injected cell. In addition, we found that the loss of mem- brane potential resulting from cytochrome c injection reported by Bhuyan et al. (6) in single oocytes could be seen also in cell pairs. After injecting one cell with cytochrome c, both cells underwent membrane depolarization, with the injected cell losing membrane potential more rapidly following injection (Fig. le). Although we have induced cell death by intracellular injection of cytochrome c, it is evident that the molecular mass of this apoptotic agent ( 13 kDa) is too great to pass through gap junctions (8). Clearly, cytochrome c itself cannot be the toxic substance carrying the death signal to bystander cells. Likewise, bystander killing is unlikely to be mediated by contact or diffusible sub- stances, since antisense to Cx38 prevented bystander cell death, but not primary cell death. Of particular interest is the fact that gap junctional coupling persists during the apoptotic process, encour- aging us to use the Xenopus oocyte and other expression systems in future experiments to identify the intercellular signals that pass between a dying cell and its coupled partners to induce bystander cell death. These studies were conducted at the Marine Biological Labora- tory, Woods Hole, Massachusetts, and were supported by grants from the NIH (HR: EY-06516 and EY-01792; KC: HL-07675); a Grass Foundation Fellowship (KC); an unrestricted award to the UIC Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness. Inc.; a Senior Research Investiga- tor Award from the RPB (HR); and an Award of Merit from the Alcon Research Institute (HR). Literature Cited 1. Robinson, S. R. 1991. Pp. 69-128 in Vision and Visual Dysfunction. Vol 3: Neuroanatomy of the Visual Pathwavs and their Development. B. Dreher and S. R. Robinson, eds. Macmillan, London. 2. Andrade-Rozental, A. F.. R. Rozental, M. G. Hopperstad, J. K. Wu, F. D. Vrionis, and D. C. Spray. 2000. Brain Res. Rev. 32: 308-315. 3. Linden, R. 2000. Brain Rcy Rev. 32: 146-158. 4. Cusato, K., A. Bosco, R. Rozental, C. A. Guimaraes, B. E. Reese, R. Linden, and D. C. Spray. 2003. J. Neurosci. 23: 6413 6422 5. Ripps, H. 2002. E.\p. Eye Res. 74: 327-336. 6 Bhuyan, A. K., A. Varshney, and M. K. Mathew. 2001. Cell Death Differ. 8: 63-69. 7 Dahl, G., T. Miller, D. L. Paul, R. Voellmy, and R. Werner. 1987. Science 236: 11.290-11,293. X Bennett, M. V. L., V. K. Verselis. R. L. White, and D. C. Spray. 1988. Pp. 287-304 in Gap Junctions. E. L. Hertzberg. and R. G. Johnson, eds. Liss, New York. Reference: Biol. Bull. 205: 199-201. (October 2003) © 2003 Marine Biological Laboratory Apoptosis in Microciona prolifera Allografls S. Tepsuporn1'*, J. C. Kaltenbach' , W. J. Kuhns2, M. M. Burger , and X. Fernandez-Busquets4 1 Mount Holyoke College. South Hadley, MA 2 Hospital for Sick Children. Toronto, Canada 3 Friedrich Miescher Institute, Basel, Switzerland University of Barcelona, Barcelona, Spain When two sponge tissue fragments from the same individual are adjoined, isogeneic recognition occurs, and the fragments fuse. On * Corresponding author: stepsupo@mtholyoke.edu the other hand, if the two pieces are from different individuals, allogeneic recognition occurs, followed by failure of fusion and. presumably, death of cells at the graft contact zone (1. 2). It has been proposed that two cell types, archaeocytes and gray cells, are involved in sponge allogeneic recognition. Archaeocytes are large 200 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS phagocytic cells with In, L-C :iudeoli; they are commonly found in wound healing :m<; • -ting areas of the sponge (1). Gray cells are motile cell' ' ai many dense cytoplasmic granules ( 1 ); they do nut . il-defined pseudopodia and are commonly found in gi . egions of the sponge but are rare in the marginal region V is (programmed cell death) in sponges was first dem- onstrated by TUNEL nick-end labeling assay in hibernating sponges that had undergone tissue regression as a naturally occur- ring winter event (4). The development of an alternate method for detecting apoptosis was the finding that a series of caspases (pro- teases) delineated sequential enzymes leading to cell death (5). Of these, caspase-3 enzyme was the final and most important pro- tease. From this have emerged antibodies for the detection of caspase-3 (6). Biochemical methods were used by Wiens et al. (7) to study apoptotic events in extracts of the marine sponge Geodia cydonium, where caspase-3 activity was found to be greatly in- creased in allograft extracts when compared with isografts. The objectives of the present research are (a) to determine by immu- nohistochemistry whether cell death at the allograft contact zone is a result of apoptosis rather than necrosis (death by injury that may result from mechanical damage to the cells) and (b) to identify the cell type or types, if any, that undergo apoptosis at the contact zone. To examine whether the death of cells at the contact zone is a result of apoptosis, we used an indirect labeling technique that provides precise tissue localization of active caspase-3 as a marker for cells undergoing apoptosis. Processed cells that were reactive were visualized by the presence of a dark brown precipitate when color was developed with diaminobenzidine (DAB). Individuals of Microciana prolifera were collected by the Ma- rine Resources Center of the Marine Biological Laboratory (Woods Hole, MA) and were maintained until use in a tank with running cold seawater. The two sponge pieces for isografts and allografts were held together with number zero stainless steel pins attached to a Styrofoam board floating in a tank of running cold seawater. The grafts were fixed at 6-h intervals from 0 to 24 h in 3.7% formaldehyde in MBL artificial seawater (MBLASW) over- night, then washed and dehydrated in a series of ethyl alcohol concentrations from 30% to 70% in MBLASW. Spicules were dissolved by overnight treatment of grafts with 4% hydrofluoric acid in 70% ethanol. The grafts were then embedded in paraffin and sectioned at 7 jum. The slides of sectioned isografts and allografts were deparaf- finized, hydrated. and treated with 3% hydrogen peroxide to re- move endogenous peroxidase activity. Tissue presumed to possess caspase-3 activity was incubated using purified rabbit IgG anti- active human caspase-3, which had been generated by affinity purification from a caspase-3 enzyme fragment (Pharmingen #559565); the antibody was used at a concentration of 0.25 jug/ml for 2 h :it room temperature. The slides were then washed with phosphate-buffered saline (PBS. pH 7.5) (Sigma), followed by treatment with appropriate biotinylated secondary antibody (Vec- tor Laboratories). After another wash in PBS, the slides were treated with avidin-biotinylated enzyme complex ( VectaStain Elite ABC Kit, Vector Laboratories) before the peroxidase substrate DAB (Sigma) was applied. The slides were washed, dehydrated. mounted, and photographed with a digital camera on a Zeiss microscope. Isograft sections treated with anti-active caspase-3 primary an- tibody did not stain, indicating that apoptosis did not occur in either the line of contact or any of the cells in the grafts during a 24-h period (Fig. 1A). On the contrary, treatment of allograft sections with the same primary antibody showed that cell death at the contact zone was indeed a result of apoptosis. Cells near the contact zone started to undergo apoptosis 6 h after grafting (data not shown). The line of contact became most apparent at 24 h (Fig. IB). The longer the period of allogeneic contact, the higher the number of large, elongated apoptotic cells accumulated at the contact zone. The morphology of these cells was consistent with their identity as archaeocytes. However, the existence of archaeocytes and gray cells in the contact area ( 1 ) prompted the use of a gray cell identity marker in S>V$V!K J'^vOii- ' -?••' -^ -'•'•'•,;•";;;•£;. > -,^'- \ ;,-.', . ' i ;^M#?. Sm:%? Figure 1. Apoptotic response of allografts prepared from the marine sponge Microciona prolifera. (A) Control isograft: fixed tissue stained with anti-active caspase-3 antibody. Arrows indicate the zone of contact. No reaction occurs in this zone or in cells in the area. Only occasionally do a few cells within the tissue show positive staining (arrowhead). Viewed under phase contrast. (B) Allograft fixed al 24 h. stained with anti-active caspase-3 antibody. A marked reaction is seen at the zone of contact (arrows}, and in cells in the neighborhood of the contact area. (C) Allograft fixed at 6 h double stained with anti-active caspase-3 antibody, visualized as a brown precipitate with DAB-H,O2. and with anti-CD44 antibodv. visualized by pink fluorescence. Brown apoptotic cells desig- nated as archaeocytes surround the contact line (arrows), and appear to be distinct from the pink fluorescent (i.e.. CD44 positive) gray cells (arrow- head). Colocalization of caspase-3 and of CD44 was not obsen'ed. Bar represents 50 [Jim. CELL BIOLOGY 201 addition to identification by morphology. CD44 has previously been demonstrated in gray cells, but not in archaeocytes ( 1 ), and therefore anti-CD44 antibodies were used in combination with anti-active caspase-3 antibodies in double labeling experiments. CD44 staining was done using a rat anti-mouse CD44 (Pharmin- gen) at I /xg/ml for 12 h at 4 °C, followed by wash and biotin- labeled secondary antibody (Vector Laboratories). Fluorescence was developed using Texas red-avidin (Vector Laboratories). The slides were treated as described above (microscope setting for single field imaging in fluorescent double staining: 647 nm filter, mercury and halogen lamp as light sources). Colocalization of anti-active caspase-3 and anti-CD44 was not observed (Fig. 1C). We conclude that cell death at the allograft contact zone is a result of apoptosis rather than tissue necrosis, and that the apop- totic cells are most likely archaeocytes. not gray cells. The ability to recognize self from non-self has been conserved throughout evolution. A better knowledge of this process in the sponge model should enhance our understanding of allogeneic recognition and its regulation in more complex species, including humans. The knowledge gained from such basic mechanisms should be of use in developing effective specific drugs that can abrogate the pathological effects of allogeneic responses (8). Acknowledgments: Mount Holyoke College HHMI Cascade Mentoring Program: Research Experience for Undergraduates of the Boston University Marine Program; Friedrich Miescher Insti- tute of the Novartis Research Foundation. X. F.-B. holds a Ramon y Cajal tenure-track position from the Ministerio de Ciencia y Tecnologfa (MCyT). Spain, and acknowledges the support of grant BIO2002-00128 from the MCyT. Literature Cited 1. Fernandez-Busquets, X., W. J. Kuhns. T. L. Simpson, M. Ho, D. Gerosa, M. Grob, and M. M. Burger. 2002. Dei. Comri. Immunol. 26: 313-323. 2. Yin, C., and T. Humphreys. 1996. Biol. Bull. 191: 159-167. 3. Simpson, T. L. 1968. P. 23 in The Structure and Function of Sponge Cells: New Criteria for tin- Taxonomy of Poecilosclerid Sponges. Pea- body Museum of Natural History. New Haven, CT. 4 Kuhns, VV. J.. M. Ho, M. M. Burger, and R. Smolowitz. 1997. Biol. Bull. 193: 239-241. 5. Kaufmann, S. H., and M. O. Hengartner. 2001. Trends Cell Biol. 11: 526-534. 6 Volm, M.. J. Mattern, and R. Koomaegi. 1999. Anricuncer Res. 19: 1669-1672. 7. Wiens. M.. A. Krasko, S. Perovic, and W. E. Miiller. 2003. Bin- chim. Biophys. Ada 1593: 179-189. s Kuhns, W. J., M. M. Burger, M. Sarkar, X. Fernandez-Busquets, and T. L. Simpson. 2000. Biol. Bull. 199: 192-194. Reference: Biol. Bull. 205: 201-203. (October 2003) © 2003 Marine Biological Laboratory The Decorated Clot: Binding of Agents of the Innate Immune System to the Fibrils of the Limulus Blood Clot Peter B. Armstrong1'2'*, and Margaret T. Armstrong1'2 1 Marine Biological Laboratory, Woods Hole, MA ~ University of California. Davis, CA The fibrillar blood clot functions as an important element of the innate immune system by its ability to entrap and immobilize bacteria that have entered the body viti wounds, thereby preventing their systemic dissemination throughout the body of the injured host (1,2). The coagulin clot of the horseshoe crab appears to play a similar role in protecting that animal from pathogenic attack. Bacteria entrapped in the coagulin clot are held so tightly as to abolish even thermal (Brownian) motion, and the clot synergizes with plasma in the killing of entrapped microbes (3). The present study investigates the proteins of the innate immune system of Limidus that bind to the fibrils of the coagulin clot, potentially supplementing the entrapment actions of the clot in two ways: first, by the lethality of clot-bound proteins for the entrapped microbes and second, by the ability of these proteins that decorate the clot fibrils to bind and inactivate the toxic products of entrapped microbes. To establish the clot, the blood cells contained in 1 drop of blood collected under sterile conditions were dispersed in 1 ml of sterile 3% NaCI (Baxter Healthcare Corp.. Deerfield. IL) in a 35-mm * Corresponding author: pbarmstrong(s'ucda\ is.edu plastic petri dish (Falcon Cat # 35-1008). After 5 min to allow attachment of the cells to the dish surface, the saline was replaced with 50% or 100% sterile Limulus plasma. Under these conditions, the blood cells rapidly degranulated. releasing the coagulin blood- clotting system. A dense coagulin clot then formed above the monolayer of attached blood cells. After 0.5-2 h of washing with several changes of wash buffer, this was either fixed directly in 4% paraformaldehyde dissolved in 3% NaCI, 10 mM CaCU or was extracted with 0.5% Triton X-100 in the same buffer and then fixed in paraformaldehyde. All of the antibodies used for this report showed identical staining patterns for the two preparations. The various proteins used to prepare antibodies were purified as fol- lows: coagulogen. the structural protein of the blood clot, as described by Srimal ci ai (4); Limulus a2-macroglobulin. as described by Armstrong el at. (5): the Limulus pentraxins, as described by Armstrong et al. (6): and hemocyanin. purified by ultracentrifugation (285,000 X g, 8 h) followed by gel filtration ehromatography (Sephacryl S-300). Antibody production in rab- bits and immunocytochemical staining utilized standard methods (7). The polyclonal antibodies were checked for specificity by Western blotting (8) and in some cases were affinity-purified on antigen-Sepharose affinity columns (7). 202 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS The fibrillar structi' • i'v coagulin clot showed to advantage both by phase • '.copy and by immunocytochemical staining with >gen antibodies (data not shown). The same fibril^ iiiunostained with antibodies prepared against highly pi .-parations of Liimilus a-,-macroglobulin (data not shown i. inilus pentraxins (Fig. I ), and hemocyanin (data not shd\' nndus os-macroglobulin functions in the binding and cle •:. uce of proteases, including, presumably, the proteases of pathogenic microbes (9). The Limn/us pentraxins show a potent cytolytic activity against foreign cells and may operate to assist in the cytolytic destruction of microbial invaders (6, 10). Hemocya- nin is the respiratory protein in solution in the blood but addition- ally shows a phenoloxidase activity that potentially functions to kill microorganisms by the generation of oxygen radicals (11). The binding of os-macroglobulin may be covalent because it was not removed by treatment prior to fixation with boiling SDS- polyacrylamide gel sample buffer containing 2-mercaptoethanol. Most of the known ligand-recognition properties of the Limulus pentraxins are Ca+2-dependent (6). In contrast, binding to the coagulin clot is Ca + "-independent, because immunostaining is not diminished by treatment of the clot with a Ca + 2-ehelating agent, ethylenediaminetetraacetic acid (EOT A— 0.1 M EDTA. 0.5 M NaCl, 10 mM Tris, pH 7.3). Most of the bound hemocyanin is removed by treatment of the clot with EDTA, but it is not certain whether this is simply a reflection of the dependence of the oligomeric structure of the hemocyanin molecule on Ca+2 ( 12) or a true Ca+2-dependent binding of hemocyanin to the coagulin fibrils. There are two potential sources for the clot-bound ar macroglobulin: the plasma (13) and the «2-macroglobulin released from the secretory granules of the blood cells (14). We have not ruled out binding of plasma a2-macroglobulin. but secretory- granule-derived os-macroglobulin does contribute to the clot- bound protein, because the clot fibrils produced by cells that degranulate in saline (0.5 M NaCl, 10 mM CaCU) in the absence of plasma are decorated with a,-macroglobulin. The fibrin fibers of the mammalian blood clot are known to bind a suite of proteins that assist in the functions of the clot. The blood clot of mammals binds fibronectin, which potentiates the immi- gration of wound-repair fibroblasts (15); FGF-2. which promotes proliferation of clot-associated endothelium (16, 17); and the ser- pins, plasmin activator inhibitor-2 (PAI-2) and os-antiplasmin. which are presumed to protect the clot from proteolysis (18). However, we are not aware of any reports of agents of the immune system binding to the fibrin clot of mammals. Thus the observation that immune effector proteins bind to the Liimilus clot suggests the novel idea that the clot is more than a passive entrapment device for invading microbes: it is potentially a delivery vehicle for proteins that are lethal to the entrapped microbes and proteins that inactivate toxic products of those microbes. Indeed, the clot syn- - •'.••' ••<.'• Ki;- I. Binding <'/ ///( LiMHilus jiciniii \ms in Ithiils >>! ihc l.muilus hlooil tl{>t in spci'itticns not c\Irticicil inY// Itifon X-llKI. The fibrils oj the clot prodiK monoluyers of Limulus blood cells me visible />\ phu.\c conlruxl niicroscn/n f.-l. O and iiiiniiinnxttiin \\-ilh tin untihtnly against the Limulus pentnnii:' 'i The (/nvnr.v in A and />' nhlicule the MI/IIC tihril r;.M/)/c />v />/«;«• cuntniM inn TOM -o/'v (A) and immunofluorescence (B). The darkish bodies H'<'" b\ /',. trasl niifn>\c<>/>\: (A. Cl arc ilie ninici n/ the hlcnj cell\ atlac/icil In the culture \iiifaee. \Vlien normal rahbit serum replaces specific antibodv. ll;c ill ,'\ that are clearly visible b\ phuse contrast micros-coin' fC) fail to slain ID), 8 and D were I'lmlo^rapheJ under identical conditions unJ were manipulated niciU'cally in Photoshop, so are directly comparable. CELL BIOLOGY 203 ergizes with factors in the plasma in effecting the active killing of clot-entrapped microbes (3). This research was supported by Grant No MCB-2677 1 from the National Science Foundation. Literature Cited 1. Dunn. I). I... and R. [.. Simmons. 1982. SurKcry 92: 513-519. 2. Rotstein. O. I). 1992. Em: J. Clin. Mk-rohiol. Intcci. O/.v 11: 1064- 1068. 3. Isakova, V.. and P. B. Armstrong. 2003. Bi,>l. Bull. 205: 203-204. 4 Srimal, S., T. Miyata. S. Ka\vabata, and S. hvanaga. 1985. J. Bio- chem. Tokyo 98: 305-3 1 S. 5. Armstrong. P. B., R. Melchior. and J. P. Quigley. 1996. ./. In.wt Pliyxiol. 42: 53-64. 6. Armstrong. P. B., S. Swarnakar. S. Srimal. S. Misquith, E. A. Hahn, R. T. Aimes, and J. P. Quigley. 1996. J. Biol. Chem. 271: 14,717- 14.721. 7. Harlow. E.. and I). Lane, eds. 1988. Antibodies, a Laboratory Man- ual. Cold Spring Harbor Laboratory. Cold Spring Harbor. NY. 8. Towbin. H.. T. Staehelin, and .1. Gordon. 1979. Proc. Nail. Acail. Sci. USA 76: 4350-4354. 9. Melchior. R., J. P. Quigley, and P. B. Armstrong. 1995. J. Bin/. Chem. 270: 13.496-13.502. 10. Swarnakar, S.. R. Asokan, .1. P. Quigley. and P. B. Armstrong. 2000. Bio, hem. ./. 347: 679-685. 11. Nagai, T., and S. Kawabata. 2000. J. Biol. Chem. 275: 29.264- 29.267. 12. Van Holde, K. E., and K. I. Miller. 1982. Q. Rev. Biorihy*. 15: 1-129. 13. Quigley, J. P.. and P. B. Armstrong. 1983. J. Biol. Chem. 258: 7903-7906. 14 Armstrong, P. B., J. P. Quiglev, and F. R. Rickles. 1990. Biol. Bull. 178: 137-143. 15. Mosher, D. F. 1975. ./. Biol. Chem. 250: 6614-6621. 16. Sahni, A., and C. W. Francis. 2(100. Blood 96: 3772-3778. 17. Sahni, A., T. Odrljin, and C. VV. Francis. 1998. J. Biol. Chem. 273: 7554-7559. 18. Ritchie, H.. L. C. Lawrie. P. \V. Crombie. M. VV. Mosesson, and N. A. Booth. 2000. ./. Biol. Chem. 275: 24.915-24.920. Reference: Biol. Bull. 205: 203-204. (October 2003) © 2003 Mamie Biological Laborators Imprisonment in a Death-Row Cell: The Fates of Microbes Entrapped in the Limulus Blood Clot Victoria Isakova1'2 and Peter B. Armstrong1' '* ' Marine Biological Laboratory, Woods Hole, MA ' Hunter College of the City University of New York, New York, NY 3 University of California, Davis, CA The fibrillar blood clot is an extracellular matrix established at sites of damage to the walls of the blood-vascular system. In humans, the clot is a polymer of the protein, fibrin. In the horse- shoe crab. Limulus polyphemus. the clot is a meshwork of fibrillar polymers of the protein, coagulin ( 1 ). The blood clot functions to seal the wound to staunch bleeding, operates as a transient extra- cellular matrix for the migration of wound-healing epithelial and connective tissue cells, and serves as a barrier to entry of microbes into the interior of the animal via the wound. This study concerns the characterization of this last function for the coagulin blood clot of Limulus. We investigated the extent of immobilization of bac- teria by the coagulin blood clot and the viability of the clot- entrapped bacteria. Plasma was collected from adult horseshoe crabs under sterile, lipopolysaccharide-free conditions by cardiac puncture. Blood cells were removed immediately after collection and the plasma was sterile-filtered through a filter with a pore size of 0.22 /j.m (Corning. Inc.. Cat # 4307691. The marine bacterium \'ihri<> a/i;i- nolyticus was grown in liquid culture on Marine Broth 2216 (Difco). Log-phase growing populations were established by cul- ture for 12 h at room temperature. Blood clots were established by plating the cells contained in 1 drop of Limulus blood collected by cardiac puncture under sterile conditions in 2 ml of sterile 3% NaCl (Baxter Healthcare Corp.. Deerfield. IL) in a 35-mm plastic petri dish (Falcon. Cat # 35-1008). Bacteria suspended in O.I M * Corresponding author: pbarmstrong@ucdavis.edu sucrose. 3% NaCl. equivalent to the bacteria contained in 250 /nl of the original culture medium, were introduced into the culture dish before the blood cells were added to facilitate direct presen- tation of bacteria to the blood cells while the latter were attaching to the culture surface of the dish. Under these conditions, the blood cells degranulate to release coagulogen and the proteases that process it into coagulin. the form that polymerizes into the fibrillar clot (2). The result is the entrapment of numbers of bacterial cells in the meshwork of fibers of the coagulin clot. The fate of the entrapped bacteria was investigated by direct light microscopic observation. The two parameters of greatest interest were the immobilization of bacteria by the clot and the killing of clot- entrapped bacteria by components of the plasma operating in synergy with the clot. In suspension. V. uli;iii<>l\ticus shows rapid flagellar-driven swimming locomotion. Bacteria entrapped in the coagulin clot are immotile. and are held so tightly as to lack even thermal (Brown- ian) motion. When bacteria were killed to eliminate swimming motility. more than 909r of the killed bacteria in suspension showed thermal (Brownian) motion: in contrast, all bacteria en- meshed in the clot were absolutely stationary, without thermal motion. Entrapped bacteria survive and proliferate in clots maintained in artificial seawater. which is isotonic to Limulus blood. In a typical trial. 87% of the bacterial clusters enmeshed in the clot contained only one cell immediately after capture, but 54% of the bacterial clusters contained two or more cells by 4 h of incubation. 204 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS "-> t^^.f *A^t „. 1&&N : ^4" Figure \. Proliferation and killing of Vibrio alginolyticus entrapped in the Limulus blood clot. Sequential views of the same microscopic field shmving individual bacteria entrapped in the clot (A. t = 0 h) survive and proliferate to establish clumps of two or more cells (B, t = 2 h) when the clot with contained bacteria is incubated in bacteriologic culture medium. Bacteria entrapped in the clot are efficiently killed when the preparation is exposed to plasma. After 4 h incubation in marine broth culture medium, the clot-entrapped bacteria show as dense phase-bright bodies when viewed by phase contrast microscopy (C). Clot-entrapped bacteria become nonrefractile ghost cells when incubated for 4 h in plasma (D). Proliferation is rapid if the clot, with its cargo of entrapped bacteria, is transferred to bacterial culture medium (Fig. 1A, B). After 4 h of incubation of the clot with entrapped bacteria in culture medium, 84% of bacterial clusters contained two or more cells. However, bacteria were killed when the preparation of clot with entrapped bacteria was transferred to sterile Limulus plasma. Killing was monitored by loss of retractility of the bacterial cells under phase -contrast microscope examination (Plan 100/1.3 NA objective) (Fig. 1C, D). The non-refractile cells ("ghost cells") are presumed to be dead or seriously damaged. Loss of bacterial refractility shows a lag period of 1.5 h, after which killing is rapid. In a typical trial, cells appeared normal and strongly retractile at 1 .5 h, but about 80% of the cells had become transparent at 2 h. We did not observe bacterial proliferation in the plasma-treated clot even after 8 h of incubation; whereas, as noted above, the seawa- ter-treated clot did support bacterial growth. The LD50 was at a 25% dilution of plasma. Interestingly, plasma-based cytotoxicity was dependent on entrapment in the coagulin clot because free bacterial cells diluted into plasma remained phase-dense and pre- sumably alive. These cells did lose the capacity for flagellar- generated motility, but showed the same capacity for proliferation when transferred to nutrient agar as did cells treated under equiv- alent conditions in plain seawater. Hemocyanin-free plasma produced by ultracentrifugation (285,000 X t-, 8 h) retained the bacteriolytic activity of whole plasma; and purified hemocyanin was not bacteriolylic at 40 mg/ ml, its concentraiion in plasma, indicating that hemocyanin is not involved in killing. The cytotoxic agent of plasma appears to be a protein, because heat-treated plasma ( 100 °C, 0.5 h) lost bacterio- lytic activity. Dialyzed plasma, which lacked all components smaller than 10-12 kDa, was fully active, indicating that small molecules are not necessary for cytotoxicity. In summary, we have defined two important contributions of the coagulin blood clot to immunity in Linutliis. The clot immobilizes microbes, which presumably impedes their dissemination through- out the animal after gaining access via a wound. Plasma also shows an immobilizing action by its inhibition of rlagellar swimming motility. Neither clot nor plasma alone kill the bacteria but the two synergize to effect the cytotoxic destruction of clot-entrapped microbes. This research was supported by a grant MCB 26771 from the National Science Foundation (PBA) and a fellowship from the Howard Hughes Medical Institute Undergraduate Science Educa- tion Program in Biology, grant 52002679 (VI). We thank Ms. Alice Child, Mr. Joseph Lee, and Drs. William Cohen and Norman Wainwright for help with the research. Literature Cited 1. Iwanaga, S., T. Miyata, F. Tokunaga, and T. Muta. 1992. Thromb. Res. 68: 1-32. 2 Armstrong, P. B., and F. R. Rickles. 1982. Ev/> Cell Res. 140: 15-24. CELL BIOLOGY 205 Reference: Biol. Hull. 205: 205-206. (October 2003) • cuticle. This research was supported by Grant No. MCB-26771 from the National Science Foundation. We thank Mr. Louis Kerr for im- portant assistance with use of the fluorometer and Dr. David Gadsby for donation of striped bass heads for the stimulation of DE production by caged horseshoe crabs. Literature Cited 1. Liebovitz, L., and G. A. Lewbart. 1987. Biitl. Bull. 173: 430. 2. Fahrenbach, W. H. 1999. Pp. 21-115 In Chelicerale Anhropoda, F. W. Harrison and R. R. Foelix, eds. Wiley-Liss, New York. 3. Harrington, J. M., and P. B. Armstrong. 1999. Biol. Bull. 197: 274-275. 4 Harrington, J. M.. and P. B. Armstrong. 2000. Bi,,l. Bull. 199: 189-190. 5. Stagner, J. I., and J. R. Redmond. 1975. Mar. Fish. Rev. 37: 1 1-19. 6 van't Hof, W., E. C. Veerman, E. J. Helmhorst. and A. V. Amer- ongen. 2001. Biol. Chem. 382: 597-619. NEUROBIOLOGY AND BEHAVIOR 207 Reference: Bio/. Bull. 205: 207-208. (October 2003) © 2003 Marine Biological Laboratory Development and Characterization of a Self-Referencing Glutamate-Selective Micro-biosensor Daniel J. Bogorff' , Mark A. Messerli', Robert P. Malchow2, ami Peter J. S. Smith1 ' Marine Biological Laboratory', Woods Hole. MA ~ University' of Illinois at Chicago, Chicago. IL Glutamate is the primary excitatory neurotransmitter in the CNS ot vertebrates, activating both ionotropic and metabotropic receptors ( 1 ) Glutamate, along with other amino acids, has also been identified as an osmoticum used to regulate the water potential across the plasma membrane of animal cells (2). The molecular mechanisms regulating the release and uptake of glutamate are consequently of considerable physiological interest. The release of glutamate from single cells can be detected with outside-out patches of glutamate receptors pulled from nerve cells (3), but these sensors lack consistency, are techni- cally arduous to construct, and lose sensitivity over time. Fluorescent detection methods have also been used, but are also difficult to make and quantitate 1 4). Our specific aim is to avoid the difficulties inherent in these other techniques, by developing a noninvasive. real-time micro-biosensor that will quantitatively measure glutamate release and uptake by single cells. High-sensitivity, real-time detection of glutamate can be achieved using electrochemical detection of H-,O2, a byproduct of the enzy- matic conversion of L-glutamate to a-ketoglutarate catalyzed by glu- tamate oxidase (5). Glutamate o.xidase itself has approximately 61- and 325-fold selectivity for glutamate over aspartate and glutamine when immobilized on macroelectrodes (5). H^O2 can be directly detected with a platinum wire electrode polarized to +0.6 V (6). However, this method can be problematic due to (a) electrical drift of the electrode response, which reduces the useful sensitivity and reli- ability of the measurement, and (b) oxidation of other compounds released by cells, most notably ascorbate. Electrical drift can be eliminated if the electrode is used in a self-referencing format (7). In this mode, measurements are made alternately, near the source of glutamate influx or efflux, and then at a second location a set distance away; the difference in the readings at the two locations is the measurement of the flux. Electrical drift is subtracted out by this process, provided that the signal generated by the electrode occurs more quickly than the rate of electrical drift: if it is too slow, the signal itself will also be subtracted. The oxidation of interfering compounds can be eliminated by using enzyme-coupled detection of H2O2 at a lower electrical potential. This is accomplished by coating the elec- trode with a redox polymer containing horseradish peroxidase (HRP) and osmium. HRP catalyzes the reduction of H2O2 to water and is itself reduced by osmium(II), which is converted to osmium! III). The electrode then donates electrons to osmium! Ill), regenerating osmi- um! II) and. in the process, producing a measurable electrical current (8. 9). Our goal in the present work is to determine if such a hydrogel-based glutamate-selective electrode could be miniaturized for use in a self-referencing mode. Microelectrodes were fabricated in a manner similar to oxygen sensors (10). except that 8-^.m carbon fiber, 12-p.m gold wire, and 10- and 25-p.m platinum wire were used at the reactive surface. Electrodes were dip-coated with Os-gel-HRP redox polymer (BAS, W. Lafayette. IN) and allowed to dry for 10 min. Electrodes were then dip-coated in 50 units/ml glutamate oxidase (Sigma) and allowed to dry for 10-30 min. Glutamate electrodes were polar- ized to either 0 or +100 mV against a Ag/AgCl reference in physiological saline. The average response of platinum-based elec- trodes was 0.45 pA ± 0. 1 5 pA/ftM of glutamate, whereas gold- based electrodes produced a weaker signal of 0.21 ± 0 pA/juAl and carbon gave the weakest signal. 0.1 ± 0.01 pA/p.M. Figure 1A shows the electrical current detected from a platinum gel electrode; it was placed 20 jum from a source pipette containing 25 /uM glutamate and moved alternately to a position 50 /urn away. The initial period of oscillation was 0.1 Hz, and the electrode com- pleted its translation to each location in 1.5 s. At the arrow, the period of oscillation of the electrode was slowed to approximately 30 s. The record clearly shows that the electrical current induced by glutamate had reached more than 90% of its maximal response within the 10-s time frame of oscillation. While this sensor could be used in a static configuration to detect glutamate, the electrical drift inherent in the electrode, which can limit detection at low concentrations of glutamate, can also be plainly seen as the slow rise in the baseline current (Fig. 1A). Figure IB shows the differ- ential responses obtained when the electrode was employed in a self-referencing mode to reduce the impact of this drift. The electrode was initially placed 20 jim from a 25 /iM glutamate source pipette, and differential recordings were made by subtract- ing responses obtained at a point 50 /im distant; the rate of oscillation was 0.1 Hz. In this condition, a steady differential signal of approximately 650 fA could be detected. The electrode was then progressively moved to positions more distant from the glutamate source, and differential recordings were obtained in the same fashion. The decline in the differential signal as a function of distance is apparent. Note also the clear, steady, small signal of approximately 50 fA that can be detected with the electrode 100 ju.m away from the source pipette — a signal that is significantly smaller than the electrical drift and noise depicted in the raw- recordings presented in Figure 1A. Our work demonstrates that glutamate-selective electrodes based on a redox polymer hydrogel system can be miniaturized sufficiently to permit detection of glutamate. It also shows that the response time of these electrodes is short enough that they can be used in a self-referencing mode, which is useful for enhancing the signal-to-noise ratio for measuring slowly changing glutamate gradients. The sensitivity of these electrodes is suitable for mea- surements of glutamate release from living cells (5, 1 1 ). This work was supported by grants from the National Center for Research Resources (P4I RR01395) and National Science Foun- dation (009-1240). Special thanks to Robert Lewis, Richard H. Sanaer. and Kasia Hammar for their efforts and assistance. 208 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS -12.8 probe movement under manual control probe movement under autematie-eontrol -13.6 20 40 60 Time (s) 80 100 120 20 urn from 25 uM glutamate source -800 0 5 10 15 Time (min) Figure 1. (A) Electrical current from a glutamate-selective micro-biosensor placed 211 IJ.IH from a 25 jjM glutamale source pipette. The electrode was moved alternately to a position 50 fjjn distant, first at a rate o/O. I H:. then ai 0.01 H:. IB) Recording of differentia/ currents obtained when the electrode is used in a self-referencing mode at increasing distances from the source. Literature Cited 1. Jahr, C. E., and R. A. Lester. 1992. Curt: O/nn. Neiirohiol. 2: 270-274. 2. Pasantes-Morales, H., R. Franco, L. Ochoa, and B. Orda/. 2002. Neiirocltcin Res. 27: 59-65. 3. Copenhagen, D. R., and C. E. Jahr. 1989. Nature 341: 536- 539. 4. Ayoub, G. Si, and D. R. Copenhagen. 1991. }. Neuroxci. Methods 37: 7-14. 5 Hu, Y., K. M. Mitchell, F. N. Albahadily, E. K. Michaelis, and G. S. Wilson. 1994. Bruin Res. 659: 1 17-125. 6. Twig, G., S.-K. Jung, M. A. Messerli, P. J. S. Smith, and O. S. Shirihai. 2001. Biol. Bull. 201: 261-262. 7. Smith, P. J. S.. K. Hanimar. D. M. Portertield, R. H. Sanger, and J. R. Trimarchi. Microxc. Res. Tech. 46: 398-417. 8. Kulagina. N. V.. L. Shankar, and A. C. Michael. 1999. Anal. Chem. 71: 5093-5100. 9. Yigzaw, Y., L. Gordon, and T. Solomon. 2002. Ctirr. Separations 19: 119-125. 10 Land, S. C., D. M. Porterheld, and P. J. S. Smith. 1999. ./. Ev/>. Biol. 202: 211-218. 1 1 Takahashi, M., B. Billups, I). Rossi, M. Sarantis, M. Hamann, and D. Altwell. 1997. ./. E.v/i. Biol. 200: 401-409. NEUROBIOLOGY AND BEHAVIOR 209 Reference: Bio/. Bull. 205: 209-21 1. (October 2003) © 2003 Marine Biological Laboratory Zinc Modulation of Hemichannel Currents in Xenopus Oocytes R. L. Chappell,1 J. Zakevicius,2 ami H. Ripps2'* ' Hunter College and The Graduate Center, CUNY, New York, NY 2 Universitv of Illinois College of Medicine, Chicago, IL Connexins are a multigene family of structural proteins com- prising gap-junctional channels, the aqueous pores that link elec- trically coupled cells in tissues throughout the body. These narrow passages (d = 16 A) allow the intercellular exchange of ions. second messengers, and other small molecules having a molecular mass <1 kDa. In the course of forming gap junctions, the con- nexins oligomerize into hexameric arrays known as "connexons" or "hemichannels" that assemble in the plasma membrane before docking with the connexons of adjacent cells ( 1 ). There is now abundant evidence that, at this penultimate stage of gap-junction formation, hemichannels can be activated both chemically and electrically (2. 3), that their properties often reflect those of fully formed gap junctions (4), and that the modulation of hemichannel activity may be of physiological significance (5). Light-induced changes in the chemical environment of the ver- tebrate retina have profound effects on both neurotransmitter-gated and electrical synapses: and these effects can, in turn, alter the sensitivity, receptive field organization, and signalling pathways that transmit the visual message to the CNS. One putative neuro- modulator that has aroused a great deal of interest in recent years is zinc, which has been found in the synaptic vesicles of glutama- tergic neurons in brain and retina (6). In the retinas of amphibia (7). fish (8). and mammals (6), zinc is present in photoreceptors, which signal second-order cells by regulating glutamate release in response to photic stimulation. Although the co-release of zinc with glutamate remains conjectural (9). the effects it exerts on retinal neurons have not been explored extensively (8, 10); and no studies have addressed the question of its effect on connexins or the channels they form. In the present study, we used the two-electrode voltage-clamp recording technique to examine the effects of zinc on the currents mediated by the connexons formed by the endogenous connexin (Cx38| of stage V-VI Xenopus oocytes, and those formed by perch Cx35, a connexin expressed in neurons of the vertebrate retina (11. 12). To study the behavior of Cx35, cells were tested 48 to 72 h after they were injected with 46 nl of a mixture of 10 ng/cell Cx35 cRNA and 10 ng/cell of an antisense oligonucleotide to Cx38. The recordings were made with the cells bathed in a Na+-free medium to eliminate the large Na* -dependent currents that are similar in time course to the hemichannel currents, but of opposite polarity (13); zinc chloride was added without substitution. Responses were elicited with a series of 10-s pulses from a holding potential of -40 mV to +60 mV (see protocol. Fig. IB inset), and were recorded with low resistance electrodes (0.7-1.5 Mfl) connected to a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA) and controlled by protocols generated in pClamp 8 (Axon). * Corresponding author: harrripplS'uic.edu Data were analyzed in ClampFit (Axon) and plotted with software programs in Origin (Microcal Inc., Northampton, MA). Membrane currents recorded in a sodium-free modified Barth's (MB) solution from Xenopus oocytes expressing Cx35 are shown in Figure 1A. The slowly developing outward currents character- istic of hemichannel activity are seen with depolarizing voltage steps > +20 mV. After changing the bath solution to one con- taining 10 n.M zinc, substantially greater hemichannel currents were elicited at these voltages (Fig. IB). When the bathing me- dium was switched to one containing 1 mM zinc, the hemichannel currents were suppressed below those recorded in MB (Fig. 1C). The I-V data obtained with an oocyte expressing Cx35 for the range of zinc concentrations tested are illustrated in Figure ID. It is evident that relatively low concentrations of zinc (1 ^M and 10 /xM) produce enhanced current responses at depolarizing voltages >40 mV, and the effect is reversed when the Zn concentration is increased to slOO juM. It should be noted that hemichannel currents recorded during experimental runs in which the solutions were delivered in reverse order (i.e.. decreasing zinc concentra- tions from 1 mM zinc through 1 /iM zinc to Na-free MB) gave rise to the same biphasic behavior. Comparable results were obtained with Cx38 (Fig. IE), where we show, in addition, that the blocking effect of 1 mM zinc could be completely reversed by coapplication of 1 mM histidine, a zinc chelator. Hemichannel currents elicited by depolarizing voltage steps from -40 mV to +40 mV from all oocytes tested were normalized to their control current in Na-free MB solution and averaged according to connexin type for each concentration of zinc applied. The results from 5 Cx35 oocytes (Fig. IF) and 11 Cx38 oocytes (Fig. 1G) were similar. Currents recorded in 1 and 10 pM zinc were greater than in the control (MB) solution, but decreased when the cells were bathed in 100 /LtM and 1 mM zinc. A return to MB (Fig. IF) following a zinc series returned the hemichannel currents to their control values. Cells injected with the antisense to Cx38 alone (controls) did not exhibit significant hemichannel activity (results not shown). The biphasic effect of zinc on membrane currents is not unique. As shown in an earlier study (8), the addition of 10 pM zinc greatly enhanced GABA-induced currents mediated by GABAA receptors (GABAAR) of skate bipolar cells; in contrast, the cur- rents were markedly reduced when the cells were exposed to 1 mM zinc. Moreover, the zinc enhancement of hemichannel currents appears to be insensitive to voltage; currents in 1 ju,A/ and 10 IJ.M zinc recorded at +40 and +60 mV were approximately 1.7 times greater than in MB. a finding consistent with that obtained for GABA.xR-mediated currents of bipolar cells. These observations suggest that zinc may interact with connexins at two external membrane binding sites, with very different affinities for zinc. The high affinity site, activated at low concentrations of zinc, gives rise 210 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS B Na-ftce MB Cx35 10 jjMZinc 1 mM Zinc *-60 -20 04uA -40 mV D 08 07 06 05 04 Io3 02 0 1 00 -0 1 E Cx35 020-, 0 18- 0 16- 0 14- 0 12- 0 10- *§. 008- 006- 004- 002- 0.00- Ci38 - Na-Fcee MB - 10 MM Zinc - 1 mM Zinc 1 mM Histidme + 1 mM Zinc mV mV 10 MM 100 MM 1 mM Zinc Concentration 10 MM 100 (iM I mM Zinc Concentration Figure 1. (Al Depolarising voltage steps from —40 mV to +60 mV in 20 mV increments (inset) elicit hemichannel currents in a Xenopus oocyte expressing Cx35. With the cell bullied in a sodium-free modified Earth's (MB) solution, in which most of the sodium is replaced by choline (Na+ reduced to <3 mM), a slowly developing nutward current, attributable to the opening of membrane hemichannels, is evident at voltages > +20 mV. The "Na-free" MB solution contained I in mM] C,,HI4NOCI (881, KCI [I], NaHCO, [2.41. N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) [15], Ca(NO,), 10.33], CaCl2 10.41], and MgSOj [0.82]: 10 mg/l gentamvcin was added, and the solution titrated with NaOH to pH 7.6. (Bl With the addition of 10 juM .-/HC" chloride, the hemichannel currents are greatly enhanced. (C) Increasing the zinc concentration to I mM suppresses the hemichannel currents to levels below those obtained in MB. (D) The effects of zinc concentration on the current-voltage relation (corrected for leakage currents) obtained for an oocyte expressing Cx35. (E) The l-V relation for the endogenous cimnexin (Cx38) displays a similar response to zinc: i.e., a large enhancement of hemicurrents in III juM zinc and suppression in I mM :inc. With the addition of I mM histidine, a zinc chelator, the suppressive effect of I mM zinc is reversed. (F-G) Bar graphs show averaged Jala obtained with Cx35 and C.\38 at different concentrations of zinc. In each data set, the currents recorded in response to a voltage step from the holding potential ( —40 mV) to +40 mV have been normalized to the value obtained initially in Na-free MB. Error bars =• ±SEM (n = i for Cx35; with Cx3S, n = 6 for I fjM ZH, and n = 12 for the higher concentrations). to an i i , -merit of hemichannel currents, whereas the low attinity SH uires high concentrations of zinc to produce its inhibitory efiV. Thepresein >. and evidence that zinc is located within the synaptic termin. Is ui i rtehrate photoreceptors (7, 8). raise the possibility that zinc modulation of hemichannel activity contrib- utes to the processing of visual information in the distal retina. These studies were conducted at the Marine Biological Labora- tory. Woods Hole, Massachusetts, and supported by grants from the National Eye Institute (EY-06516 and EY-01792), a PSC/ NEUROBIOLOGY AND BEHAVIOR 211 CUNY grant (65711-0034) to RC. an unrestricted award to the UIC Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, Inc., a Senior Research Investiga- tor Award from the RPB (HR), and an Award of Merit from the Alcon Research Institute (HR). Literature Cited 1. Musil, L. S., and D. A. Goodenough. 1991. J. Cell Biol. 115: 1357_1374. 2. DeVries, S. H., and E. A. Schwartz. 1992. /. Physiol. 445: 201-230 3. Malchow. R. P.. H. Qian, and H. Ripps. 1994. J. Gen. Physiol. 104: 1039-1055. 4. Ebihara, L., V. M. Berthoud, and E. C. Beyer. 1995. Biophys. J. 68: 1796-1803. 5. Kamermans, M., I. Fahrenf'ort. K. Schultz, II. Janssen-Bien- hold. T. Sjoerdsma, and R. Weiler. 2001. Science 292: 1178- 1180. 6. Ugarte, M., and N. N. Osborne. 2001. Prog. Neurobiol. 64: 219-249. 7. Wu, S. M., X. Qiao, J. L. Noebels. and X. L. Yang. 1993. Vision Res. 33: 2611-2616. 8. Qian. H.. L. Li, R. L. Chappell, and H. Ripps. 1997. J. Neuro- physiol 78: 2402-2412. 9. Kay, A. R. 2003. J. Xeurosd. 23: 6847-6855. 10. Rosenstein, F. J., and R. L. Chappell. 2003. Neurosci. Lett. 345: 81-84. 1 1 O'Brien, J., M. R. Al-Ubaidi, and H. Ripps. 1996. Mot. Biol. Cell 7: 233-243. 12. White, T. W., M. R. Deans, J. O'Brien, M. R. Al-Ubaidi, D. A. Goodenough, H. Ripps, and R. Bruzzone. 1999. Em: J. Neurosci. 11: 1883-1890. 13. Ripps, H.. H. Qian, and J. Zakevicius. 2002. J. Neurosci. Melh. 121: 81-92. Reference: Biol. Bull. 205: 211-212. (October 2003) © 2003 Marine Biological Laboratory Transient Use of Tricaine to Remove the Telencephalon Has No Residual Effects on Physiological Recordings of Supramedullary/Dorsal Neurons of the Gunner, Tautogolabrm adspersus S. J. Zottoli, O. T. Burton, J. A. Chambers, R. Eseh, L. M. Gutierrez, and M. M. Kron Williams College, Williamstown. MA When used as a general anesthetic, tricaine significantly alters physiological parameters recorded from supramedullary/dorsal cells of the cunner. Tautogolabrus adspersus. Specifically, tricaine reduces spike height, increases the current needed to elicit an action potential, and blocks afferent input ( 1 ). In contrast, equiv- alent recordings from locally anesthetized fish are not altered in this way. However, although the use of local anesthetic is a better alternative to tricaine general anesthesia for physiological record- ings ( 1 ), there are limitations. Local anesthetics have a limited lifetime, are difficult to reapply while recording, and can enter the bloodstream. As in other vertebrates, the telencephalic hemispheres of fish are considered to be the "highest" brain centers. For example, the telencephalon of goldfish has been implicated in spatial and avoid- ance learning (e.g.. 2, 3). The removal of the telencephalon under transient tricaine anesthesia is used in lieu of general anesthesia in many laboratories (e.g.. 4. 5). However, no studies have been conducted to determine whether tricaine — after its removal — af- fects physiological parameters of neurons whose somata lie within the central nervous system. We have therefore studied whether either the transient use of tricaine or telencephalon removal have any residual effects on resting potential, spike height, and current needed to elicit a spike. Responses of supramedullary/dorsal cells to depolarizing cur- rent pulses and to electrical stimulation of the skin on the right operculum were recorded from cunner, 10.5 ± 1.2 cm (mean ± SD: n = 22) in body length. The fish were either transiently anesthetized with tricaine during the removal of the telencephalic hemispheres, or were not anesthetized. In the first condition, the fish were initially anesthetized in tricaine (ethyl-w-aminobenzoate; 300 mg/1. Sigma-Aldrich) in sea- water adjusted to pH 8. When respiration ceased, the fish were transferred to an operating chamber where tricaine (100 mg/1) in chilled seawater adjusted to pH 8 was recirculated through the mouth and over the gills. Ice packs were placed in the operating chamber on either side of the fish. The skull was removed to expose the telencephalic hemispheres, and these structures were removed. The fish were injected with tubocurarine chloride (0.1 mg/kg) to block neuromuscular transmission, and then the seawa- ter with anesthetic was replaced with anesthetic-free, chilled sea- water. Finally, the rostral spinal cord was exposed. In the second condition no anesthetic was used. Fish were injected with tubocu- rarine chloride and placed in an operating chamber. Their telen- cephalic hemispheres and the rostral spinal cord were exposed. In seven experiments, the telencephalon was stimulated to determine whether any input to the dorsal cells could be elicited. Single microelectrode recordings (3 M KCl-filled, 5-20 MO initial resistance) were made from somata of supramedullary/ dorsal cell neurons in both anesthetic and anesthetic-free condi- tions. The recordings were all made within 78 ± 43.4 ^m (mean ± SD; n = 26) of the surface of the brain. After tricaine was used transiently to remove the telencephalic hemispheres. action potentials 102.1 ± 9.4 mV (mean + SD, n = 16) in amplitude could be evoked by 4.5 ± 2.9 nA current (initial recordings were started 68 ± 55 min after removal of tricaine). In anesthetic-free experiments neither the spike height ( 103.3 ± 12.5 mV, n= 11) nor the current needed to evoke the spike (2.4 ± 1.7 nA) was significantly different (P > 0.05; Bonferroni's Multiple Comparison Test). In addition, there were no significant differ- ences in resting membrane potential (transient use of tricaine = -74.1 ± 5.8 mV; anesthetic free = -72 ± 7.1 mV). Post-synaptic potentials (PSPs) were readily evoked by stimulation of the skin of 212 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS A \ B \ Figure 1. Comparison of PSPs evoked hy electrical stimulation of the skin in supramedullary/dorsal cells. (A, B, C> A calibration pulse of 80 in \ '. 2 ms is present at the beginning of each recording. Cells fired action potentials in response to a short intracellular depolarising pulse in one of the ni'o superimposed traces. This pulse was followed bv electrical stim- iilution of the skin of the right opeiriili/in (to provide a baseline, in one of the nm superimposed traces this stimulus was not given: stimulus artifacts are designated with arrowheads). I A) Recordings from a fish in which tricaine was used transiently to remove the telencephalon. A PSP (arrow) gives rise to an action potential. IB I Recordings from an unanesthetized p\h. A PSP farrow) gives rise to an action potential. (C) Recordings from a fish under general tricaine anesthesia. No PSPs could be evoked to electrical stimulation of the right operciilitm. the right operculum in both conditions. This was not the case in animals under general anesthesia (1; Fig. 1). In anesthetic-free experiments, stimulation of the telencephalic hemispheres at only the high voltages (80-100 V) activated the dorsal cells. To test whether this activation was the result of direct or indirect telencephalic input to the supramedullary/dorsal cells and not current spread to adjacent nervous tissue, in three exper- iments the telencephalic hemispheres were removed and then replaced in their original positions. The disconnected telencephalic hemispheres were stimulated again at roughly the same location and the same voltages. The response to stimulation persisted. These results indicate that activation was through current spread to adjacent structures such as the trigeminal nerve, which is known to contain processes of dorsal cells. General anesthetics, such as tricaine, are known to reduce so- dium currents, and their effects are reversible (6). The transient use of tricaine and the removal of the telencephalic hemispheres in this study appear to have had no residual effect on spike height, on the current needed to elicit an action potential, or on the ability to elicit PSPs from the supramedullary/dorsal cells. Although Rose (7) provides a compelling argument that it is implausible for fish to experience pain, implausible is not conclu- sive. Nociceptors have been identified in the trout (8, 9). Endog- enous opioid peptides (10) and opioid receptors in fish (e.g., 11, 12) may serve an anti-nociceptive function as in mammals (11). If there is no telencephalic influence on the system being studied, then the transient use of tricaine followed by removal of the telencephalon serves as a reasonable precaution against the possi- bility that fish experience pain. This work was supported in part by Howard Hughes Medical Institute and Essel Foundation grants to Williams College. All experimental procedures were approved by the MBL IACUC. Literature Cited 1 Arnolds, D. E. W., S. J. Zottoli, C. E. Adams, S. M. Dineen, S. Fevrier, U. Guo, and A. J. Pascal. 2002. Binl. Bull. 203: 188-189. 2. Overmier. J. B., and M. R. Papini. 1986. Behav. Neiirosci. 100: 190-199. 3. Portavella, M., J. P. Vargas, B. Torres, and C. Salas. 2002. Brain Res. Bull. 57: 397-399. 4. Duman, C. H., and D. Bodznick. 1996. J. Comp. Phvsiol. A 179: 797-807. 5 Rohregger, M., and N. Dieringer. 2002. J. Neurophvs. 87: 385- 398. 6. Frazier, D. T., and T. Narahashi. 1975. Eur. ./. Pharmacol. 33: 313-317. 7. Rose, J. D. 2002. Rev. Fish. Sci. 10: 1-38. 8. Sneddon. L. V. 2003. Brain Res. 972: 44-52. 9. Sneddon. L. II., V. A. Braith»aite, and M. J. Gentle. 2003. Proc. R. Soc. Loiul. fi 270: 1115-1121. 10. Gonzalez-Nunez, V., R. Gonzalez-Sarmiento, and R. E. Rodriguez. 2003. A/,./. Bruin Rc\. 114: 31-39. 11 Darlison, M. G., F. R. Greten, R. J. Harvey, H-J. Kreienkamp, T. Sliihmer. H. Z \viers, K. Lederis, and D. Richter. 1997. Proc. Natl. Acud. Sci. 94: 8214-8219. 1 2. Rodriguez, R. E., A. Barrallo. F. Garcia-Malvar, I. J. McFadyen, R. Gonzalez-Sarmiento, and J. R. Traynor. 2000. Neiirosci. Lett. 2X8: 207-210. NEUROB1OLOGY AND BEHAVIOR 213 Reference: Biol Bull. 205: 213-214. (October 2003) © 2003 Marine Biological Laboratory Zinc Chelation Enhances the Sensitivity of the ERG b-Wave in Dark-Adapted Skate Retina S. Redenti1 ami R. L Chappc'Il'"* 'The Graduate Center. CUNY, New York, NY -Hunter College, CUNY, New York, NY A decade ago, Wu et at. reported evidence of a dense band of ionic zinc in the region of the photoreceptor terminals of the salamander retina ( 1 ). They speculated that zinc may play a neuro- modulatory role in the outer retina, including possible feedback onto photoreceptors to down-regulate transmitter release, as well as feed- forward onto second order cells. Subsequently, a high ionic zinc concentration was identified in a similar region near the base of the photoreceptors in the all-rod retina of the skate (2). In addition, Ugarte and Osbome (3) have reported recently that a dense band of ionic zinc, located in the photoreceptor region of the light-adapted rat retina, is redistributed under dark-adapted conditions. Zinc has been known to affect the response of receptors on a number of retinal cell types to various neurotransmitters (4). In the skate, zinc regulates both GABA (2) and glutamate (5) receptors of isolated cells. Furthermore, the zinc chelator histidine (an amino acid endogenous in the retina, where it may play various roles in cell metabolism and disease (6, 7, 8. 9, 10)) has been shown to enhance the size of the b-wave of the electroretinogram (ERG) of the skate (5) and zebrafish (11). Histidine can also increase the membrane currents recorded postsynaptically from horizontal cells during voltage-clamp in the skate retinal slice preparation (12). The effects of histidine support the notion that endogenous zinc may be playing a role in the physiological response of the retina to light. Recent studies of the effects of histidine on the zebrafish ERG have demonstrated an increase in sensitivity of its mixed rod-cone retina in the presence of a zinc chelator (11). Here, we report studies of the effect of histidine on the retina of the skate. Raja erinacea. indicating that application of this zinc chelator enhances the sensitivity of the b-wave of the ERG in an all-rod retina. Skates were obtained through the Marine Resources Center of the Marine Biological Laboratory (Woods Hole. MA). The ani- mals were allowed to dark-adapt for at least one hour prior to an experiment. After approved euthanasia, the eyes were enucleated and dissected under dim red light. The anterior portion of the eye, including cornea and lens, was removed; and the remaining retinal eyecup preparation was used for ERG recordings. Eyecups were placed into a chamber over a silver chloride reference electrode within a Faraday cage. The active silver chloride electrode was connected to superfusion solutions in the eyecup via a glass capillary containing Ringer/agar. ERG responses to increasing intensities of illumination were recorded while the preparation was superfused (-0.5 ml/min) with skate-modified Ringer's solution (2) alone, or to which 200 fj.M picrotoxin (to block GABAergic receptors in the retina known to be zinc-sensitive (2)), and then 100 /i/W histidine plus 200 p,M picrotoxin had been added. We had found that responses in picrotoxin did not increase after the first 10 : Corresponding author: rchappellCs'gc. cuny.edu min, but that ERG responses in histidine could continue to increase for up to 30 min. Therefore, the preparation was kept in the dark for 30 min in Ringer, at least 10 min in picrotoxin. and at least 30 min in histidine plus picrotoxin prior to recording an intensity- response series. One-second flashes were used to elicit ERG re- sponses from which b-wave amplitudes were measured. The in- tensity of the unattenuated beam (Log 1=0) was 260 /nW/cnr. Individual ERG responses recorded from one skate eyecup prepa- ration in response to stimulation at an intensity of Log I = -3.0 are shown in Figure 1A. Note that in addition to increased b-wave amplitude observed when picrotoxin, or histidine plus picrotoxin, were applied, an increased a-wave as well as a more prominent OFF component were often observed, as described previously by Chappell and Rosenstein (13) using picrotoxin alone. Similar data at all intensities were obtained from five prepara- tions. Intensity-response data from one such preparation are plot- ted in Figure IB. A Vmax under normal Ringer conditions for each eye was determined by obtaining the best fit of a Naka-Rushton curve (14) to the intensity-response data obtained in the control Ringer solution. The value of Vmax obtained was used to normalize the data before averaging it and plotting the results, which are shown in Figure 1C and ID. Figure 1C is a bar graph of the averaged data from five preparations at the intensity Log I = -4, an intensity near the half amplitude intensity (a) for the intensity- response curve obtained in Ringer. The curves plotted with the intensity-response data in Figure 1 D are the best fit of the data in Ringer, picrotoxin, and histidine plus picrotoxin. respectively, to the Naka-Rushton relation, using the least-squares approximation in Oriain. Using this approach, we could obtain the value of the intensity corresponding to the half amplitude (a) of the Naka- Rushton curves, as well as the Vmjx for each of these curves. The values of Vm.lx obtained increased from 0.95 in Ringer, to 1.09 in 200 nM picrotoxin. and 1.4 in 100 ;uM histidine plus 200 \j.M picrotoxin, while the values of u shifted from a Log I of -4.3 to -4.52 to -5.13. respectively. The shift of a toward dimmer intensities represents an increase in sensitivity of 0.8 log units (roughly a factor of 6) in the presence of histidine. Such an increase in sensitivity is consistent with the feedback model proposed by Wu et til. ( 1 ). in which they suggest that Zn2 + may feed back onto photoreceptor terminals to reduce Ca'* entry and thereby reduce vesicular transmitter release from the photoreceptor terminals. It would also be consistent with a reduced inhibition of glutamate receptors on second-order retinal neurons (5), or possibly even an action of zinc on hemichannels (15). since hemichannels have been suggested as a means of feedback from horizontal cells onto cones in the carp retina, where the hemichannel blocker cambenoxolone has been shown to alter feedback-mediated responses (16). Whatever mechanism is in- 214 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS B 100 jiM Hislidine i- 200 nM 1'n rutcmn 200 .1 M I'll roti. MM Ringer 40 MV L^_ 80- 60- > 40- 3. 20- 0- - Ringer - 200 (JV1 Picrotoxin - 100 nM Histidine +200 niM Picrotoxin 1.2 1.0 .2 04- 5 02 Log 1 = -3 Log I = -4 D 20-, 1.8- Ringer !• Picrotoxin H Picrotoxin Control I -5 Log I Ringer 200nM Picrotoxin 1 00 nM Histidine +200 (iM Pic; Figure 1. The c/iir chelntor histidine increases sensitivity of the skate electroretinogram (ERG) b-wave response. (A) ERG responses recorded from a dark-adapted skate eyeciip preparation in response to a I -x flash of white light at an intensity of Log I = -3 (Unattenuated beam intensity. Log 1 = 0, was 260 nW/cnr). The amplitude of the b-wave ("b". upper trace) of the ERG recorded from a dark-adapted skate eyecup preparation increased when 100 /J.M histidine (in the presence of 200 u.M picrotoxin to block :inc-sensitive GABA receptors} was added to the superfusate. A small increase in the a-wtive < "a ". upper trace) was sometimes seen as well. IB) Intensity-response data from one e\ecup preparation in Ringer. 200 juM picrotoxin. and 100 p.M histidine plus 200 fj.M picrotoxin. ( C) Nonna/i-ed data at Log I = -4 from 5 preparations. (D) Intensity-response data, averaged and plotted (mean ± SEM). with curves representing the best fit to the Naka-Rushton equation fur each condition. The Log I of the half-amplitude intensity (a) of these curves determined in Ringer. 200 fjM picrotoxin, and in 100 /xM histidine plus 200 p.M picrotoxin were —4.3, —4.5. and —5.7. respectiveh: Thus, in addition to a 50% increase in V,,,,M. a for the histidine intensity-response curve irm shifted C..S' lot; units to the left, representing a 6-fold increase in sensitivity in the presence of histidine. volved. the evidence that removal of extracellular zinc by chelation alters the sensitivity of the physiological response to light in an all-rod retina suggests that zinc may be playing an important role as a neuromodulator of rod afferent pathways in the vertebrate retina. Supported by PSC/CUNY grant 6571 1-0034. Literature Cited I Wu, S. M., X. Qiao, ,1. L. Noebels, and \. L. Yang. 1993. Vision Res. 33: 261 l-2hl/ivs;,./. 78: 2402-2412. 3. Ugarte, M., and N. N. Oshorne. 1999. Ev/>. Eye Res. 69: 459-461. 4. Ugarte, M.. and N. N. Osborne. 2(H)1. ProK. NeuroNol. 64: 214-249. 5. Rosenstein, F. J., and R. L. Chappell. 2003. ,\'cnrosci. Lett. 345: ! S4. 6. Jia,i», Y., V. C. Vu, F. Buchholz, S. O'Connell, S. J. Rhodes, C. Camltloro. V. R. Xia, A. J. Lusis, and M. (;. J. Rosenfeld. 1996. Bio/. ( ft, , 271: 10.723-10.730. 7 Kusakari. .. S. Nishikawa. S. Ishiguro. and M. Tamai. 1997. CHIT. Eye Res. 16: 600-604. 8. Lewin, A. S., K. A. Drenser, \V. \V. Hauswirth. S. Nishikawa, D. Yasunuira, J. C. Flannery, and M. M. La Vail. 1998. Mi;. Med. 4: 467-971. 9. Mondal, M. S., A. Ruiz, J. Hu. D. Bok, and R. R. Rando. 2001. FEBS Lett. 489: 14-18. 10. Wistow, G., S. L. Bernstein, M. K. Wyatt, R. N. Fariss, A. Behal. J. VV. Touchman. (.',. Bnuffard, D. Smith, and K. Peterson. 20(t2. Mol. Vis. 8: 205-220. 11 Rrdenti, S., and R. L. Chappell. 2002. Hiol. Hull. 203: 200- 202. 12 Chappell, R. L., and S. Redenti. 2001. Hiol. /lull. 201: 265-267. 13. Chappell. R. L., and F. J. Rosenstein. 1996. ./ Gen. Pliysiol. 107: 535-544. 14 Naka, K. L, and W. A. H. Rushton. 1966. J. Pliysiol. (Loud.) 185: 587-599. 15. Chappell. R. L., J. Zakevicius, and H. Ripps. 2003. Hiol. Hull. 205: 2IW-21 1 Id. Kumermans, M., I. Fahrenfort, K. Schultz, II. Janssen-Bienhold, T. Sjoerdsma. and R. \\eiler. 2001. Science 292: 1 178-1 180. NEUROBIOLOGY AND BEHAVIOR 215 Reference: Biol. Bull. 205: 215-216. (October 2003) £> 2003 Marine Biological Laboratory Intracellular Release of Caged Calcium in Skate Horizontal Cells Using Fine Optical Fibers Anthony J. A. Molina1, Katherine Haminar, Richard Sanger~, Peter J. S. Smith2, and Robert P. Malchow1 1 University of Illinois at Chicago, Chicago, IL 2 Marine Biological Laboratory; Woods Hole, MA Horizontal cells are second order retinal neurons that receive direct input from photoreceptors and are involved in establishing a number of key features of visual perception. These cells mediate the formation of the inhibitory surround portion of the classic center-surround receptive fields of retinal neurons ( 1 ). The center- surround receptive fields are important for enhancing the contrast of visual objects and are also involved in color perception. The molecular mechanisms by which horizontal cells send lateral in- hibitory signals to photoreceptors and bipolar cells are still under debate, but protons released from horizontal cells have been hy- pothesized to alter the flow of visual information within the outer retina (2). Indeed, small changes in extracellular pH can dramat- ically alter neural signals within the retina, in part because photo- receptor calcium channels are highly sensitive to protons. When protons bind to photoreceptor calcium channels, the voltage acti- vation range of the channels shifts to more depolarized potentials and the overall conductance of the cell to calcium is reduced, which significantly reduces neurotransmitter release (3). Our pre- vious work has shown that glutamate, the neurotransmitter re- leased by photoreceptors onto horizontal cells, modulates the flux of hydrogen ions from skate retinal horizontal cells (4). Glutamate- induced changes in H* flux depend on the presence of extracel- lular calcium and likely reflect the activation of plasma membrane calcium/H* ATPases. These transporters extrude intracellular cal- cium in exchange for extracellular hydrogen ions, decreasing the concentration of protons at the extracellular face of the horizontal cells (5). We would like to know whether local changes in calcium cause localized alterations in proton flux from horizontal cells. In the experiments reported here, we sought to develop methods to lo- cally raise intracellular calcium levels in horizontal cells. We explored the use of small optical fibers that can apply focal ultraviolet stimuli to cells loaded with the caged calcium buffer NP-EGTA. This compound contains a UV-sensitive calcium bind- ing site that releases its bound calcium upon absorption of ultra- violet light (6). Horizontal cells were isolated from skate retinas by enzy- matic dissociation, as described by Malchow et al. (7). The dissociated horizontal cells were placed in primary culture, where they were readily identified due to their distinct mor- phology and large size, about 150 /am. Cells plated on Falcon 3001 35-mm culture dishes were loaded with the cell membrane permeable AM ester forms of NP-EGTA and the calcium- sensitive dye Oregon Green, prepared as follows. NP-EGTA (50 /ag) and Oregon Green (50 /ag) were dissolved in DMSO with 2(}(7c pluronic acid and added to 8 ml of skate Ringer's solution, yielding final concentrations of 5 /aM Oregon Green-AM and 8 p.M NP-EGTA-AM. The cultured horizontal cells were then incubated with this solution for 30 min at 14 °C, washed twice with skate Ringer's solution, and allowed to stand for a minimum of 1 h. During this time, endogenous esterases cleave off the AM portion of the dye and the caged calcium compound, thereby trapping them inside the cell. Because NP- EGTA enters the cell unbound to calcium, it is important to pre-expose the preparation to a brief rise in intracellular cal- cium. Application of 100 /aM glutamate for 20 s permits cal- cium entry and loading of NP-EGTA compounds trapped within the cell. The cells were then thoroughly washed with fresh Ringer's solution, and experiments were typically conducted 10-30 min after calcium loading. Imaging of intracellular cal- cium concentration was performed with a Zeiss Attofluor im- aging system. Multi-mode optical fibers. F-MCB-T. were obtained from the Newport Corp.; they have a core diameter of 100 /am, a cladding diameter of 100 /am, a coating diameter of 140 /am, and a numer- ical aperture (NA) of 0.22. which gives them a high coupling efficiency. We prepared these fibers as follows. First, the coating and cladding were burned off the fibers with a heated tungsten coil, leaving a 5-cm portion bare. This portion of the fiber was then pulled to a final tip diameter of 1-2 /am with a Sutler P-2000 laser puller. The fiber was inserted into a borosilicate capillary tube previously pulled to a 5-/am tip diameter and placed so that the tip of the optical fiber protruded about 5 /am from the tip of the glass. The fiber was then glued in place with cyanoacrylate glue. Fibers were coupled to another multi-mode fiber, which was coupled to an ultraviolet laser. Figure 1A shows the ultraviolet light output from one such pulled fiber. In this experiment, the dish was filled with a fluores- cent solution, and the fluorescence of the solution, caused by ultraviolet light stimulation, was examined. The fluorescence was cone-shaped, with the region of highest intensity localized at the aperture of the optical fiber. A Narashige hydraulic manipulator was then used to place the fiber in a dish with the tip positioned 5 /am away from the membrane of a horizontal cell. Figure IB shows the change in Oregon Green fluorescence measured from a single horizontal cell upon the photolytic release of calcium from NP- EGTA-loaded cells. When the cell is stimulated for 5 s with ultraviolet light from the pulled optical fiber, a significant increase in fluorescence is detected, indicative of a rise in intracellular calcium concentration. The stimulus was then repeated two more times, which induced calcium increases that were smaller with each subsequent stimulation. In control experiments, with cells loaded only with Oregon Green, application of ultraviolet stimuli led to increases in measured fluorescence. Note that these increases were about 85% smaller (;; = 2) than those on cells containing NP-EGTA. Moreover, the control fluorescence disappeared imme- 216 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 60 i § 8 40 - B 50 100 seconds 150 200 Figure 1. . Tricas, T. C., and S. M. Highstein. 1991. J. Com/'. Physiol. A. 169: 25-37. 7. Montgomery, J. C., and D. Bodznick. 1994. Neurusci. Lett. 174: 145-148. Reference: Biol. Bull. 205: 218-219. (October 2003) © 2003 Marine Biological Laboratory Memory Reconsolidation in Hermissenda F. M. Child1*. H. T. Epstein', A. M. Kirjrian1 . ami D. L. Alkoif 1 Marine Biological Laboratory, Woods Hole, MA 02543 ~ Blanchette Rockefeller Neiiroscience Institute, Rock\'ille, MD 20850 Remembering seems a priori to be composed of three main processes: acquisition (or input), storage (or consolidation), and retrieval (or recall) ( 1 ). In this study the acquisition of memory is the form of Pavlovian conditioning elaborated by Lederhendler er m\cin (ANI). Animals were trained with nine training events ana1 then tested for recall at 4 h post-training. For three groups of animals. ANI was added either immediatel\ after (curve ani4), or at 10 inin (curve ani4 + 10) or 30 inin after the testing at 4 h Iciin'e ani4+30). Animals were then relested at 8. 24. 48. and for some. 72 h for recall or retention of the training. Controls (con) were not treated with ANI. Data points below the -em line indicate foot contraction and positive recall of the training: positive numbers indicate body elongation as in normal locomotion with recall inhibition and blocked memory. (B) Combined results of the four inhibitors. actinom\cin-D (act-D). anisomycin (anil the tripeptide CAM inhibitor (rgd). and the sensory block (sh). Animals were treated as described in A. with the inhibitors added at the times indicated (on the graph) after the initial testing at 4 h. The animals were tested again for recall at 5. 6. or 8 h post-training. hihiting translation. It was administered at 1 ju,g/ml. The translation-inhibition was not checked. 3. Actinomycin-D (Act-D) interferes with mRNA synthesis by inhibiting transcription. It was administered at O.I jug/ml. The transcription-inhibition was not checked. 4. ROD (arginyl-glycyl-aspartate) inhibits bond formation be- tween cell adhesion molecules (CAMs). It was administered at 10 /xg/ml. The bond formation inhibition was not checked. In the first experiment, specimens of Hermissenda were given nine TEs and tested at 4 h; this is the time at which our previous studies have shown recall to become insensitive to all four inhib- itors we are using, thereby showing that the stored memory is consolidated long-term memory (CLTM). After testing to verify arrival at CLTM, ANI was added either immediately, 10 min later, or 30 min later. The data in Figure 1A show that adding anisomy- cin immediately or 10 min after testing for recall resulted in the loss of recall; adding the anisomycin after 30 min showed that recall has been re-established. We next studied whether the other three inhibitors had similar effects (Fig. IB). All three inhibitors wiped out recall if added immediately after testing at 4 h, whereas waiting for 30 min before using the inhibitors revealed that recall had been re-established, as had been found for ANI (Fig. 1A). Thus, these results demonstrate the existence of reconsolidation in Hermissenda. Reconsolidation was triggered by testing for recall after 4 h and then probing with each of the four inhibitors. The reconsolidation was found to be reached by about 30 min after recall: thus it is a much more rapid process than the initial acqui- sition and consolidation phase, which has been shown to take nearly 4 h. The sensory block (SB) was previously shown by Epstein el nl. (3) to inhibit short-term memory (STM) as well as long-term (LTM) and consolidated long-term memory (CLTM). The fact that it works on both consolidation and reconsolidation raises again the question of what steps it is affecting. Molecular and physiological studies will be needed to get at this question. Finally, both consolidation and reconsolidation deserve study in that there may be ramifications for learning and schooling. If the first consolidation study by Miiller and Pilzecker (3) is correct, teaching additional novel material within 6 to 10 min after having taught a primary point that is meant to be remembered could well weaken retention of that point. Similarly, if recalling something weakens the memory, then new information might prevent recon- solidation and thus should be avoided. These aspects need serious study by researchers in the field of education. Literature Cited 1. Abel, T., and K. M. Lattal. 2001. Curr. Opin. Neurohiol. 11: 180-187. 2. Lederhendler, 1. 1., S. Gart, and D. L. Alkon. 1986. J. Neurosci. 6: 1325-1331. 3. Epstein, D. A., H. T. Epstein, F. M. Child, A. M. Kuzirian, and D. L. Alkon. 2000. Biol. Bull. 199: 182-183. 4. Miiller, G. E., and A. Pilzeckvr. 1900. Z. Psychol Erganzungsband 1: 1-300. 5. Riccio, D. C., E. W. Moody, and P. M. Millin. 2002. Integr. Physio/. Behav. Sci. 37: 245-253. 6. Sara, S. J. 2000. Learn. Mem. 7: 73-84. 7. Nader, K. 2003. Trend', Neurosci. 26: 65-72. 8. Pedreira, M. E., and H. M. Maldonado. 2003. Neuron 38: 863- 869. 9. Kuzirian. A. M., H. T. Epstein, D. Buck, F. M. Child, T. Nelson, and D. L. Alkon. 2001. J. Neurocytal. 30: 993-1008. 10 Epstein, H. T., F. M. Child, A. M. Kuzirian, and D. L. Alkon. 2003. Netirobiol. Learn. Mem. 79: 127-131. 220 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull. 205: ^0-222. (October 2003) © 2003 Marine Biologu.il Moratory ii ing Alone, Not the Tripeptide RGD, Modulates Calexcitin in Hermissenda A. M Kuzirian,1-* F. M. Child,1 H. T. Epstein,1 M. E. Motta,2 C. E. Oldenburg.3 and D. L Alkon4 1 Marine Biological Laboratory, Woods Hole. MA ' University' of New Hampshire, Durham. NH "? Carnegie Mellon University, Pittsburgh, PA 4 Blanchette Rockefeller Neuroscience Institute, Johns Hopkins University, Rockville, MD In the nudibranch mollusc Hermissenda erassieomis. the inten- sity of immunostaining for the Ca2 VGTP-binding protein calex- citin ( 1 ) correlates positively with the degree of learning obtained and the level of memory expressed after Pavlovian conditioning (2. 3). When inhibitors of transcription (actinomycin-D; Act-D) and translation (anisomycin; ANI) are applied after training, they affect the animal's ability to recall the learned behavior. Epstein el a/. (4) have recently described the time windows for these effects. They noted that there are two phases of sensitivity to the inhibitors: an early phase, immediately after training (0-13 nun), and a later phase (70-160 min for Act-D, and 70-220 min for ANI). Subse- quently. Epstein et al. (5) reported that long-term memory also has two distinct biological configurations: long-term memory (LTM). which lasts about 24 h. and consolidated long-term memory (CLTM), which persists up to 6 days. The amount of memory retained by conditioned animals appears to depend, in part, on how much consolidation has been completed; and the transition from LTM to CLTM is sensitive to a cell adhesion molecule (CAM) inhibitor, the tripeptide arginyl-glycyl-aspartate (RGD). One working hypothesis predicts that increased calexcitin levels are needed to establish LTM. and that RGD-sensitive CAMs are subsequently involved in the transition from LTM to CLTM. What was unknown was the possible relationship between calexcitin levels and the effects of RGD during this critical transition period. Thus, as part of a continuing study to describe and define the memory stages demonstrated in Hermissenda (6. 4. 5), we under- took an immunocytochemical study to investigate possible corre- lations between calexcitin levels and the observed effects of RGD during the transition from LTM to CLTM. Hermissenda were purchased from Sea Life Supply (Sand City. California), and the tripeptide CAM inhibitor, arginyl-glycyl-as- partate (RGD), was obtained from Calbiochem (San Diego, CA). Animals were acclimated to laboratory conditions for a minimum of 3 days. The training procedure used was adapted from a Pav- lovian conditioning regime developed by Crow and Alkon (7), elaborated later by Lederhendler, Gart. and Alkon (8), and modi- fied by Kuzirian el ul. (9) and Epstein (10). Before training or testing, animals were dark-adapted for 10 min in a transparent acrylic plastic tray with 16 lanes ( 15 cm long and 0.9 cm by 0.9 cm in cross section) in an 1 I C incubator. In paired training, animals were exposed to a bright, white light (650-700 lux) for 6 s (the conditioned stimulus, CS) coincident with, after a 2-s delay, 4 s of vigorous agitation (the unconditioned stimulus, LIS). Animals re- spond to the agitation by contracting lengthwise (unconditioned * Corresponding uuilmr: akuziria@mbl.edu response. UR). The two stimuli are designated a paired training event (TE), which was repeated at 1-min intervals for nine repe- titions. Recall of training was tested by four presentations of the CS alone at 1-min intervals. Behavioral recall was assessed by cal- culating the percentage by which the animal's length changed be- tween light-on and light-off. Untrained (naive) animals show no recall and typically lengthen as a normal response to light, as do animals given light and agitation in an unpaired or random fashion ( 10). Experimental conditions shown by Epstein et ill. (5) to demon- strate the RGD-sensitive transition between LTM and CLTM were repeated to test for possible correlations between different times of RGD application after training and changes in calexcitin intensi- ties. RGD was diluted to an effective working dosage of 10 jag/ml (29 /iA/) with Tris-buffered (20 mA/) natural seawater (NSW-Tris) (pH 8.0). All solutions were administered post-training, at desig- nated times, by bath application using a training tray with a modified injection-port cover (4). After training, the RGD-exposed animals were tested for retention of recall at 4 and 24 h. then rapidly decapitated. The central nervous systems (CNS) were immediately fixed in 4% paraformaldehyde/NSW-Tris to preserve, as accurately as possible, the expressed calexcitin levels. To discern the temporal effects of training alone on the immuno- staining intensities of calexcitin, and to test for possible effects from testing with the CS alone, animals were similarly conditioned with 9 TEs in natural seawater. but they were not exposed to RGD or tested for recall. They were decapitated and fixed at time points similar to those for the RGD-exposed animals. Naive, untrained animals were used as overall experimental controls and were tested after 24 h. All fixed CNSs were then processed for embedding in polyeth- ylene glycol-400-distearate and were sectioned (6 jum). Calexcitin was immunolabeled with a primary polyclonal antibody (25U2; cloned from squid optic lobe and raised in rabbit: 1:1000 dil) (2, 3); subsequently, color was developed with a biotinylated second- ary antibody, and avidin-bound microperoxidase reacted with the chromogen 3-amino-9-ethylcarbazole (AEC). Gray-scale intensity was measured (using NIH Image software) from digital photomi- crographs of serial sections of the B-photoreceptors in the eyes of each animal. Intensity differences between conditions were statis- tically analyzed using ANOVA and Student's t tests. The data indicate that the levels of calexcitin remained steady from 40 to 200 min post-training, whether the animals were exposed to RGD or not (Fig. 1 ). Levels then fell to a baseline about 240 min after training. ANOVA and pairwise Student's / tests among and between intensities measured during the first phase (40-200 min post-training) indicated no significant differences between all intensity values (/•" = 0.67, tx < 2.0; P = >0.7; NEUROBIOLOGY AND BEHAVIOR 221 I Post-Training Treatment Times Figure 1. Immune/staining intensity levels for calexcitin under riro experimental conditions. First, specimens of Hermissenda were exposed to 10 fig/ml of RGD. a tripeptide. cell adhesion molecule inhibitor, at the designated post-training (nine training events) application times through to 240 min (designated RGD.x (mint: donhlc-hatched columns). At 240 min. the animals were tested, the RGD removed, and the animals transferred in the training tray to a nih with running setiwater heing siphoned through each lane until they were tested again at 24-h post-training. Second, replicate sets of animals (solid black columns) were similarly trained but not exposed to RGD or rested, and then decapitated and fixed at time points similar to those for the RGD-exposed animals. An overall control group (single-hatched column) consisted of naive, untrained animals held under similar conditions until they were also decapitated and filed. All central nen'ous svstems were processed collectively for immunocytochemistry. Calexcitin initnunostaining intensity was measured from digital photomicrographs using NIH Image sofnvare. Results were statistica/lv compared using ANOVA and painvise Student 's t tests (see text). n = 4-10 eyes measured), whether animals were treated with RGD and tested at 4 and 24 h. or simply trained and fixed at similar time points. The same was true for the second phase (240 min to 24 h post-training). Again, the intensities were statistically identi- cal, whether animals were tested or not. at 240 min or at 24 h ( F = 0.07. ts < 2.0: P = >0.9; n = 4-10 eyes measured). However. the overall ANOVA between all conditions did show significant differences between the first and second phases (F = 7.49: P = <0.001). The principal results obtained from this study indicate that the working hypothesis must be redefined to indicate that, to preserve a memory, calexcitin levels must remain elevated through the transition of LTM to CLTM. Also, the previously demonstrated RGD inhibition of this LTM/CLTM transition does not appear to operate by affecting calexcitin levels, but rather, as predicted by Epstein et til. (5). through the competitive inhibition and function- ing of CAMs. Calexcitin itself may contribute to the LTM/CLTM transition through the initiation of early gene transcription and translation of CAMs. The calexcitin immunostaining intensities observed during this study were similar to levels reported previously for LTM (3). The initial study by Kuzirian et al. (3) showed that the rise in ealexcitin intensity occurred by 90 min post-training, the earliest time point sampled. However, this study indicates that the intensity rise begins earlier, by 40 min or even sooner. The results of this study further demonstrated that the treatment of Hermissenda with the CAM inhibitor RGD is independent of the expressed levels of calexcitin. Therefore, the behavioral effects related to RGD expo- sure reported by Epstein et al. (5) were not mediated directly through changes in calexcitin levels. The similarity of intensity levels in tested and nontested animals also suggests that calexcitin levels may be insensitive to the immediate perturbation of recall caused solely by testing: a phenomenon known as reconsolidation (11) (also described as extinction). Memory generated by an as- sociative conditioning regime can be weakened by testing with the conditioned stimulus (CS) alone and must be reconsolidated to remain fixed. The data indicated quite clearly that there were no differences in calexcitin intensity related to testing alone. Previous reports (3. 12) show that calexcitin is involved in establishing LTM. This was accomplished by exposing Hennis- senda to a sensory block (an additional vestibular input) at time points before LTM is established (at 60 min post-training) (5) and afterward. The sensory block suppressed this rise in calexcitin levels until LTM was fixed. However, the current data indicate that calexcitin levels remain high through to the period coinciding with the establishment of CLTM (4. 5), and thus calexcitin may also be involved in some way with the consolidation of LTM to CLTM. Calexcitin was also elevated during the time period designated by Epstein et al. (4) as potentially being an intermediate-term memory phase, a phenomenon known to occur in Hermissenda and Aplysia, 222 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS among other animals (1? U> Since calexcitin, once activated by PKC, is known t> > i e<, • i lie release of internal calcium stores by binding to ryam • ;>l"is on the ER, and is responsible for early gene aci all of which are involved in memory acqui- sition, storat recall (15), it would be logical to broaden this study b\ ::,mg the effects of transcription and translation inhibik)'.' on the immuno-expression levels of calexcitin under experimental conditions similar to those just described. Indeed, such studies are underway. Drs. Daniel Alkon and Thomas Nelson provided Hennissenda and calexcitin antibodies for this study. MEM and CEO acknowl- edge the assistance of Beth Linnon. program coordinator, and the student internship program sponsored by the Marine Resources Center, Marine Biological Laboratory. Literature Cited I Nelson, T. J., S. Cavallaro, C. L. Yi, D. McPhie, B. G. Schreurs, P. A. Gusev, A. Favit, O. Zhar, J. Kim, S. Beushausen, G. Ascoli, J. Olds, R. Neve, and D. L. Alkon. 1996. Proc. Natl. Acad. Sci. USA 93: 13808-13813. 2. Kuzirian, A. M., H. T. Epstein, T. J. Nelson, N. S. Rafferty, and D. L. Alkon. 1998. Biol. Bull. 195: 198-201. 3 Kuzirian, A. M., H. T. Epstein, D. Buck, F. M. Child, T. Nelson, and D. L. Alkon. 2001. J. Neurocytol. 30: 993-1008. 4. Epstein, H. T., F. M. Child, A. M. Kuzirian, and D. L. Alkon. 2003. Neurobiol. Learn. Mem. 79: 127-131. 5. Epstein, H. T., A. M. Kuzirian, F. M. Child, and D. L. Alkon. 2003. Neurobiol. Learn. Mem. (In press). 6 Epstein, D. A., H. T. Epstein, F. M. Child, A. M. Kuzirian, and D. L. Alkon. 2000. Biol. Bull 199: 182-183. 7. Crow, T., and D. L. Alkon. 1974. Science 201: 1239-1241 8. Lederhendler, I. I.. H. S. Gart, and D. L. Alkon. 1986. J. Neuro- sci. 6: 1325-1331. 9. Kuzirian, A. M., F. M. Child, H. T. Epstein, P. J. S. Smith, and C. T. Tamse. 1996. Biol. Bull. 191: 260-261. 10. Epstein, H. T. 1997. Biol. Bull. 193: 255-257. 11. Sara, S. J. 2000. Learn. Mem. 1: 73-84. 12. Borley, K. A., H. T. Epstein, and A. M. Kuzirian. 2002. Biol. Bull. 203: 197-198. 13 Crow, T., J. J. Xue-Bian, and V. Siddiqi. 1999. J. Neurophysiol. 82: 495-500. 14 Sutton, M. A., S. E. Masters, M. W. Bagnall, and T. J. Carew. 2001. Neuron 31: 143-154. 15. Alkon, D. L., T. J. Nelson, W. Zhao, and S. Cavallaro. 1998. Trends Neurosci. 21: 529-537. Reference: Biol. Bull. 205: 222-223. (October 2003) © 2003 Marine Biological Laboratory Neurochemical Modulation of Behavioral Response to Chemical Stimuli in Homarus americanus Anna Savage1'2 and Jelle Atema2'* 1 Ainherst College, Amherst, MA ~ Boston University Marine Program, Woods Hole, MA Serotonin (5-HT) and octopamine have been implicated in reg- ulating the behavioral phenotype of lobsters by altering levels of aggression in agonistic interactions ( 1 ). High-dosage injections of these amines consistently evoke extension (5-HT) and flexion (octopamine) postures in both lobsters and crayfish. These postures have been likened — probably erroneously — to aggressive and sub- missive postures, suggesting a specific role for these neuromodu- lators in social behavior (2). Dopamine injections into freely moving lobsters have also induced motor activity, including ex- tension of claws, legs, and tail, originally interpreted as an ago- nistic posture (3). However, the relationship between neuromodu- lator treatment and overall crustacean behavior is not simple, with responses varying with the stimuli presented and the aminergic manipulations performed (4, 5). Serotonin is known to regulate feeding behavior in other animal species (6), but its effects on lobster feeding behavior and responses to odors have not yet been investigated. As lobsters are known to rely heavily on chemical signals for food and social information (7), we examined the reaction of H. americanus to food and social odors after the animals li.i 1 been injected with serotonin and two other biological amines o .union to the lobster central nervous system (CNS). Seven adui ntermolt male lobsters (81-93 mm carapace length) * Corresponding author itema@bu.edu were injected with serotonin, octopamine, dopamine (Sigma Chemi- cals), or lobster saline. Each injection of amine was administered in random order over 4 consecutive days of testing, and consisted of 0.3 mg of neuromodulator per kg of animal dissolved in 1 ml lobster saline. Injections were made intramuscularly into the first abdominal segment to the right of the ventral nerve cord, following Peeke el al. (4). After injection, each lobster was placed in an observation tank (36 X 46 X 72 cm) with a constant flow of unfiltered seawater. Each tank had a plastic, two-entrance shelter in front of the window and a constantly flowing air-lift water circulation system with funnel inter- ruption (7). Food odor and body odor were injected into the funnel, which delivered an irregular flow of tank water into the shelter at a mean rate of 4.8 ml/s. Lobsters were not fed for the 4-d duration of the experiment. Tanks were kept dark during observations except for a 25-watt bulb covered in black plastic with a small hole cut through that allowed a narrow beam of light to illuminate the shelter interior. Body odor stimuli were prepared by collecting water samples from one male and one female lobster isolated in 10 1 of aerated standing seawater for at least 3 h. Food odor consisted of store-bought clam juice. Fifteen minutes after drug injection, lobsters were presented with 0.5 ml of each stimulus at 5-min intervals, and the resulting behaviors and their durations were recorded to the nearest second. The order of stimulus introduction was held constant over the four days of injections but varied randomly among the animals. We selected be- haviors from Atema and Cowan (7) based on frequency and quanti- NEUROBIOLOGY AND BEHAVIOR 223 Serotonin Octopamine Dopamine Saline 0 50 100 150 200 250 Time after injection (min) Figure 1. The effect of injections with 0.3 mg/kg of serotonin, octopamine. dopamine. or saline on the duration of lobster dactyl clasping behavior in response to clam juice. Mean duration ± SE shown for 7 lobsters or 6 time points post-injection. The serotonin-affected response was significantly higher, and the dopamine-affected response was significantly lower, than the saline control (see te.\t>. liability. They included "locate source," turning toward the glass at the front of the shelter and probing the stimulus inflow tube; "check entrance." walking over to. and standing still at, one of the two entrances: and "dactyl clasping," a typical feeding response marked by opening and closing the dactyls of the first two pairs of walking legs. Stimulus introductions were repeated every 15 min for the first hour, and then again 1 20 and 2 1 0 min post-injection to test for a time delay in response. The study was conducted blind for six of the eight animals tested, with the observer unaware of the amine injected from day to day. Lobsters injected with serotonin and presented with clam juice displayed a mean duration of dactyl clasping that was twice that observed after saline injections. Statistical analysis showed that this difference in duration was significant (ANOVA post-hoc t test, t = 6.69. P = 7e " ). The serotonin effect peaked at 60 min post- injection, when the duration of clasping was over 5 times that of the saline control; the effect then returned to initial levels by t = 120 min (Fig. 1 ). The I = 60 min response was also significantly longer than the initial response at / = 15 min (t = -2.69, P = .0082). In contrast to serotonin, dopamine induced a shorter duration of clasping in response to clam juice at all times examined: the mean response was less than 507c that of saline. This difference was statistically signifi- cant (t = -2.68. P = .0078). Octopamine-induced clasping in re- sponse to clam juice varied widely between animals, and the overall difference in duration for octopamine injections compared to saline was not significant (/ = 1.254. P = 0.21). Dactyl clasping was not observed in response to body odor. The occurrence of "locate source" and "check entrance" behaviors, infrequent for all three chemical stimuli, appeared unrelated to treatment condition. The significant increase in length of dactyl clasping after injection with serotonin and the decrease after injection with dopamine suggest that these neuromodulators are priming lobsters toward certain be- haviors, making them more (5-HT) or less (dopamine) motivated to feed when given the opportunity. This serotonin-induced persistence has been demonstrated in agonistic encounters, where 5-HT-treated subordinate lobsters are less willing to retreat (8). However, seroto- nin's effects are not limited to aggression. Rather, it appears to act as a general motivator of lobster behaviors, including both agonistic encounters and feeding responses. Additionally, prolonged dactyl clasping requires sequences of rapid flexion and extension: thus the behaviors induced by serotonin cannot be attributed to simple exten- sor effects. The observed inconsistency of dactyl clasping follow ing octopamine injection reflects the complex relationship between neu- romodulator action and lobster behavior (4). Dopamine-induced de- pression of response is a novel result and merits further behavioral and neurochemical investigation. Financial support is acknowledged from NSF-REU (OCE- 0097498 site awarded to Boston University Marine Program). We thank Sean Sapcariu for overseeing blind injection schedules. Molly Steinbach for design advice, and Gabi Gerlach for her aid in statistical analysis. Literature Cited 1 Huber, R.. and E. A. Kravitz. 1995. Brain Behav. Eml. 46: 72-83. 2. Livingstone, M. S., R. M. Harris-Warrick, and E. A. Kravitz. 1980. Science 208: 76-79. 3. Barthe. J. Y., N. Mons, D. Cattaert, M. Geffard, and F. Clarac. 1989. Brain Res. 497: 368-373. 4. Peeke. H. V. S., G. S. Blank, M. H. Figler, and E. S. Chang. 2000. J. Comp. Phyxiol. 186: 575-582. 5. Doernberg, S. B., S. I. Cromarty. R. Heinrich, B. S. Beltz, and E. A. Kravitz. 2001. J. Comp. Physinl. 187: 91-103. 6. Mancilla-Diaz, J. M., R. E. Escartin-Perez, V. E. Lopez-Alonso, and S. E. Cruz-Morales. 2002. Eur. Neitropsychophannacoi. 12: 445- 451. 7. Atema. J., and D. F. Cowan. 1986. /. Chem. Ecol. 12: 2065-2080. S. Huber. R., K. Smith, A. Delago. K. Isaksson, and E. A. Kravitz. 1997. Proc. Natl. Acad. Sci. 94: 5939-5942. 224 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Bio/. Bull. 2(!-: -225. (October 2003) © 2003 Marine Bu •!.-• .!ory /i Recognition in Juvenile Zebrafish (Danio rerio) Based on Olfactory Cues A'. D. Mann', E. R. Tnrnell1, J. Ateimr, ami G. Gerlach1'" ' Marine Biological Laboratory; Woods Hole, MA ' Boston University Marine Program, Woods Hole, MA Genetic analyses of numerous fish species have shown that shoals formed by larvae often consist of closely related kin ( 1 ). Aggregating with kin may be an altruistic trait that evolved through kin selection (2). Individuals would increase their inclu- sive fitness by sharing the benefits of shoaling among related individuals (3). Laboratory experiments on recognition of kin v\. non-kin groups of Atlantic Salmon (Salnw sular) (4) demonstrated possible advantages: kin groups had fewer aggressive interactions, used a greater proportion of "threat" behavior as opposed to fighting, and subordinates especially had improved growth. The mechanisms by which these kin groups develop and stay separate from each other are not known. The genes of the major histocompatibility complex (MHC) are a source of individual odors released into the water via urine (5). Such pheromones might be involved in olfactory kin recognition. Here, we tested the hypothesis that zebrafish can recognize kin based on olfactory cues. Zebrafish (Danio rt-rin) live in freshwater streams and rice paddies in the Ganges River of East India, Bangladesh, and Burma. Although this species is widely used as a model in genetic and developmental research, little is known about its natural behavior. Zebrafish spawn up to several hundred eggs at a time, and these develop in the substrate without any parental care. Larvae (G.G. pers. obs.), and sometimes adults, can be observed in shoals (6. 7), but their genetic relatedness is unknown. We observed wild-type juvenile zebrafish. aged 6-8 weeks; the fish were kept in 2.5-1 aquaria under a day/night cycle of 14/10 h and fed on a standard diet of brine shrimp nauplii and dry fish food. Twenty-four hours before an experiment started, 2 separate kin groups consisting of 12 full siblings each were placed into two 9-1 aquaria with standing water. From each kin group, 3 fish were tested in the flume, one at a time, for a total of 6 fish. This procedure was repeated 3 times for a total of 6 kin groups and I X test animals. Water from each of the two aquaria was used as the two stimuli in an olfactory preference test. Single individuals of either group were used as test fish and were placed into a choice flume (20 cm long X 4 cm wide, water level 2.5 cm) that main- tained two separate water columns (Fig. 1) (8). Uniform and unidirectional water flow was maintained at a constant rate of 40 ml/min ( = 3.5 mm/s). Periodic dye tests showed that the two water columns remained well separated. Prior to each trial, formulated fresh water was run through both channels of the flume for 5 in in to allow l he subject to acclimate. Each trial consisted of four 3-min periods, during which water from kin and non-kin aquaria was presented alternate sides of the flume to correct for the possi- bility of um >Y-d side bias. Everv 10 s, we recorded which side of ' Corresponding autho/: ggerlach@mbl.edu B a b c d Figure 1. Diagram of the choice flume, and a xraplt ofte.il result, (a) Water inflow area: (b) collimator to hoinogcni-e turbulent flow; (c) bar- rier-separated channels: (d) area of flume where water columns remain separated without the harrier (fine dotted center line): fe) screen to contain test subjects: (/) outflow channel. Black bar in graph indicates preference [% ± SEM] for kin over non-kin hy test subjects (* = P < 0.05). the flume (A or B, Fig. 1 ) the fish was swimming on. The number of times each animal was recorded on the side with kin stimulus was expressed as a percentage of the total number of recorded observations (i.e., kin plus non-kin). A score greater than 507r (random distribution) indicated a preference for the kin stimulus; and when the percentages of all the fish were compared using a Wilcoxon matched-pairs signed-ranks (WSR) test, the preference for kin was significant (WSR = 45.0, P = 0.050) (Fig. 1). Our study is the first to show that juvenile zebrafish can recognize and prefer their siblings to unrelated conspecifics based on olfactory cues. There are two general categories of kin recognition mechanisms, both based on learning processes as Tang-Martinez (9) empha- sized. The first ("indirect") mechanism is based on familiarity, where individuals behave nepotistically to conspecifics with whom they grow up. The second ("direct") kin recognition mechanism allows individuals to identify even unfamiliar kin. Direct recogni- tion is thought to be based on 'phenotype matching', in which an individual must learn cues, either from the phenotypes of close relatives (familial imprinting) (10), or from itself, to form a tem- plate for comparison with the phenotype of other individuals. Our results cannot distinguish between these recognition mechanisms. Literature Cited 1 Krause. J., D. J. Hoare, D. Croft, J. Lawrence, A. Ward, G. D. Ruxton, J. G. J. Godin. and R. James. 2000. Prot: K. Sot: Land. B Biol. Sci. 267: 2011-2017. 2. Pitcher, T. J. 1986. The Behaviour ofTc/eosr Fishes. Croom Helm, London. 3. Hamilton. \V. D. 1964. ./. Theoi: Biol. 1: 1-16. 4. Brown, G. E., and J. A. Brown. 1993. Belui\: Ecol. Sociobiol. 33: 225-23 1 . NEUROBIOLOGY AND BEHAVIOR 225 5. Apanius, V., D. Penn, P. R. Slev, L. R. RulT. and W. Potts. 1997. Crit. Rev. Immitnol. 17: 174 -224. h. Pritchard, V. I.., J. Lawrence. R. K. Butlin, and J. Krause. 2001. Anim. Belitu: 62: 1085-1088. 7. Delaney, M., C. Follet, N. Ryan, N. Hanney. J. Lusk-Vablick, and G. Gerlach. 2002. Biol. Bull. 203: 240-24 1. 8. Atema, J., M. Kingsford. and G. Gerlach. 2002. Mar. Ecol. ProK. Set: 241: I5I-IM). 9. Tang-Martinez, Z. 2001. Hclmv. Process. 53: 21-40. 10. Sherman. P. W., H. K. Reeve, and D. W. Pfennig. 1997. Pp. (•>')-% in Bi'liuvionil Ecology: An Evolutionary Approach, J. R. Krebs and N. B. Davies. eds. Blackwell Scientific, Oxford. UK. Reference: Biol. Bull. 205: 2:5-226. (October 2003) © 2003 Marine Biological Laboratory Mate Choice in Zebrafish (Danio rerio) Analyzed With Video-Stimulus Techniques E. R. Turnell', K. D. Mann', G. G. Rosenthul2, and G. Gerlach'-* 1 Marine Biological Laboratory, Woods Hole, MA 2 Boston University- Marine Program, Woods Hole. MA In many species, individuals of both sexes have developed a variety of visual signals and behavioral patterns with which to broadcast their quality as mating partners ( 1 ). The complexity of these signals makes it difficult to distinguish those that are most important in mate selection. Animated models offer a solution to this problem by allowing for the alteration of single parameters in the complex stimulus presented. In this study, we have tested the use of computer animated three-dimensional models to analyze mate choice criteria in zebrafish; we applied this tool to examine the roles played by two visual characteristics in mate selection. Sexually mature wild-type zebrafish. aged at least 8 months, were kept in 9-1 aquaria under a day/night cycle of 14/10 h. Subjects were tested in a 50 cm X 32 cm tank: a vertical line drawn on the wall of the tank divided it into two equal sections. A and B. The tank was placed between two 17-inch Dell computer monitors on which animated models of swimming zebrafish were displayed. The models were created using 3D Studio Max 1.0 (Kinetix) on a Dell Optiplex GXPro computer and a Targa 1000 board for digital/analog conversion of video signals, as described by Rosenthal (2, 3). Before each trial, an individual fish was placed in the tank and allowed to acclimate for 5 min. During each trial, the subject was simultaneously shown two different animated stimuli. Each trial consisted of four 5-min viewing periods separated by 1-min inter- vals during which black covers were gently slid in front of the monitors. Three pairs of stimuli were shown to the subjects: [1] male versus female body shape (both with natural horizontal stripes). [2] vertical versus horizontal stripes (both with female shape), and [3] vertical versus horizontal stripes (both with male shape). The vertical and horizontal stripe patterns had equal amounts of blue coloration. Female-shaped images differed from male-shaped images only in that their bellies were 10% larger in side view. Stimulus pairs [1] and [2] were presented to both male and female subjects, while stimulus pair [3] was presented to female subjects only. In addition, stimulus pair (1] was shown to females who had spawned on the day of the trial. (The females * Corresponding author: ggerlach@mbl.edu used in all other trials did not spawn on the day of the trial.) The two animated stimuli were alternated between the monitors to balance for side effects. During each viewing period, the location of the fish was recorded every 10 s. The percentage of time the subject spent in proximity to each stimulus (presence on side A or B) was calculated and compared using a Wilcoxon matched-pairs signed-ranks (WSR) test. The results and statistical evaluations are shown in Figure 1. Males did not differentiate between the male and female-shaped images but showed a significant preference (19.6%) for the horizontal stripe pattern over the vertical stripe pattern when the images were female-shaped. Female zebrafish preferred a male-shaped stimulus over a female-shaped stimulus by 20.3%. However, females that had just spawned eggs on the morning of the trial did not show a preference for either the male or the female shape. Females also showed a significant preference (10.7%) for the horizontal stripe pattern over the vertical stripe pattern when the images were male-shaped, but did not differ- entiate between stripe patterns when the images were female- shaped. The preference of females for the male-shaped stimulus over the female-shaped stimulus indicates that belly size alone allows fe- male zebrafish to distinguish between sexes. The indifference of the females who hud recently laid eggs suggests that females' interest in males correlates with their reproductive stage. The significant bias of males and females against vertical stripe pattern in the opposite-sex animation may result from selection against mating with heterospecifics. The failure of males to distinguish between the male- and female-shaped images has at least two possible explanations: (a) the belly of the female image may not have been large enough to realistically simulate a fecund female zebrafish: or (b) male ze- brafish may rely less on visual cues than on olfactory cues to select mates. Prior studies have shown that male zebrafish are attracted to the pheromones released by females (4-6). This study shows that video-stimulus techniques can be used to further study mate choice and visual preferences in zebrafish. 226 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 50 Figure 1. Preference (% ± SEM) of males (white bars) and females (black bars) for each stimulus image, as measured by time spent in proximity to each one. [la] Females significantly preferred the male-shaped stimulus (M) over the female-shaped stimulus (F) ( WSR = 27.0, n = 12, P = 0.034); males showed no preference (WSR = 3.0. n = 15. P = 0.868). [Ib] Females who had spawned the day of the trial also showed no preference behveen images M and F ( WSR = 17.5, n = 75, P = 0.288). [2] Males significantly preferred the horizontal to the vertical stripe pattern when both images were female (F, FV) (WSR = 37.0. n = /5. P = 0.017); females showed no preference (WSR = 2.0. n = 15. P = 0.923). 13] Females significantly preferred the horizontal to the vertical stripe pattern when both images were male (M. MV) (WSR = 38.0. n = 16. P = 0.049). * = P < 0.05. Literature Cited 1. Dugatkin, L. A., and G. J. FitzGerald. 1997. Pp. 266-291 in Be- havioral Ecology ofTe/eost Fishes. J.-G. J. Godin, ed. Oxford Univer- sity Press, Oxford. 2. Rosenthal, G. G. 1999. Environ. Biol. Fishes 56: 307-316. 3 Rosenthal, G. G., W. E. Wagner, and M. J. Ryan. 2002. Aniin. Bchav. 63: 37-45. 4 van den Hurk, R., and J. G. D. Lambert. 1983. Can. ./. Zoo/. 61: 2381-2387. 5. Bloom, H. D., and A. Perlmutter. 1977. J. Exp. Zoo/. 199: 215-226. 6 Delaney, M., C. Follet, N. Ryan, N. Hanney, J. Lusk-Vablick, and G. Gerlach. 2002. Bio/. Bull. 203: 240-241. MOLECULAR BIOLOGY. PATHOLOGY, AND MICROBIOLOGY 227 Reference: Biol. Bull. 205: 227-228. (October 2003) © 2003 Marine Biological Laboratory Expressed Sequence Tag Analysis of Genes Expressed in the Bay Scallop, Argopecten irradians S. B. Roberts ami F. IV. Goetz Marine Biological Laboratory', Woods Hole. MA The bay scallop (Argopecten irradians) is a marine bivalve found along the eastern United States. As in other pectinids, the bay scallop has a single, large adductor muscle which acts to open and close the shell with great force. This muscle is a prominent feature observed when the shell is removed, and is a favorite of both scientists and consumers, as it offers an excellent model for understanding muscle physiology and provides a healthy, high- protein food source. Consumer demand has caused the increase in aquaculture of bay scallops for direct marketing or to seed local waters. The objective of the present study was to better understand the factors that contribute to the growth and development of the scallop adductor muscle. Because little is known about these processes in scallops, we have started by isolating and identifying genes from adductor muscle tissue. The results from this work could help facilitate future research. One example is in aquacul- ture. where these genes could be used as markers for a selective breeding programs. The use of such techniques could result in increasing the yield of adductor muscle or altering the size and density of the muscle fibers. To identify the factors or genes involved in scallop muscle structure and function, a cDNA library was constructed from bay scallop adductor muscle, and expressed sequence tags (ESTs) were sequenced. ESTs are small pieces of DNA sequence (usually 100 to 800 nucleotides long) generated by sequencing randomly se- lected cDNA clones from a library. In this paper, the sequences of genes expressed in the bay scallop adductor muscle are reported. To construct the cDNA library, adductor muscle tissue was dissected from four adult bay scallops obtained from Woods Hole. Massachusetts. Total RNA was extracted with Tri-Reagent (Mo- lecular Research Center Inc.). and mRNA was isolated using the Poly-A-Tract mRNA Isolation System (Promega). The cDNA library was constructed using the A Zap Express cDNA/Gigapack cloning kit (Stratagene), starting with 5 /ng of mRNA as previously described ( 1 ). To obtain ESTs. the library was mass excised to pBK-CMV phagemids and plated. From these plates. 454 randomly chosen cDNA clones were picked and sequenced from the 5' region by using the dideoxy chain termination method, with Big Dye Ter- minator (Applied Biosystems) and a vector-specific primer. The reactions were precipitated and resuspended in Hi-Di Formamide with EDTA (Applied Biosystems) and run on an ABI Prism 3730 automated sequencer (Applied Biosystems). Gene identification analysis was performed using Phred (base-calling) and Cross_ match (vector removal) software (http://www.phrap.org/); then EST sequences were compared with those in the NCBI database (nr) using the Blast-X and Blast-N programs (http://www.ncbi. nlm.nih.gov/BLAST) (2). Of the 454 cDNA clones, 20 yielded no sequence and an additional 9 produced sequences of less than 150 bp; these were not used for sequence analysis. The average length of the remain- ing 425 sequences was 792 bp. Based on top Blast hits, 137 distinct sequences were observed and 90 of these genes were putatively identified; 47 lacked similarity with known genes and could not be identified. Of the latter 47 distinct sequences. 38 were most similar to unidentified products {e.g., hypothetical protein, unnamed protein product) and 9 produced no hits in the database. Only 16 of the distinct sequences had previously been sequenced from the genus Argopecten and were either myosins. ribosomal proteins, or mitochondria! sequence. The mitochondrial sequence appeared 54 times (13%) in the ESTs and does not necessarily code for a protein; the presence of a poly-A region in the sequence leads to its isolation along with mRNA. Of the genes identified based on sequence similarity, 33% are involved in cell structure (Fig. 1 ). The two genes most prevalent in the cDNA library were actin and myosin because they are the Cell Structure 33% Cell Signaling 8% Binding Proteins 7% Unclassified 11% Gene / Protein Expression 3% Immune Related 7% Metabolism 31% Figure 1. Percentages of identifiable ESTs sequenced that could he grouped together based on gene function or that could not be classified. 228 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS major components of liscle tissue. Of the 425 cDNA clones, actin (2 forms) occur- i 80 times (19%) and myosin (11 forms) was found 68 tin ' >. The remaining ESTs could be classified as being involved in gene/protein expression (3%). immunity (7%). met;" (31%), protein binding (7%). or cell signaling (8% ) (Fig. 1 ). Some identified genes (11%) could not be classified into any of these groups. The sequences of all the ESTs generated from the bay scallop cDNA library and the putative identifications determined to date are available on a Bay Scallop EST project website (http://www. mbl.edu/goetz/EST.html) as well as in NCBI's GenBank database (http://www.ncbi.nlm.nih.gov/) [GenBank Accession numbers CF197421-CF197787]. The ESTs generated offer a valuable re- source to scientists in a wide range of disciplines including muscle physiology, growth and development, immunity, genetic identifi- cation, and aquaculture. Funding for this research was provided by USDA grant #2002- 03633 — Program in Growth and Nutrient Utilization. Literature Cited 1 Garczynski, M. A., and F. W. Goetz. 1997. Binl. Kcpmd. 57: 856-864. 2 Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. J. Mol. Bio/. 215: 403-410. Reference: Binl. Bull. 205: 228-230. (October 2003) © 2003 Marine Biological Laboratory Scanning Electron Microscopy Investigation of Epizootic Lobster Shell Disease in Homams americamis A. C. Hsu' and R. M. Smolowitz2'* ' Boston University Marine Program, Woods Hole, MA ~ Marine Biological Laboratory, Woods Hole, MA The American lobster, Homarus americamis, represents an im- portant fishery for much of the New England coast as well as several coastal provinces in Canada. Yet during the past decade. New England has reported dramatic decreases in the catch value in this lucrative industry ( 1 ). Shell disease is the deterioration of the crustacean exoskeleton by chitinoclastic organisms occurring in both marine and freshwater environments (2). In the past 6 years, the prevalence and severity of shell disease has markedly increased (K. Castro, Rhode Island Sea Grant, pers. comm). Predominately occurring in areas from Buzzard's Bay (Massachusetts) to eastern Long Island Sound (New York), it has been termed epizootic lobster shell disease (ELSD). Recently ELSD lobsters have been observed in Cape Cod Bay (Massachusetts), Kittery (Maine), and in offshore waters of New England ( 1 ). For the present study, carapace lesions from wild-caught spec- imens of H. wm-riciiiiiis were examined for the etiological agent responsible for ELSD. Although previous studies on lobster shell disease have used histology and molecular techniques to define the organisms involved in lesions (3, 4) no study has used scanning electron microscopy (SEM) to observe the progression of lesion development. For this study, SEM was used to produce three- dimensional views of ELSD development from geographically distinct areas along the New England coast for site comparisons. During 2002-2003, lobsters with lesions (11 = 22) and without lesions (n == 14) were collected. The sites sampled were the inshore waters of eastern Long Island Sound (n = 4). Rhode Island (» = 13). Buzzards Bay (n = 7). Cape Cod Bay (n = 3), and Maine (n = 4), and the offshore waters of New Hampshire (11 = 5 ). Animals were defined as "healthy" or "infected" depend- ing on the presence of noticeable lesions on the cephalothorax. ' Corresponding author: rsmol@mbl.edu Carapace pieces were collected, fixed in 10% formalin in sterile seawater (5), and dehydrated in increasing concentrations of eth- anol on ice. Samples were trimmed, critical-point-dried, and sput- ter-coated with gold palladium (6). The surface of cuticle lesions in early disease phases and the deeper interface between lesions and normal cuticle (leading edge of lesions) were compared and analyzed. To compare the presence of morphologically distinct bacteria identified in lesions, images were analyzed using Sigma- Scan 4.0 (Jandel Scientific). Gross examination of carapace pieces showed that the carapaces of healthy animals showed no degradation, while samples from infected animals were severely eroded. Microscopic analysis of healthy carapace revealed minimal bacterial buildup (Fig. 1A). In contrast, carapace lesions of infected lobsters were covered with bacterial cells. Setal cores and natural abrasions were consistently filled with bacteria embedded in the cuticle at the lesion surface (Fig. IB). Additionally, bacteria were abundant at the leading edge of the lesions (Fig. 1C). Overall, healthy carapace samples had substantially fewer bacteria on the carapace surface. These obser- vations were consistent for all sampling sites. The role of bacteria in the progression of lesion development was indicated by bacteria found embedded in shallow pits along the epicuticle (Fig. ID). In other areas on the surface, halo-like holes surrounded several bacterial types that were associated with shallow erosions. These holes were not observed at the deep leading edges of the lesions. Bore holes appeared to match the length/width ratio of associated bacteria and, therefore, were be- lieved to be caused by bacteria secreting chitinase. lipase. or protease (7). Diatoms, algae, and fungi/actinomycetes were noted typically in low abundance within the cuticle filaments, but bac- teria were consistently the dominant organisms on both the cara- pace surface and the leading edge of lesions. MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY 229 Figure 1. SEM images from infected and healthy lobsters collected from various sampling sites. (A) Healthy setae with minima/ bacterial cells at base of core {arrow) from a non-diseased Maine lobster. (B) Infected setae with a high abundance of bacteria (arrow) from a diseased New Hampshire lobster. iCl Bacterial buildup (arrow) in the leading edge of a lesion from an infected Buzzard's Bay lobster. (D) Enzymatic digest by coccoid, rod, and rod linked bacteria from an infected Buzzard's Bay lobster. Bars represent 10 pun in A, B, and C, and I p-in in (). Five morphologically distinct bacterial types were observed on both healthy and infected animals. The majority of cells were either rods ( 1 x 0.4 /xm). coccoid rods (0.8 x 0.5 /^m), or cocci (0.5 X 0.45 /j.m). Segmented rod links (each piece 1.5 X 0.5 /urn) and coccoid links (each piece 1.5 X 1.0 /urn) were less abundant and found on infected animals only. Most bacterial types were found on animals from all geographical areas, but coccoid links were observed only on infected Rhode Island and Cape Cod Bay samples, indicating that they may be secondary invaders. Bacteria are ubiquitous in the marine environment, so identify- ing the causative agents in a disease can be difficult. Examination of the interface between healthy and necrotic tissue provided the evidence necessary to identify bacteria as the disease-initiating organisms. Scanning electron microscopic imagery cannot speciate bacteria, so additional techniques such as those used in molecular biology are needed to identify the bacteria responsible for ELSD. Findings from the present study revealed the complexity of this disease in its progression and development and demonstrated the necessity for further molecular strides, including bacterial specia- tion and infection studies, in ELSD research. Literature Cited 1 . Dean, M. J., K. A. Lund) . and T. B. Hoopes. 2002. Massachusetts Division of Marine Fisheries, Technical Report TR-13 (Online). Avail- able: http://www.state.ina.us/dfwele/dmf/index.html [accessed August 2003]. 2. Stewart, J. E. 1980. Pp. 32 1 -324 in The Biology and Management of Lobsters, Vol. 1. J. S. Cobb and B. F. Phillips, eds. Academic Press, New York. 3. Chistoserdov, A., R. Smnlowitz, and A. Hsu. 2003. Pp. fil-f)4 in Connecticut Sea Grunt Extension, Third Long Island Sound Lobster Health Symposium. University of Connecticut, Storrs. 230 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 4. Smolowitz, R. M., R '• liis, and D. A. Abt. 1992. Biol. Bull. l»3: 99-112. 5. Luna, L. G. 1'Wi. r 107 in Histopathologic Methods and Color Alias ofSin t ii» on the basis of their similarity to characterized genes in GenBank as part of the ongo- * Corresponding author: sdyhrman@whoi.edu ing Trichodesmium annotation effort (unpubl. data). Multiple primer pairs were designed to amplify internal fragments (referred to as internal) or the complete plioA, pstSl, or pstS2 gene (referred to as external) (Table 1). DNA was extracted following the method described by Orcutt et al. (6). PCR amplification conditions were performed with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) using conditions optimized for each gene and species (Table 1 ). To optimize amplification, the annealing temperature was var- ied for each gene and species over a range of at least 25 °C. Other variables included magnesium and enzyme concentration. We were able to amplify the complete phoA and pstSl gene from T. erythraeum and T. teiuie, and the complete pxtS2 gene from T. erythraeum. We amplified gene fragments from pstSl in T. thiebautii and A', xpirulix, and from pstS2 in T. tenue and K. spimlis (Table 2). With the entire 3.5 kb of the phoA gene amplified from T. erythraeum, we were able to clone it into the pBluescript II SK ( + ) plasmid in Escherichia coli DH5a, in order to identify the activity associated with the putative gene. However, activity and expression work is still ongoing. All PCR products were sequenced at the Josephine Bay Paul Center of the Marine Biological Laboratory (Woods Hole, MA) using the facility's protocols. With the T. erythraeum genome as a guide, internal primers were designed to obtain full coverage of the entire gene on both strands. Genes were aligned between species to compare their sequence divergence using Sequencher 4.1 software (Gene Codes Inc. Ann Arbor, MI). Preliminary sequencing data for the genes and gene fragments indicate that pstS], pxtS2, and phoA in T. tenue as well as pstS2 in Table 1 Primer sequences and annealing temperatures for PCR reactions Gene External 5'-.V Annealing temp. Internal 5'-3' Annealing temp. PhoA ATGCGTGGGGACTTAACAGTAA 61.2 TTCGCTATCATCATACACCATAATTCCACC 60 TCTAATCACAAAATCATCTGTTGTGAGAG AGAACCTAATGATGACTATACTAACGACCC PstSJ \GCACAAACTAAAACCAG 58.5 TTTCCATCACCTATATATCAAC 51.2 GACGAATCAGCAGTGACAAG CATAACCATACTCAACATATCC PstS2 AA AT FAGTATCGTTGCCTAAAT 59 ATCTTTTCCAGCTCCACTATAC 50.2 TTTGTGCTTATACATTTATTATC CTCCTACTTTCTTTTTCCACTC MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY 231 Table 2 Amplified genes from Trichodesmium and Katagnyniene spiralis PhoA PstSl PstS2 /. erythraeum XXX XXX XXX T. teiute XXX XXX X A', spiruln XXX X X I tliichiiutii X XXX — entire gene X — gene fragment Kategii\nii'iic are approximately 98*^ identical to the correspond- ing genes in T. erythmeum. Given the morphological distinction of the Triclwdesmium species, this high degree of similarity was surprising. However, coverage of the entire gene in each species may reveal regions within the genes that are less highly conserved. This hypothesis is consistent with the results for the only other functional gene to be sequenced from multiple Trichtnlexniiiiiii species. lictR. which shows a lower degree of similarity. 9 1 %-95% (7). Despite extensive efforts to amplify all the genes by varying annealing temperature, primers, and concentrations of magnesium, enzyme, and template, we were unable to amplify pslS2 or phoA from T. thiehautii. This is particularly interesting because T. thic- bautii has been shown to have alkaline phosphatase activity (2), and because of the degree of similarity of plwA among the other species. Additionally. T. thii'huiiTii's evolutionary relationship, in- ferred from Triclwdesmium ITS sequences (6), indicates T. tenue and K. spinilis are more closely related to each other than to 7". thiehautii. It may be that plwA in T. iliiehautii is very different compared to the other species or that a different gene and gene product are used in the hydrolysis of dissolved organic phosphorus for this species. Future work is needed to determine whether pstS2 and plioA are present in T. tliichuutii. Although other species and isolates of Trichodesmium should be analyzed, our initial identification of putative P-regulated genes in these Trichodesmium species has improved our knowledge of phosphorus scavenging mechanisms in this important genus. This work was funded by NSF grant OCE-0220945, and by NSF Research Experience for Undergraduates grant OCE-0097498. Literature Cited I. Capone, D., J. Zehr, H. Paerl, B. Bergman, and E. Carpenter. 1997. Science 276: 1221-1229. 2 Dyhrman, S.. E. Webb, and D. Anderson. 2002. Limnol. Oceanogr. 47: 1832-1X36. 3. Sanudo-\Vilhelmy. S. A., C. Gobler, D. Hutchins, M. Yang. K. Lwiza, J. Burns, D. Capone, J. Raven, and E. Carpenter. 2001. Ntiture 411: 66-64, 4. Scanlan, D., and N. West. 2002. FEMS Micmhiol. £<•«/. 40: 1-12. 5. Scanlan, D., and W. H. Wilson. 1999. Hnlrobi,>loKui 401: 144 175. 6. Orcutt. K. M.. U. Rasmussen. E. A. Webb, J. B. Waterbury, K. Gundersen, and B. Bergman. 2002. 4/>/>/ Environ. Microhinl. 68: 2236-2245. 7 ,|. ins, ,n. S.. B. Bergman. E. Carpenter, S. Giovannoni, and K. Vergin. 1999. F EMS Micwbiol. Ecol. 30: 57-65. Reference: Biol. Bull. 205: 231-232. (October 2003) © 2003 Marine Biological Laboratory Molecular Quantification of Toxic Alexandrium fundyense in the Gulf of Maine Madeline Galac1 , Deana Erdner2, Donald M. Anderson', and Sonya Dyhrman' ' ' State University of New York at Stony Brook, Stony Brook, NY 2 Woods Hole Oceanographic Institution, Woods Hole, MA The toxic dinoflagellate Alexandrium fundyense is widespread in the northeastern part of North America, including the Gulf of Maine, and is responsible for seasonal harmful algal blooms in these regions. Even at low cell densities, A. fundyense produces toxin that can accumulate in shellfish and cause paralytic shellfish poisoning (PSP). PSP can be debilitating or lethal to humans and other shellfish consumers and is a public health concern ( I ). Accurate measurements of A. fundyense distributions, at a low cell density, are critical to continued PSP monitoring and mitigation efforts. Traditional assessments of -4. fundyense cell number rely on microscopic counts of species abundance. However, it is difficult to visually distinguish A. fundyense from other dinoflagellates and other species of Alexandrium. as there are often only subtle mor- phological differences between taxa. Oligonucleotide probe-based 'Corresponding author: sdyhrman(S'whoi.edii methods (2) help to distinguish between Alexandrium and other genera and are commonly employed in the Gulf of Maine to map cell distribution. Although these approaches are useful, we devel- oped and applied an assay that does not require microscopy. Various studies have used quantitative PCR (qPCR) to assay cell numbers of dinoflagellates such as Pfiesteria piscicidn (3). In this study we mapped A. fundyense distribution using a qPCR assay that appears to be specific and sensitive. Primers were designed to amplify a 174 base pair region of the large ribosomal subunit (LSU) gene (4). This gene was chosen because it has been sequenced from many species ofAlexamlrinm. is commonly used for phylogenic analyses (5). and is present in high copy number. Building on previous work (4), the specificity of the LSU primers was tested against DNA extracted from phytoplankton cultures with Qiagen's DNEasy Kit. according to the manufactur- er's instructions, using standard hot start amplification conditions 232 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 500 A 43° 45'N - 43° 10'N- -69° 30'W -68° 55'W _l i -68° 20'W . 14 4 ' •'''•' /••?",-VV Cell Number cells/I 0 0 or non-detectable Figure 1. Spcci/iiit\ <>l ' Uirtic rihosomal snbunil ILSU) gene primer?, ami a imii> «t AlcxanJrium fundyense distribution. (At The arrow indicates a 174-hp fragment of the LSU gem: as shown In tin- Illtl-hp laddci Amplification occurred nnl\ in strains of A. fundyense that are present in the Gulf of Maine (GTCA28. CB30I, GTPPOIl. Amp/iln ation c/icl nut incur in Alexandrium \tiams I rum other pun-. «f the world (SP3B5. AL8T. TN9A): nor did amplification occur in oilier Alexandrium species \nch n\ A. ostenfeldii ami A. andersoni that could occur with A. fundyense in the Gulf of Maine (A. ostenfeldii, TC02). Other Jinoflagellate species also did not amplify (CCMPIV37, SA2. Karenia breve, Prorocentrum minimum, GPES22). (B> A map of A. fundyense distribution in surface water of the Gull "/ Manic during a cruise from 2V May to 6 June 21103. and a 60 °C annealing temperature. Under these conditions ampli- fication occurred only with A. finnlvfiisc isolated from the Gulf of Maine (Fig. 1A). Field samples were collected from surface water along transects in the Gulf of Maine from 29 May through 6 June 21)03. At each station, 4 1 of surface water was collected, prescreened through a 64-ju.m sieve, and collected on a 15-jum filter. The samples were each extracted with Qiagen DNEasy kit. Using qPCR, the number of cells in a field sample was deter- mined. In this study, qPCR was performed using Strutagene Bril- liant SYBR Green QPCR Master Mix. and a fluorescence thresh- old was set by the analytical software for the BioRad iCycler. The PCR cycle during which this threshold was crossed for each sample designated the Cr. Sample C , can be compared to the Cr of standards with a known cell count to specify the number of cells present in the sample (4). To ensure that different strains of A. fundyense have the same copy number of the LSU gene, standard curves were built from two Gulf of Maine strains in culture (GTCA28 and CB301). Both strains resulted in similar standard curves (v = -2.5281.V + 30.664, R2 = 0.4514 for the first strain and v = -2.0291* + 27.6 1 , R2 •= 1 for the second strain). A dilution series of a known number of cells was also analyzed with a field sample matrix. No significant PCR inhibition of the target was detected from extra- neous DNA in the field samples. The number of cells detected in the Gulf of Maine ranged from I to 35 cells per liter (Fig. IB). This range is similar to the range of cell numbers calculated from other methods (live counts done during the cruise and oligonucleotide probe counts [Anderson, unpubl. data]), which we did not expect to be exactly the same since the samples were taken from different Niskein bottles and processed differently. Of the stations tested, station 86 and off- shore stations 104 and 105 had the highest concentrations of cells. To confirm amplification of A. fundyense from the field samples. PCR products generated from samples 89 and 1 28 were sequenced at the Marine Biological Laboratory. Woods Hole. Massachusetts, using the facility's protocols and were shown to be A. fundyense. In summary. qPCR appears to be a specific and sensitive ap- proach to monitoring the abundance of A. fundyense. This method shows great promise for mapping the A. fundyense populations in the Gulf of Maine. This research was supported by an EPA Star Award through the ECOHAB Program (R-83041501-0) and a NSF-REU site grant (OCE-0097498) to the Boston University Marine Program. Literature Cited 1. Anderson, D. M. 1997. Lininol. OccanoKr. 42: 1009-1022. 2 Anderson, D. M., D. M. Kulis, B. A. Kealer, and E. Berdalet. 1999. / Phycul. 35: S70-883. 3. Bowers, H. A., T. Tengs, H. B. Glasgow, J. M. Burkholder, P. A. Ruhlee, and D. W. Oldach. 2000. Appl. Environ. Microhio/. 66: 4641-4648. 4. La Du, J., D. Erdner, S. Dyhrman, and D. Anderson. 2002. Biol. Bull. 203: 244-245. 5. Scholin, C. A., M. Herzog, M. Sogin, and D. M. Anderson. 1994. ./. Pin-col. 30: W9-10I1. MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY 233 Reference: Biol. Bull. 205: 233-234. (October 2003) © 200? Marine Biological Laboratory Description of Vibrio alginolyticus Infection in Cultured Sepia officinalis. Sepia apaina, and Sepia pharaonis C. R. Songster1 and R. M. Smolowitz2'* 1 Western College of Veterinary Medicine, Saskatoon, Canada ' Marine Biological Laboratory, Woods Hole, MA Cuttlefish of the genus Sepia (S. officinalis, S. apama. and S. phanumisl have been cultured at the Marine Resources Center (MRC) of the Marine Biological Laboratory since 1992. The objectives of this retrospective study were to identify common causes of morbidity and mortality in the cuttlefish populations maintained at the MRC. and to describe the histological appear- ance of those lesions. Such information can be used in developing more effective methods of diagnosis, prevention, and treatment. Necropsy cases were selected for inclusion in this study if bacterial cultures had been obtained at necropsy. Bacterial cultures had been obtained from 53 necropsies performed between March 1999 and June 2003. Those culture samples were taken from the digestive gland, kidney sac. or gonad and were plated on marine brain heart infusion medium ( 1 ). Retrospective examination of archived necropsy cases of Sepia spp. showed that mortality was commonly associated with bacte- rial cultures positive for Vibrio alginolyticus, a marine bacterium routinely found in coastal waters, sediment, and culture systems (2. 3). Of the 53 cases in which bacterial cultures were taken. 33 were positive for V. alginolyticus. Of the 33 animals with cultures positive for the bacterium, archived histological sections were available in 19 cases. The sections were paraffin-embedded fixed tissues, sectioned at 6 /xm and stained with hematoxylin and eosin (4). Examination of these sections showed that the most commonly affected tissues were the kidney, branchial heart appendage, branchial heart, and gill (42%). A high incidence of infection (47%) was noted in the reproductive organs (nidamental gland, accessory nidamental gland, and go- nads). All animals with reproductive lesions were older than 9 months. Of the 19 included cases. 16 (84%) had some form of epidermal ulceration. with 7 classified as moderate to severe, and 9 as mild. No reaction was detected in the digestive gland of any animal. Examination of Gram-stained tissue sections (3) confirmed the presence of gram-negative bacteria in the infected foci. Histopathological examination of sections showed that, based on the appearance of the response as identified in the tissues, the sepiod inflammatory reaction to V. alginolyticus occurred in one of three distinct forms. The first was multifocal, necrotizing, granu- loma-like lesions (0.95-1.4 mm diameter), most often seen in reproductive tissues such as the testicular ducts and accessory nidamental glands (Fig. I A. B). The second form consisted of multifocal. necrotizing, granulomatous-like inflammation that re- sulted in subtle lesions (0.1-0.25 mm diameter) containing small numbers of hemocytes and bacteria, and was predominately found in the gills of affected animals. Finally, multifocal. necrotizing. 'Corresponding author: rsmol@mbl.edu granulomatous-like inflammation of intermediate size was com- monly found in branchial heart and its appendage (0.2-0.7 mm diameter) (Fig. 1C). Severe, necrotizing hemocytic inflammation also occurred in the serosa of various organs including the branchial heart and stomach, which are anatomically suspended in large vascular sinuses. Sim- ilar hemocytic inflammation of the kidney fronds was unique in that the bacteria and associated necrosis occurred primarily at the periphery of the fronds (suspended in the renal sac), while the hemocytic response was located in the vascular/connective tissue core (Fig. ID). Bacteria causing the observed lesions may have accessed the circulation via epidermal ulcerations, but ulceration was not rec- ognized in all animals and was only mild in others, so the potential for other routes should not be ignored. In the circulation, the bacteria lodged in the branchial heart, resulting in foci of moderate inflammation and necrosis. Most other lesions appear to have been spread by bacterial seeding of organs that receive circulation from the branchial heart. Thus, bacteria-laden hemolymph that passed from the heart directly to the gill and branchial heart appendage resulted in foci of inflammation. The branchial heart appendage forms an ultrafiltrate that is discharged into the kidney sac and bathes the kidney fronds; the epithelium of the kidney fronds modifies the filtrate before it exits the body through a renal pore. Bacteria can proliferate in the filtrate and attack the suspended kidney fronds. Hemocytes. which mount the inflammatory re- sponse, migrate from vessels within the connective core of the kidney fronds to the site of infection. These results show that V. alginolyticus is a pathogen of signif- icance in sepoids cultured at the MRC. \'. alginolyticus is common in coastal waters and is therefore likely to be present in the facility's seawater supply (2). This pathogen has been associated with tank surfaces in culture systems (5). Comparisons between bacterial populations on wild-caught and laboratory-reared squid. Lolligiincula hrt-vis. showed that animals reared in the laboratory had higher total numbers of bacteria. The increase was primarily due to Vibrio spp.. including V. alginolyticus (6). In that study, the increase in bacteria was not linked to the ulceration of the epider- mis. Similarly, V. ulginolvticus has caused disease in juvenile red abalone, Haliotis rufescens, but large numbers can be found on the foot of otherwise healthy individuals (5). Such findings suggest that direct infection of the epidermis is an unlikely pathogenesis for V. alginnlvticiis in cuttlefish. Routes of infection probably include infection secondary to ulceration. especially if the injury is caused by jetting into tank walls colonized by the bacteria. V. a/giiiol\tii'iis has also been found on the carapace of copepods (7). 234 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS :-l *1 "i Figure 1. Wn/o/o.ij/Vi// rctutiim n/1 Sepia .v/'/i. M Vibrio alginolylicus. f/Aj Granuloma-like fornnitioti in lite accessory nUlu/ni'iituI xluiiil. (B) Coagulative necrosis and bacteria in the center of the granuloma (1) are surrounded by a layer of dying heinocytcs (2). which is in mm surrounded by a fibrotic heiiiocvtic renction 13). Id Moderate inflammation in the branchial heart is typified by large numbers of bacteria and cellular necrosis (It, and hemocytic infiltration (2). (Dl The periphery of the kidney frond.', are necrotic and bacteria-laden ( I ). while a front of heinocytes infiltrates the tissue from vessels in the core (2). A normal tissue frond is also present (3). Crustaceans, including amphipods, are the primary food fed to cuttlefish cultured at the MRC. and thus may be an important source of infection. Finally, over half of the studied cuttlefish with reproductive-associated lesions were at or near the upper limit of their life span (8), suggesting that senescent tissues are more susceptible to this pathogen and are a potential point of initiation for a systemic infection. This retrospective study shows that systemic V. alginolyticus is a common pathogen in Sepia spp. cultured at the MRC. A number of potential routes of infection exist but, as in most aquatic animal diseases, stress and husbandry likely play an important role in the occurrence of disease. Better sepoid husbandry methods may help reduce or prevent this disease from occurring. Additionally, the data collected in this study indicate that, when systemic bacterial disease is suspected in Sepia spp., the branchial heart and branchial heart appendage should be examined histologically, and bacterial cultures should be obtained from the renal sac. Literature Cited 1 Wenuganen, S., R. Bullis, and R. Smiilowitz. 1997. Biol. Bull. 193: 269-27(1. 2. Bulows, A., H. G. Truper, M. Dworkin, \V. Hardner, and K. Schlei- fer. 1992. Pp. 2995-2996 in The Proka notes. 2nd ed.. Springer- Verlag, New York. 3. Hormansdorfer, S., H. \Ventges, K. Neugebaur-Buchler, and J. Bauer. 2(100. Int. .1. H\K. Environ. Health 203: 169-175. 4. Humanson, G. L. 1962. Pp. 3-126 in Animal Tissue Techniques. W. H. Freeman, San Francisco. 5 Elston, R.. and G. S. Lockwood. 1983. / Fish Dis. 6: 1 1 1-128. 6 Ford, L. A., S. K. Alexander, K. M. Cooper, and R. T. Hanlon. 1986. ./. Im-ertehr. Puthol. 48: 13-26. 7 Dumontet, S.. K. Krovacek, S. B. Svensnn, V. Pa.squale, S. B. Balnda, and G. Figliuolo. 2000. Coin/), linnntnol. Microbiol. Infect. Dis. 23: 53-72. 8 Forsythe, J. W., R. H. De Rusha, and R. T. Hanlon. 1994. / Zoo/. Loud. 233: 175-192. MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY 235 Reference: Bio/. Bull. 205: 235-236. (October 2003) 1 21103 Marine Biological Lahoiaioi\ Detection of Edwardsiella Infections in Opsanus tan by Polymerase Chain Reaction Kn-stal D. Baircl' ', Hemant M. Chikarmane1'3, Roxanna Smolowitz1'*, and Kevin R. Uhlinger' ' Marine Biological Laboratory, Woods Hole, MA : Barnstable County AmeriCorps Cape Cod, Barnstable, MA "' Cape Cod Community College, W. Barnstable, MA Opsanns tan. the oyster toadtish. is an important laboratory animal used in hearing and balance research at the Marine Bio- logical Laboratory (MBL) (1). Wild-caught and cultured fish are maintained year-round in both recirculation and flow-through tanks in the Marine Resources Center (MRC) at the MBL. A major cause of disease in toadfish held at the MRC is Edwardsiella tarda ( 1. 2). A member of the Enterobacteriaceae, E. tarda is a natural inhabitant of fresh and marine water and causes gastrointestinal and extraintestinal disease in humans (3). Published reports of fish disease caused by E. tarda involve cultured \\ arm-water fish, but the disease can also affect cold-water salmon (4). Poor water quality, high water temperatures, feces. and decaying organic matter likely contribute to the onset and severity of the disease and probably allow for occurrence and proliferation of the bacteria in the fish's environment. These factors, combined with capture and holding-induced stresses, may account for the high levels of dis- ease caused by E. tarda in the toadfish in our facility. Currently, the only diagnostic test available for identification of E. tarda in diseased fish is bacterial culture and identification using traditional biochemical tests. At the MRC. these tests are sent to an outside laboratory, and the process takes about three weeks. Treat- ment is often attempted before verification that the lesions identi- fied at necropsy were caused by E. tarda. Using the PCR methods described here, we can specifically and rapidly identify E. tarda, resulting in timely and appropriate management procedures and treatments for infected fish. The polymerase chain reaction (PCR) is a DNA-based method that can be used for the quick and sensitive detection of microor- ganisms in both antemorteni and necropsied tissues. PCR primers specific for E. tarda isolates from Japanese eels have been derived from an anonymous species-specific sequence (5) or from the hemolysin gene (6). Here we describe PCR primers based on Eilmirdxiella small subunit (ssu) RNA genes for direct identifica- tion of E. tarda. Primer development will be described in detail elsewhere. The type strains E. tarda ATCC 15947 and E. icrahiri ATCC 33202 were obtained from the American Type Culture Collection. E. ictalnri is an important pathogen of cultured catfish and pro- duces very similar lesions to those caused by E. tarda in toadfish. In addition. E. tarda has been shown to cause disease in catfish (7). Cultures were grown in Difco marine brain heart infusion broth. One milliliter of the liquid broth culture was transferred aseptically to 1.5-ml microfuge tubes, which were then centrifuged at 2790 X g for 4 min to pellet cells. Pellets were resuspended in 0.6 ml of lysis buffer ( 10 mM Tris-Cl pH 8.0. 5 mM EDTA. and \c7c SDS). * Corresponding author: rsmol@mbl.edu. Proteinase K (50 p.g) was added, and tubes were incubated over- night at 50°C. Genomic DNA was precipitated by the addition of 0.55 ml of isopropanol: the liquid portion was removed com- pletely. The DNA pellet was rinsed twice with 0.8 ml of 70% ethanol. After the ethanol was evaporated, the DNA was dissolved in 0.8 ml of TE (Tris-EDTA: 8). Forward primer Eta 1 -363 f (5'-GTG TRC GTG TTA ATA GCA-3') was designed to amplify E. tarda from human sources (biotype 1). represented by the type strain ATCC 15947. Forward primer Eta2-351 (5'-TAG GGA GGA AGG TGT GAA-3') was designed to amplify E. tarda strains isolated from fish (biotype 2). An Edwardsiella genus-specific reverse primer Edwsp-780r (5'- CTC TAG CTT GCC ACT CTT-3') was used with the forward primers. PCR amplification was performed with an Applied Biosystems GeneAmp 9700. The reaction mixture (10 /n.1) contained IX Taq polymerase buffer (Promega). 1.5 mM MgCK. I mM dNTP mix. 100 nM each of forward and reverse primer. 0.5 units Taq poly- merase (Promega) and template DNA. The thermal cycle profile commenced with an initial denaturation for 1 min at 94°C; 30 cycles of denaturation ( 1 min at 94°C). annealing ( 1 min at 58°C), and extension ( 1 min at 72°C); and a final extension at 72°C for 7 min. Amplification products were electrophoresed in a 1 .5% aga- rose gel (Fisher Scientific) in 0.5 X Tris-Borate-EDTA (TBE) buffer (8). Gels were stained with 1 /ng/ml ethidium bromide and digitally photographed. Images were manipulated in Adobe Pho- toshop. Some amplifications were carried out directly from bacte- rial colonies, in which case the initial denaturation time in the PCR profile was increased to 5 min at 94 °C. The results, as determined by electrophoresis. showed that DNA of E. tarda and E. ictaluri could be distinctly amplified by the appropriate primers (Fig. 1). The Etal-363f-Edwsp-780r primers amplified E. tarda ATCC 15947. yielding a product of 216 bp. and did not amplify E. ictalttri ATCC 33202 (Fig. 1A. lanes 1-4) or MRC fish isolates biochemically identified as E. tarda (not shown). As would be expected. Eta2-351f-Edsp-780r failed to amplify both type strains (Fig. 1A, lanes 5-8). However. Eta2- 351f-Edwsp-780r primers amplified the E. tarda fish isolates (Fig. IB. lanes 1-6). demonstrating that the strains isolated at the MRC were clearly of fish origin, and biotype 2. The biotype 2 amplifi- cations were performed directly from bacterial colonies, showing that direct identifications were indeed possible. Future work w ill involve development of a direct assay method for E. tarda from both postmortem toadfish tissues and. more importantly, antemortem tissues. The ability to amplify E. tarda strains from toadfish is the first step to being able to understand, isolate, and control future outbreaks of E. tarda. 236 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS A B M1234M5678 M123 45 678 Etal-363f Eta2-351f Eta2-351f Figure 1. Gel electrophoresis of PCR amplification products from Edwardsiella. (Panel A) Amplifications from purified genomic DNA. Lanes 1. 3. 5, 7 are E. tarda ATCC 15947. Lanes 2, 4, 6, 8 are E. ictaluri ATCC 33202. (Panel B} Amplifications from bacterial colonies. Lanes 1-6, E. terdafish isolates. Lane 7. Vibrio sp. negative control. Lane 8, no template control. Ed\\'sp-780r was used as the reverse primer in all cases. Foncard primers are indicated. R. Smolowitz acknowledges support from NIH/NIDCD grant 5P01 DC001837-09. H. Chikarmane acknowledges support from the Wilkens Foundation and the Cape Cod Community College Educational Foundation. Literature Cited 1 Smolowitz, R., and R. Bullis. 1997. Biol. Bull. 193: 270-271 2 Wenuganen, S., R. Bullis, R. Smolowitz, and E. Barbieri. 1997. Biol. Bull. 193: 269-270. 3. Janda, J. M., and S. L. Abbott. 1993. Ctin. Infect. Dis. 17: 742-74X 4. Plumb. J. A. 1999. Pp. 479-490 in Fish Diseases and Disorders Vol. 3, Viral, Bacterial, and Fungal Infections, P. T. K. Woo and D. W. Bruno, eds. CABI Publishing, New York. 5. Aoki, T., and I. Hirono. 1995. Asian Fisheries Society Special Pub- lication. 10: 135-146. 6. Chen, J. D.. and S. V. Lai. 1998. Zo,,l. Stud. 37: 169-176. 7. Darwish, A., J. A. Plumb, and J. C. Newton. 2000. J. Aquat. Anim. Health 12: 255-266. S. Sambrook, J., and I). Russell. 20(11. Molecular Cloning: A Labora- ton Manual. 3td ed. Cold Spring Harbor Laboratory Press. New York. Reference: Biol. Bull. 205: 236-237. (October 2003) © 2003 Marine Biological Laboratory Catalase in Microsporidian Spores Before and During Discharge Earl Weidner1 and Ann Findley2 1 Louisiana State University, Baton Rouge, LA • University of Louisiana at Monroe, Monroe, LA The noted parasitologist Horace W. Stunkard characterized mi- crosporidians as among the most widespread of parasites, possess- ing significant survival adaptations, a consequence of their long- term host associations ( 1 ). One of the more obvious adaptations within this group is the extrusion apparatus of the infective spore stage. This apparatus consists of an aperture, a polaroplast mem- brane, a polar filament, and a posterior vacuole, or swelling or- ganelle. The energy source for the firing of this apparatus is thought to reside in the posterior vacuole (2). In an earlier report (3), we inuicawd that the posterior vacuole had properties of peroxisomev which function primarily to process very long chain fatty acids (VLCFA) with the assistance of key enzymes, acyl- CoA oxidase (ACOX) and catalase. Although peroxisomes are characteristically found in aerobic cells with mitochondria, there is evidence that peroxisomal enzymes occur in the amitochondriate microsporidian Spragttea lophii. In particular, biochemical assays and western blot analyses indicate that ACOX and catalase are discharged from the spores of S. lophii (Findley. unpubl. data). Here, we report the localization of catalase within the spore cell. We used a cytochemical protocol described by Angermuller and Fahimi (4). In brief, spores were prefixed in 2.5% glutaraldehyde (to eliminate resident peroxiase activity) and were subsequently incubated in Tris buffer (pH 10.5) in the presence of diaminoben- zidine (DAB) and hydrogen peroxide. The cells were then post- fixed in 29r osmium tetroxide and processed for transmission electron microscopy. MOLECULAR BIOLOGY. PATHOLOGY. AND MICROBIOLOGY . 237 % ***N *«**$ ! '•£* • **» %>?*$•• B Figure \. Linuli:; Spraguea lophii ctitalase activity. (A} Electron micrograph of alkaline-DAB reaction confined to the posterior \ucnole. Arnws v/iou reaction product in vacuole. IB> Similar ima^c o\ DAB reaction extended to discharged spore tube (arrows). Discharged tube at /our;- ni;lit portion of unaxe has DAB reaction on outside. lC> Control preparation without DAB or hydrogen peroxide s/iuir.s no reaction in the spore posterior vacuole. Bar scales represent 0.5 fj.ni. Electron micrographs revealed DAB reaction product in the posterior vacuole of the spores before and during its discharge (Fig. 1A). As the invasion tube emerges from the tiring spore, the DAB reaction is associated with the inner surface of the tube (Fig. 1 B ) and also along the outer surface of the discharged tube. In the control preparations without DAB and hydrogen peroxide, there was no catalasc-induced reaction product in the spore posterior vacuole (Fig. 1C). In addition to the peroxisomal enzymes, spores of S. lophii extrude the VLCFA nervonic acid into the external medium. Nervonic acid is a characteristic peroxisomal component, and the data indicate that it is discharged from the spores along with the catalase and ACOX enzymes. Of particular interest is the sizeable drop in nervonic acid levels that occurs during and utter discharge of 5. lophii spores (Findley, unpubl. data). These data indicate that DAB-labeled catalase is initially re- stricted to the posterior vacuole. subsequently moves to the ex- truding polar tube during spore firing, appears on the outside of the discharged tube, and finally diffuses to the extracellular medium that bathes the discharged sporoplasms (not shown). The results reported here also support the Lorn and Vavra model of spore discharge (2). In this model, the posterior vacuole swells signifi- cantly after the spore is activated, the extrusion apparatus everts, and a membraneous sac forms at the end of the discharged tube to accommodate the exiting spore cell, or sporoplasm. If the Lom and Vavra model is correct, the membrane of the extruded sporoplasm may be derived from the extrusion apparatus and may therefore be reversed. Indeed, studies with cytoplasmic protein probes indicate that in discharged sporoplasms. the mem- brane orientation is reversed as proposed (DeGiorgis and Weidner. unpubl. obs.). Literature Cited 1. Stunkard, J. W., and F. E. Lux. 1965. Biol. Bull. 129: 371-385. 2. Lom, J., and J. Vavra. 1963. Ada Protocol. 1: 81-92. 3. Weidner, E., and A. Findley. 20(12. Iliol. Bull. 203: 212 4. Angermuller, S., and H. I). Fahimi. 1981. Histochem. 71: 33-44. 238 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Binl. Bull. 205: 238-239. (October 2003) © 2003 Marine Bioloj.'f_-:!l !,.iUir:uory <;<• ivvth of a Salt Marsh Invertebrate on Several Species of Marsh Grass Detritus A. M. Agnew', D. H. Sltull''*. and R. Bitchsbainir ' Gordon College, Wenhain, MA 2 Massachusetts Audubon Society, Wenluuu. MA Salt marshes are important and productive ecosystems. Marsh grasses fuel coastal ecosystem production, and marsh invertebrates convert abundant decomposing marsh grasses into biomass avail- able to higher trophic levels. Changes in climate, land use. nutrient input, and introduced species potentially threaten this ecosystem, however. An accelerated rate of sea-level rise has allowed cord grass (Spartina alterniflora) to migrate shoreward ( 1 ). Meanwhile, the high-marsh invasive reed Phragmites australis has expanded seaward, reducing the extent of indigenous high-marsh grasses such as Spartina patens and Distichlis spicata (2). Although changes in invertebrate community structure have been observed following Pliragiiiitex invasion (3), less is known about its effect on ecosystem function. How would changes in the species com- position of marsh grasses affect food supply for higher trophic levels? Are all species of marsh grass equally nutritious for the invertebrates that feed on them? These questions are closely re- lated to some of the primary goals of the Plum Island Ecosystem Long Term Ecological Research (PIE-LTER) program, which is concerned with the processing of organic matter within the salt marsh. To address some of the above questions, our study investigated the growth rates of the salt marsh amphipod Orchestia grillus. one of the most abundant and best-studied detritivores at our study site (4), feeding on four species of marsh grass. Two sites were studied. Clubhead Creek and Greenwood Creek; the latter is nutrient en- riched due to its proximity to a sewage outfall. The purpose of our research was to examine the nutritional quality of the detritus in terms of O. grillus growth rates and nitrogen and carbon content. The four plant species studied were Spartina alterniflora (cord grass). Spartina patens (marsh hay). Distichlis spicata (spike grass), and Phraginilex uustralis (common reed). S. alternifli>ra is the dominant low-marsh species. S. patens and D. spicata are native high-marsh species, and P. australis is a high-marsh inva- sive. Samples of one-year-old standing dead grasses and young new- growth grasses were collected from each site from 2 to 6 June 2003. We sampled two differently aged grasses to assess how the quality of detritus changes over time. Old samples were sorted, cut, and frozen at -20 °C. Young grass samples were dried at 70 'C for 24 h, soaked in seawater for 63 h, and then dried again at 70 °C for 24 h before freezing, to simulate the formation of fresh detritus. Organisms collected from the marshes were allowed to acclimate to laboratory conditions in a large tank containing marsh wrack for 3 days. At the start of the experiment cm 22 June 2003, single organisms were weighed and placed into petri dishes containing a single type of grass Irom each site. Ten replicates were designated * Corresponding iiutht-r: dshull(5'gordon.edu for each grass-species/age/site treatment. These were placed at random over a numbered grid on a laboratory bench. Grasses were changed about once a week, and dishes were kept wet by the addition of filtered seawater from the marsh every few days. Growth data for O. grillus were collected weekly by removing, patting dry, and weighing individuals, and then returning them to their petri dish. These data were normalized by dividing measure- ments by the initial size of each individual. Mortality data were collected daily, but all organisms that died before the end of the experiment were eliminated from growth data sets. The experiment was allowed to run for 38 days. Within a few weeks, nearly 80% of the organisms feeding on fresh grass were dead, and it became clear that those individuals remaining alive in these treatments were not growing. This pattern may have been due to the presence of high phenolic concentrations in the fresh marsh grass detritus. Phenolic concentrations (deter- mined by absorbance of methanol extracts at 320 nm) were sig- nificantly higher in the fresh detritus, as demonstrated by two- factor ANOVA with grass species and age as fixed factors (F, 1.241 = 33 1 . P < 0.000 1 ). and Orchestia mortality rates were significantly correlated with phenolic concentrations (R2 = 0.56. P = 0.01 ). For these reasons, we focused our analysis of growth rates on the year-old detritus. The mean growth rates for O. grillus consuming year-old detri- tus varied with marsh grass species. Rates were highest for am- phipods consuming D. spicata (O. grillus increased in size by 40% to 50%) and decreased in the order of S. alterniflora, S. patens, and P. australis (Fig. 1A). Growth rates were determined from the slope of organism size versus time plots by linear regression, and rates among sites and species were compared by two-factor anal- ysis of variance, with collection site and marsh grass species as fixed factors. Data were log-transformed prior to analysis to cor- rect for heteroscedasticity. No differences in growth rates between the two sites were found (/•", , 4l,, = 0.088. P = 0.77). Variation in the growth rates of O. grillus on different marsh grass species was highly significant (F,,^,,, = 5.7. P = 0.002), with no significant interaction (F|141)| = 0.12. P = 0.95). Post hoc comparisons indicated that the growth rates of O. grillus on P. australis were significantly lower than those on S. alterniflora and D. spicata (Scheffe F-test. P < 0.01 ). Variation in O. grillus growth rate was not correlated with the percent carbon content of the detritus (R~ = 0.052, P = 0.59). However, linear regression analysis indicated that nitrogen content in year-old detritus accounted for approximately 70%> of the vari- ation in growth rates (Fig. IB, F,,.,,, = 13.7. P = 0.01 ). Our results indicate that, when compared to the three native marsh plant species studied, the invasive species, P. australis. is a relatively poor food source for O. grillus. A diet of salt marsh ECOLOGY AND POPULATION BIOLOGY 239 1.7 1.6 H fl> 15- N '-a '35 TJ I | 1.3 i 1.2 - 1.1 - 10 20 Time (d) A • S. alterniflora o S. patens • D. spicata o p. australis / Orchestia grillus/cfrfmg OTI year-old detritus over the course of the growth experiment. Data from the n\-o sampling sites were pooled. Locations of points along the \-a\i.\ were shivered to improve readability: Error bars represent 9.5% confidence limits. (B) Averaged growth rates for O. grillus consuming \ear-old detritus species from both sites versus nitrogen content of the detritus. species with higher nitrogen content, such as D. spicata. resulted in significantly higher growth rates for this organism. Furthermore, the observed seaward encroachment of P. australis is slowly pushing out D. spicuui. the species that we found to have the highest nutritional quality (2). Given a continued shoreward mi- gration of S. altemiflora due to sea-level rise and the seaward spread off. australis. the overall food quality of marsh detritus for this invertebrate could decline. This suggests that changes in marsh grass species composition could affect higher trophic levels in this salt-marsh ecosystem. This research was supported by an NSF-REU fellowship through the Plum Island Ecosystem LTER to AMA. Literature Cited I Donnelly. J.. and M. Bertness. 2001. Proc. Nail Acad. Sci. USA 98: 14218-142:3. 2. \\indham. U and R. Lathrop. 1999. Estuaries 22: 927-935. 3. Talley, T. S.. and I,. A. Levin. 2001. Biol. Invasions 3: 51-68. 4. Lopez, G., J. S. Levinton. and L. B. Slobodkin. 1977. Oecologia 30: 111-127. Reference: Bi»l. Bull. 205: 239-241. (October 2003) © 2003 Marine Biological Laboratory Patterns of Sedimentation in a Salt Marsh-Dominated Estuary Jason R. Cavatorta1'*, Morgan Johnston2, Charles Hopkinson3, and Vinton Valentine'" ' Ainherst College. Amherst. MA ' The Pennsylvania State University, University Park. PA ' Marine Biological Laboratory, Woods Hole. MA Salt marshes survive sea-level rise by an accompanied increase in elevation. This increase in elevation results from the accumu- lation of organic matter produced by marsh plants and of sediment transported to the marsh platform by tidal activity, storm events, and rafts of ice ( 1 ). Sea level is currently rising and is predicted to continue doing so (2): if marshes cannot increase in elevation at an equal or greater rate, inundation may eventually occur (3). Marsh inundation initially occurs by an increase in internal marsh ponds (4). We traced changes in internal marsh ponds and identified the spatial distribution of suspended solids in tidal waters and marsh sedimentation in the Parker River estuary in northeastern Massa- chusetts. Changes in marsh ponds were qualitatively assessed by com- paring a 1953 topographic map of the area, compiled from early 1950s aerial photography, with 2001 color orthophotography. The topographic map was obtained from the National Oceanic and Atmospheric Administration (NOAA) and georeferenced (i.e.. ' Corresponding author: JRCavatortafs amherst.edu 240 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS given a geographic location) using ArcGIS (version 8.3.0) soft- ware. We determined 'iition of total suspended solids (TSS) along the main a> :c estuary as well as along three third-order tidal creek-, v iihm marshes of the lower estuary. We collected known v< s of water at high water on a spring tide adjacent to where s limentation was examined and filtered it through pre- weighed 0.7-/j,m glass tiher filters. We deployed 94 sediment traps along the estuary; these consisted of pre-weighed 9-cm glass fiber filters secured with rubber bands to upside-down plastic petri dish covers (5). Marsh grass was cut away, and a galvanized nail was inserted through the petri dish covers to secure the structures to the marsh. At West Creek. Club Head Creek, and Nelson Island Creek, we set up transects of sediment traps perpendicular to a mosquito ditch and a first-, second-, and third-order stream (Fig. 1, inset). Each transect consisted of three sites: 4, 20, and 50 m in from the creek edge to investigate the amount of sediment reaching the marsh interior. Eleven sites were also selected along the Parker River (Fig. 1 ). We deployed two replicate sediment traps at each site. We recovered two sets of sediment traps on 2 and 23 July 2003, after they were exposed to several spring tidal cycles. Several samples (n = 5) with anomalously high weight increases were moderated by rinsing with distilled water to reduce high salt concentrations. Comparisons were made using a single factor analysis of vari- ance (6). Post-ANOVA pairs were analyzed using Student's / test with a Bonferroni adjusted alpha value to account for multiple comparisons. Internal marsh ponds have increased in number and area since the early 1950s. Many areas that are largely ponded today (several of which cover thousands of square meters) were not ponded on the 1953 topographic map. Few ponds appear (during either year) in the upper 10 km of the estuary. Broad marshes adjacent to the lower third of the estuary are densely ponded. Pond drainage and vegetative recovery seem to have occurred only rarely. TSS along the main axis clearly show the influence of Parker River runoff: solids in the water column are most concentrated in the upper estuary and decrease towards the sound (P = 0.003), which receives regular exchange with the ocean (Fig. 2B). TSS were also low at the mouths of tidal creeks adjacent to the sound and increased in the second- and third-order reaches, where TSS is about double the amount in the sound (Fig. 2 A). Nelson Island Creek received 0.050 g of sedimentation per 9-cm filter, significantly more than West Creek, which is located farther upstream (P = 0.009). Club Head Creek, which is located between them, received an intermediate amount of sediment (Fig. 2E). We did not observe a strong relationship between TSS and sediment accumulation: their maximum values, however, overlap somewhat in the creeks (Fig. 2A. C). The slight overlap is not surprising since TSS measurements were taken only once. More TSS measurements would perhaps yield mean values more strongly correlated with sedimentation. We did not observe a significant difference between sediment accumulation 4 m and 50 m from the creek. The three- creeks considered in this study are fed from water with relative's low TSS. Our data indicate that sediment accreting on the marsh platform may be generated within the creeks them- selves, presumably from eroded riverbanks. Slumping banks de- liver large amounts of sediment to stream channels, where it may become suspended and deposited on the marsh surface during flood tides. Examination of 1953 and 2001 imagery supports this conclusion, as creek bank erosion is pronounced near the mouths of these creeks, especially at Nelson Island Creek where the most sedimentation was observed. The spatial distributions of sedimentation and TSS along the main axis of the estuary are complex, but generally decrease with distance down the estuary. Parker River sediments seem to origi- nate at the headwaters of the river and to remain in its system. This sediment source may explain why internal ponds are rare along the river's upper reaches (although a higher concentration of mosquito ditches, dug to drain the marsh, also prevent pond formation). Greater sedimentation may not have been observed along the Parker because of the brevity of the study and the fact that summer is typically the season with the lowest sediment transport (5). Generally low sedimentation may also explain why decreasing sedimentation was not measured farther from the creek bank. Sediment sources from other nearby creeks may also have inflated interior marsh accretion. Because of limited sediment source in the creeks, belowground plant production may be more important than sedimentation in marsh accretion, even though softer organic sediments may be greatly compacted. This supposition is corroborated by the fact that the Parker River marsh sediments consist of 55% organic matter or more. Because plant productivity is higher on creek banks (7). depressed internal areas may develop because subter- ranean accretion rates are slower away from the creek bank. The increase in size and number of internal ponds, which coincides with observed patterns of sedimentation, may be a useful indicator that marshes are not maintaining elevation relative to a using sea level. Because the marshes studied are typical of New England macro-tidal marshes, similar marsh degradation is likely occurring in other areas. This study received support from The Woods Hole Marine Sciences Consortium, the Plum Island Sound LTER NSF #OCE- 9726921. and the Atlantic Coast Environmental Indicators Con- sortium EPA grant number R828677. Special thanks to Hap Garritt and W. McDonald Lee for their assistance. Literature Cited 1 Gleason, M. L., D. A. Elmer, N. C. Pien, and J. S. Fisher. 1979. Estuaries 2(4): 271-273. 2 Houghton, J. T., Y. Ding, D. J. Griggs, and M. Noguer. 2001. Climate Change 2001: The Scientific Basis. Cambridge University Press. Cambridge. England. 3. Orson, R., W. Panageotou, and S. Leatherman. 1985. J. Coastal Res. 1: 29-37. 4 Kearney, M. S., R. E. Grace, and J. Stevenson. 1988. Geogr. Rev, 78: 205-220. ?. Reed, D. J. 1989. Esruanes 12(4): 222-227. 6. Rooth, J., J. Cornwell, and J. Stevenson. 2003. Extuaries 26: 475- 483. 7. Frev, R., and P. Basan. 1985. Pp. 222-301 in Coaxial Setlimentaiy Environments, R. Davis. Jr.. ed. Springer- Verlag, New York. ECOLOGY AND POPULATION BIOLOGY 241 14 i 12 - t- T 10 - 1 01 g E "f fi "*" e/j D (/) -±- — D 1 2 3 Stream Order M Figure 1 Figure 2A 50 T c 0 06 -| . 45 • ^ 40 "a 35 — ja ft — EO) n AC . _/\ \ ^ ~o 0 04 T f T 2P*»i^ \ E 30 •O 25 "V^^JZ ^y" y> ™ rh 1 CO 20 CO '5 |_ 10 5 0 I \ ^** c 3 0.03 • | | 0.02 - o 75 ° 001- — rTT ^^!>^^ Vn"^^^i 1 9 14 19 24 Distance Down Estuary (km) ° 0.00 -I — ' • — i — • • — i — • • — i — ' • — i D 1 2 3 Stream Order Figure 2B Figure 2C 9 14 19 Distance Down Estuary (km) 0.07 3 0.06 i" °-°5 re 0.04 | 0.03 3 0.02 % 0.01 1 0.00 we CHC Creek Name NIC Figure 2D Fimire 2E Figure 1. Map with while crosses showing locations of sedimentation traps. Inset is a close-up of West Creek. Figure 2. Total mean \etlimeni accitnutlateit on seilimeni irap.\. uiul total suspended solids iTSSl of selected locations along the estuary. "D" as an a.\i.\ title refers to mosquito ditch, "M" refers to creek mouth. (A) TSS of creek order, averaged from three creeks adjacent to estuary. IB) TSS down estuary starting at the dam on Central Street in Byfield. Mean total of nonorganic sediment accumulated plotted aizuimt individual creek, by (C} order of creek and iDl distance down Parker River Estuary. (E) Mean total of nonorganic sediment accumulated in each of the three creeks adjacent to estuary. Error bars represent standard error. 242 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biot. Bull. 20v 242 !43 (October 2003) © 2003 Marine Biolo ':> uory Mu!- Approaches to Tracing Nitrogen Loss in the West Falmouth Wastewater Plume T. Thoins' , A. E. Giblin'. and K. H. Foreman2 1 Colorado College, Colorado Springs, CO ' Marine Biological Laboratory, Woods Hole. MA Wastewater transported through groundwater to receiving estu- aries is a major contributor of nitrogen (N) in densely settled watersheds, and contributes to coastal eutrophication (1-3). Al- though nitrate removal by microbial denitrification, adsorption, and uptake by plants is well documented, little is known about the nitrogen loss in the vadose zone and aquifer (1, 4). This project focused on the fate of nitrogen leaving the Fal- mouth Wastewater Treatment Plant (FWTP) in Falmouth, Massa- chusetts. The plant lies on the Snug Harbor sub-watershed of West Falmouth Harbor (WFH). and the wastewater it discharges perco- lates through 25-30 m of vadose zone before meeting the aquifer and traveling in groundwater to WFH (3). This site provides a good model for a study of N loss due to the well-documented history of wastewater discharge and previous research involving N loading and removal. The two principal objectives of this study were ( 1 ) to assess the efficiency of N removal as wastewater travels from FWTP to WFH, and (2) to determine where N loss is occurring. To satisfy our first objective, we quantified N loss using two independent methods: a mass balance calculation, and a conservative tracer technique. We then compared these results to a previous study conducted in 1999 (3). The site of N removal was examined using the same conservative tracer technique applied to samples within the path of the plume in the aquifer. We further investigated whether denitrification was the primary mechanism responsible for N loss in the aquifer by using dissolved N^ and Ar gas analysis (5). With the development of the membrane inlet mass spectrometer, it has recently become possible to measure dissolved N2/Ar ratios with sufficient precision and accuracy to estimate denitrification (6). We measured dissolved inorganic nitrogen (DIN; nitrate + ammonium), and boron (B) in wastewater effluent at FWTP and in monitoring wells located within and outside of the treatment plant. Boron, a component of laundry detergent, can be used as a con- servative tracer for wastewater (7). We also sampled groundwater about to enter WFH within the boundaries of the Snug Harbor watershed. Samples were taken from 0.5 to 1.5 m below the sediment surface, using well-points. These shoreline samples were analyzed for DIN. B, and dissolved N-, and Ar gas concentrations. Samples with elevated salinity, indicating seawater intrusion, were rejected. Nitrate, ammonium, and B concentrations were measured using standard colorimetric techniques. We compiled discharge data for the FWTP from its establish- ment in 1987 to the present, and observed that loading increased during the first .7 years of operation (Fig. 1A). Based upon an estimated 1 ; ear groundwater travel time from the center of the plant to WFh 3), we used the 1993 discharge value of 13,177 kg N/y to compare \\:th the loading now reaching the harbor. To 16000 14000 12000 10000 5 >, 1 2000 P .. • . • • • = 1500 z 01 1000 500 0 6000 4000 2000 0 Shoreline Samples. South to North 200 ui 150 5 100 50 0 • Samples Atong Rume D Mean of Multi-depth wells * n » D n * ) 200 400 600 800 1000 1200 Distance from FTP(m) Kigure 1. Loading Jala from the Falmonth Wastewater Treatment Plant tFWTP), and data from the wells and shoreline well points down gradient of the plant. (A) Monthly (line) and annual (filled squares) discharge (DIN concentration in effluent * flow), in kg N. from FWTP. 1987-2002. (B) Comparison of measured DIN to expected DIN at each shore \\ell-poinl based IHI dilution correction estimated from the DIN/B ratio. (C) DIN/B ratios from the effluent in the last holding pond from FWTP, front monitoring wells located 100 to 460 in down gradient, and from average of shoreline samples. Squares represent the average value for replicate samples of the effluent in the last holding poiul and multi-depth wells. ECOLOGY AND POPULATION BIOLOGY 243 calculate the DIN entering WFH, we used the average DIN con- centration ill' shoreline groundwater. 208.5 H.M (n = 28, se = 27.8). multiplied by a literature value for the groundwater Hux (1737 * 103nrVy) (3). Thus. 5070 kg/y of DIN is currently dis- charging from the Snug Harbor sub-watershed. To isolate DIN originating at FWTP. we subtracted 554 kg of DIN reaching the shore derived from fertilizer, atmospheric deposition, and septic wastewater (3). Thus, we calculate that 66% of the DIN discharged from the FWTP in 1993 was lost en route to the harbor. The conservative tracer method produced similar results. Changes in the DIN/B were used to calculate N loss based on the following formulas: DIN expected (at each wellpoint) = (DIN effluent * B measured )/B effluent %DIN loss = (1 - (Average (DIN measured)/ Average (DIN expected))) * 100 Expected and measured concentrations refer to shoreline samples (Fig. IB), and DIN and B effluent concentrations (2017 juM and 13.3 fj.M respectively) were measured from the last holding pond at FWTP. This method estimates that 56% of DIN is lost. The method corrects for dilution, but it does not account for N input from fertilizers or atmospheric deposition and thus may slightly underestimate DIN loss. Using the DIN shoreline load measured in 1999 (3) compared with 1989 effluent data, we calculated an 81% DIN loss. The lower percent removal that we found in 2003, in addition to the two-fold increase in DIN concentration in shoreline samples over 4 years ( 109 fxM in 1999, 208 pM in 2003). suggests that the efficiency of N removal may be declining over time. Alternatively, we may have underestimated groundwater travel time, thus underestimat- ing losses (Fig. 1 A). To ascertain where the loss occurred, we used DIN/B ratios from the monitoring wells in the wastewater plume (Fig. 1C). The DIN/B data show that about 60% of the DIN is lost between the point of effluent discharge and the first monitoring well. There- after. DIN/B ratios decrease little with distance traveled in ground- water, suggesting that the primary loss due to denitrification and retention occurs either in the vadose zone, at the interface of the aquifer and vadose zone, or during the initial 100 m of travel in the aquifer. A second approach to examining the loss of DIN within the aquifer was to use N2/Ar ratios to calculate the excess N2 gas present in the shoreline samples. The Ar content of the water was used to calculate the temperature of the water entering the aquifer. We assumed that when the water entered the aquifer, both gases were in atmospheric equilibrium at that temperature. The amount by which the N, measured in the water exceeded the amount expected with atmospheric equilibration was used to estimate denitrification within the aquifer. Values of excess N2 along the shore averaged 46.8 ^M N (n = 18, se = 3.9), indicating that only 8% of the DIN in the effluent leaving the FWTP was denitrified within the aquifer. Because high rates of denitrification within the aquifer should have been detected as N,, we believe that denitri- fication is not a major process in the initial 100 m of the aquifer. However, we cannot ignore the interface, where N2 gas can escape, as a possible site of N removal. These results, coupled with the DIN/B data from within the plume, suggest that denitrification within this aquifer is small and most of the removal may occur in the vadose zone, as has been reported previously ( 1 ). This is consistent with the idea that dis- solved organic carbon, a component of the denitrification process, is largely consumed in vadose zones thicker than 5 m, therefore allowing for little denitrification activity in the aquifer (8). This research was supported by a grant from the NSF Research Experience for Undergraduates (OCE-0097498). Literature Cited 1. Valiela, I., G. Collins, J. Kremer, K. Lajtha, M. Geist, B. Seely, J. Brawley, and C. H. Sham. 1997. Ecol. Appl. 7(2): 358-380. 2. Kroeger, K. D., J. Bovven, I). Corcoran, J. Moorman, J. Micha- lowski, and I. Valiela. 1999. Em-iron. Cape Cod 2: 15-26. 3. Kroeger, K. D. 2003. Controls on Magnitude and Species Composi- tion of Groundwater-Transported Nitrogen Exports From Glacial Out- wash Plain Watersheds. Ph.D. Dissertation. Boston Marine University Program. Boston. 4. Jordan, M. J., K. J. Nadelhoffer, and B. Fry. 1997. Ecol. Appl. 7(3): 864-881. 5. Vogel, J. C., A. S. Talma, and T. H. E. Heaton. 1981. J. Hytlrol. 50: 191-200. 6 Kana, T. M., C. Darkangelo, M. D. Hunt, J. B. Oldham, G. E. Bennett, and J. C. Cornwell. 1994. Ami/. Cliem. 66: 4166-4170 7. Westgate, E. J., K. Kroeger, W. J. Pabich, and I. Valiela. 2000. Biul. Bull. 199: 221-223. 8. Pabich, J. W., I. Valiela, and H. F. Hemond. 2001. BioKi-oclwinistry 55: 247-268. 244 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull. 205: 244-245. (October 2003) © 2003 Marine Biolor ory Nitro^c md Speciation Through the Subterranean Estuary of Waquoit Bay, Massachusetts J. M. Talbol' . K. D. Kroeger, A. Rago2, M. C. Allen2, and M. A. Charette2'* Boston University, Boston, MA 2 Woods Hole Oceanographic Institution, Woods Hole, MA Fresh groundwater discharge is an important vehicle for nitro- gen transport to coastal waters ( 1 ). In near-shore aquifers, mixing of fresh groundwater with saline pore waters produces groundwa- ter of intermediate salinity that fluxes to sea as submarine ground- water discharge (2). Discharge of this mixed water, known as a subterranean estuary (3), may affect the rate of delivery of nitrogen to the water column. In this study we estimated the flux of dissolved inorganic nitrogen (NO," and NH4+) to the head of Waquoit Bay through both the fresh and intermediate-salinity portions of the near-shore aquifer and investigated the behavior of N during transport through the subterranean estuary. To quantify nitrogen concentrations in the aquifer, we collected groundwater samples from four locations on the shore of Waquoit Bay along a 12-m transect that was perpendicular to shore and extended 6 m above and below the mean tide line on the beach. At each location we collected groundwater samples at depth intervals of 0.15 to 0.6 m tn a depth of 7 to 8.5 m below the land surface. Samples were collected using an AMS Retract-a-Tip piezometer with a peristaltic pump and filtered through an in-line 0.45-/nm polyethersulphone filter. About 20 groundwater samples from each profile were obtained and stored frozen until analysis. Salinity was measured by salinometer (4); pH. temperature, dissolved oxygen, and redox potential were measured oil-site with a YSI 600 multi- probe. Samples were analyzed colorimetrically for dissolved am- monium and nitrate concentration with a QuickChem FIA+ La- chat nutrient auto-analyzer (Zellweger Analytics, QuickChem 8000 Series) within 3 weeks of collection. In each profile, salinity (Fig. 1A) increased with depth beneath an area of fresh groundwater. This follows the Ghyben-Hertzberg model of high-density seawater intrusion beneath the lower density fresh groundwater (5). Nitrate was the most abundant DIN species present in the relatively oxidizing fresh groundwater (Fig. IB). This N traveled as a plume and apparently discharged to the bay within the intertidal zone. DIN was present almost exclusively as ammonium in the reducing intermediate-salinity portion of the aquifer (Fig. 1C). Low concentrations were found near the salt- fresh groundwater interface and approached a uniform concentra- tion with depth. These results are consistent with collections from across the head of the bay (Kroeger and Charette. unpubl.). Low concentrations of NH4 + in Waquoit Bay surface water and in fresh groundwater (Fig. ID) suggested that ammonium is trans- ported into the subterranean estuary by advection through marine sediments where organic N is mineralized. Our data show that ammonium moved conservatively through the intermediate-salin- ity zone (Fig. ID). High concentrations of NH4 + in saline ground- water may be explained by cation exchange and ion pairing, but * Corresponding author: mcluireiteis whoi.edu considering the linear relationship with salinity, such activity is negligible. This conservative transport suggests that the NH4+ must travel with intermediate-salinity groundwater discharging to sea. Absence of NO, ~ within the subterranean estuary, low concen- trations in bay surface water, and high concentrations in fresh groundwater (Fig. IE) suggest loss of NO3~ by denitrification within the reducing conditions of the saline groundwater. How- ever, it may be that much of the fresh groundwater containing high NO,~ discharges without mixing with the subterranean estuary. Assessing the amount of NO3~ that is mixing with the saline groundwater is difficult since nitrate tends to travel as a plume and concentration in the freshwater endmember is not clear. To estimate relative groundwater flux of N to estuarine surface waters, we multiplied the average DIN concentration in each portion of the aquifer by estimated rates of groundwater advection through each zone. We calculated average DIN concentrations using our collections plus previously collected samples from an along-shore transect across the head of the bay (Kroeger and Charette, unpubl.). Freshwater flux of N was calculated using a freshwater discharge rate of 6500 m /day, determined from hydrological measurements of head gradient and hydraulic conductivity at our sampling sites (Mulligan and Hutchinson, unpubl.). A rate of intermediate-salinity groundwater discharge of 3400 m Vday was calculated as the difference between an estimated total (fresh + saline) groundwater discharge rate of 9900 m Vday and the fresh groundwater discharge rate. Total groundwater discharge to the head of the bay was based on mass-balance-derived estimates of submarine groundwater discharge using measurements of radon activity in groundwater and bay surface waters (6). Total DIN flux to the bay was calculated as the sum of the DIN flux through each portion of the aquifer. On the basis of these calculations, the freshwater portion of the aquifer discharged 6.2 kg N/day to the head of the bay. DIN flux through the intermediate-salinity zone was 1 .5 kg N/day. which accounts for 20% of the total DIN flux to the head of the bay of 7.7 kg N/day. These results suggest that DIN flux through the subterranean estu- ary of Waquoit Bay may be significant relative to DIN flux due to freshwater discharge. The DIN transported is composed entirely of regenerated ammonium, as opposed to the terrestrially derived (7) nitrate transported by fresh groundwater. Despite mixing of fresh and saline groundwater masses (Fig. 1A). the subterranean estuary is not a site of net biogeochemical transformations of ammonium. This research was funded by a National Science Foundation Research Experience for Undergraduates site Grant (OCE- 0097498) to I. Valiela, NSF grant (OCE-0095384) to M.A.C., and Woods Hole Oceanographic Institution Coastal Ocean Institute Postdoctoral Fellowship to K.D.K. We thank the Waquoit Bay National Estuarine Research Reserve staff for use of their facili- ties. Special thanks to Dan Abraham, Rachel Hutchinson, Ann Mulligan, and Craig Herbold for their assistance with this project. RCOLOGY AND POPULATION BIOLOGY d) 245 8 m 12m 60 50 40 f.30 20 10 ol- 0 Ammonium • PZ1 OPZ2 • PZ3 OPZ4 » Bay Water 10 20 Salinity 30 - 2 C) Ammonium I I ,M 1 1 _6 - s e) 250 200 I 150 8 100 , 50 . I 0 Nitrate • PZ1 DPZ2 • PZ3 OPZ4 • Bay Water 10 20 Salinity 30 Figure 1. Behavior of nitrate anil ammonium in near-shore aquifer. Cross-sectional contour plots based on depth profiles collected in a transect beginning <> in above mean title line for (A) salinity. IB) NO,~ concentration, and(C) NH4+ concentration. Concentrations of(D) ammonium and (E) nitrate \ersus \alinity fur each profile. Different symbols indicate samples collected al pic:nmeter locations I, 2. 3, and 4 from 0-12 m. depicted in panels A. B. and C of this figure. Filled diamond \\mhol\ indicate average ammonium (.1.3 /xMj and nitrate (1.2 JU.M) concentrations in Waqnoil Bay sin-face water. Literature Cited 1. Valiela, I., K.. Foreman, M. LaMontagne, I). Hersh, .1. Costa. C. D'Avanzo, M. Babione, P. Peckol, B. DeMen-Andrcsun, C. Sham el al. 1992. Estuaries 15: 443-457. 2. Li. L.. D. A. Barry, F. Stagnitti. and J.-Y. Parlange. 1999. \\atcr Kcso,,r. Res. 35: 3253-32.^). 3. Moore, W. S. 1999. Mar. Chem. 65: I I 1-12?. 4 Knapp. G. P., M. C. Stalcup, and R. J. Stanley. 199(1. Technical Report WHOI-90-35, Woods Hole Oceanographic Institution. Woods Hole. MA. 25 pp. 5. Freeze, R. A., and J. A. Cherry. 1979. Grouiuhmter. Prentice Hall, Edgewood Cliffs. N.I. Pp. 375-378. 6. Abraham, D. M., M. A. Charette, M. C. Allen, A. Rago, and K. D. Kroeger. 2003. Radiochemical estimates of submarine goundwaiei discharge to Waquoit Bay. Massachusetts. Kiol. Bull. 2(15: 246-247. 7. Valiela, I., G. Collins, J. Kremer. K. Lajtha. J. Geist, B. Seely. J. Bra«ley, and C. H. Sham. 1997. Ecol. Appl. 1: 358-380. 246 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Bio/. Bull. 205: M6-247. (October 2003) 0 and often peak at intermeJi salinity (15-20) due to a desorption reaction * Corresponding author: mdiarette&whoi.ecJu Falmouth, MA (A) Waquoit Bay (B) Distance (m)(c) * 0 4 8 12 ** -~ 0 E a a a Figure 1. Lucullan of It tii/uoir Bay. Massachusetts, and our sampling site during the summer of 2003. Wai/noil Ba\ is located activity, a continuous measurement at radan-222. iiiul a transect a/ four depth profiles oriented perpendicular lo the heach u-ere used to quantify SGD (B, A = --''Ra, O = 222Rn, * - ** = transect of 4 depth profiles). A verticil/ cross .sectiim of ' xroiintlwater revealed a sharp increase in salinity 111 the freshwater-saltwater interface tC. contour plot from * to **). related to ion exchange (7). The pattern we observed could be explained by an increased 22<1Ra source with increasing depth and distance from shore, a function of either higher sediment content of thorium-230 (radiogenic parent of 22(1Ra) or a decrease in the grain size of aquifer sediments. This latter scenario would result in greater sediment surface area, thereby increasing the availability of radium for desorption. However, sediment sampling at the head of Waquoit Bay is necessary to determine which of these two possi- bilities is the nmst likely explanation for the observed pattern. The pattern of groundwater 222Rn with salinity was similar to that of ~2"Ra, with ~3-fold increases in activity at salinities higher than 20 (Fig. 2a). 222Rn also displayed an increase in activity with increasing depth and distance from the berm (Fig. 2b). Because Rn is a noble gas, its distribution throughout the aquifer should be uniform, and not affected by changes in salinity. Also, because the residence time of fresh groundwater beneath the bay is several years (8), the activities of 222Rn in this zone are likely to be in equilibrium with the sediment activity of its parent isotope 22"Ra. Therefore, the higher observed activities of 222Rn in the saline portion of the aquifer must be a function of a greater 22"Ra source, which is consistent with the observed 22<1Ra distribution. ECOLOGY AND POPULATION BIOLOGY 247 8000 1t^ 222 A _— 6000 ° 226Ra . . ' — 10 "•— E • E Q. J3. 4000 o Q. i o * _ - 5.0 DC 8! 2000 .. • Oo o *ei>» a . • . • ».* ° * *„ ° o •„ «'o ,• • > •• 8^ nn 0 10 20 30 Salinity Distance (m) 4 8 12 0.25 Figure 2. TVu- distributions of radiwn-226 and radon-222 in groiind- water displayed a patient of increasing activity with increasing salinity, depth, and distance from the beach benn. The non-conservative distribu- tion of-'"Ra and ---Rn is displayed in a plot of isotope activities, incasio-cd as disintegrations per minute per liter of water (dpm I ~ ' ). as a function of increasing salinity IA). A vertical cross section of Rn activities through the fresh, intermediate, and saline portions of the aquifer IB, contour pint. individual samples represented h\ diamonds (4)) reveals increasing ac- tivity from the surface to 8 m, and distance from the henn in 12 in. A 5-point running average of the lime-series record of groundwater veloci- ties is plotted alongside tidal height above sea floor (C). To quantify SGD to Waquoit Bay. we applied a non-steady-state mass balance model to our time-series 222Rn record collected over 3 days at the head of the bay. Corrections for atmospheric evasion, loss due to advection to the lower bay. and changes in inventory due to water column depth were all applied to the time-series data using the approach of Burnett and Dulaiovu (5) to calculate the excess 222Rn flux (dpm m"2 d~') during each 30-min interval. Dividing by the average groundwater 222Rn activity (dpm m 3). we estimated SGD rates ranging from 0 to 0.16 m d '. with a mean of 0.08 ± 0.02 m d"1 (n = 132. 1-cr). In general. SGD was related to tidal stage (Fig. 2cl. whereby SGD was highest at low. tide and lowest at hiiih tide, a result consistent with other time- series estimates of SGD in coastal systems (6. 9). We suggest that both a change in hydraulic head gradient, modulated by the rise and fall of the tide, and the effects of subsurface recirculation due to tidal pumping are the likely explanation for this observation. To calculate a volumetric flux of SGD (m3 d"1). we must first estimate the area of the seepage face at the head of Waquoit Bay. The length of the seepage face (1760 m) was obtained from a false-color aerial infrared image ( 1-m resolution) taken at low tide in the fall of 2002 (Charette. unpubl. data). A seepage meter study by Michael et a/. (10) found the width of the seepage face to be 70 m. Based on this effective seepage surface area of 123.200 m2, we estimated a volume discharge of 9900 m3 d~'. Following the mass balance model of Charette et al. (4) and using the same effective seepage area, we estimated a "''Ra-derived volumetric flux of SGD of ~ 1200m3 d ' (n = 4). Since high levels of 22"Ra are present only in the saline portion of the aquifer and 222Rn is high in both fresh and saline groundwater (relative to surface waters), it is possible that the difference between these two esti- mates represents the fresh component of SGD. However, an alter- native explanation is greater uncertainty in the 226Ra estimate due to the limited data compared to the 222Rn record. Recent applications of 222Rn and 22"Ra to estimate SGD have assumed that groundwater discharge is static over time and space. The flux estimate derived from the continuous 222Rn record, how- ever, suggests substantial temporal variability in groundwater dis- charge across a tidal cycle. This method would be useful for longer-term studies, where variability in SGD may be driven by the spring/neap tidal cycle and seasonal to interannual changes in aquifer recharge. Also, high-resolution sampling (cm scale) of :22Rn and 22flRa in groundwater revealed that these isotopes do not behave as predicted across a salinity gradient, probably because of an increased source term across the freshwater-saltwater interface. We thank the Waquoit Bay National Estuarine Research Re- serve (WBNERR) for the use of their field site and research facilities during the summer of 2003. This study was funded by a NSF-Research Experience for Undergraduates (REU) site grant (OCE-0097498) and a NSF grant (OCE-0095384) to M.A.C. Literature Cited 1. Zektzer, 1. S., V. A. Ivanov, and A. V. Meskheteli. 197.1. ./. Hydro!. 20: 1-36. 2. Johannes, R. E. 1980. Mar. Ecol. Prog. Ser. 3: 365-373. 3. Moore, W. S. 1999. Mar. Client. 65: 1 1 1-125. 4 Charette, M. A., K. O. Buesseler, and J. E. Andrews. 2001. Lim- nol. Oceanogr. 46(2): 465-470. 5. Burnett, W. C., and H. Dulainva. 2003. ./. Environ. Radioact. 69: 21-35. 6. Moore, W. S. 1984. Nitcl. lustrum. Methods 222: 407-41 1. 7. Moore, VV. S., H. Astwood, and C. Lindstrom. 1995. Geochim. Cosmochim. Aau 59(2(1): 42X5-4298. 8. Cambareri, T. C., and E. M. Eichner. 1998. Ground Water 36(4): 626-634. 9 Sholkovitz, E. R., C. VV. Herbold, and M. A. Charette. 2003. Limnol. Oceanogr. Methods 1: 16—28. 10. Michael, H. A., J. S. Lubestky, and C. F. Harvey. 2003. Geophys. Res. Lett. 30(6): 1-4. 248 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Bio/. Bull. 205: 2- 249. (October 2003) © 2003 Marine Biological 1 ;., oratory importance of Metabolism in the Development of Salt Marsh Ponds M. E. Johnston' *, J. R. Cavatorta2, C. S. Hopkinson3, and V. Valentine^ ' The Pennsylvania State University, University Park, PA - Amherst College, Amherst. MA ^Marine Biological Laboratory, Woods Hole, MA Ponds are a common feature on the salt marsh surface and are typically found in depressed regions of the high-marsh platform ( 1 ). They are widely held to be valuable habitat for larval and juvenile fish as well as for avifauna. Pools of standing water develop in areas that do not receive a regular supply of sediment with flooding. This standing water often becomes hypersaline and anoxic. thus inhibiting marsh grass production and further exac- erbating marsh accretion (2). In areas where the rate of marsh accretion is less than the rate of sea-level rise, the formation of interior marsh ponds is expected to increase, thus contributing to marsh degradation (3). This study focuses on pond metabolism, measured by dissolved oxygen changes, as a possible mechanism of marsh pond expan- sion, as well as an indication of habitat quality. A network of ponds that formed in the past 50 years in about a 1 -hectare (ha) area of intertidal salt marsh was examined along the Rowley River of the Plum Island Sound estuary in northeastern Massachusetts (4). Pond metabolism was investigated using two techniques: free- water diurnal changes in dissolved oxygen, and oxygen consump- tion of sediment cores. In the free-water technique, dissolved oxygen (DO) was measured in three ponds at '/2-h intervals for durations of 3 to 5 days during June and July of 2003. DO was measured using a pulsed, polarographic O: electrode, which is corrected for temperature and conductivity (YSI, Inc.). Rates of change in DO attributable to gross primary production and respi- ration were corrected for diffusion across the air-sea interface using a gas transfer velocity proportional to wind speed (5). Net ecosystem production was calculated as the balance between gross primary production and respiration over 24-h intervals. Sediment cores ( 15.5 X 50 cm) were taken in the field from the edge and the center of a pond and returned to the laboratory to measure the respective benthic respiration rates. Another set of cores (10 X 50 cm) was used to determine the average carbon content of the peat in the pond environment by drying and com- busting peat samples of known depth and volume and assuming a ratio of carbon to organic matter of 0.5:1. To investigate the relative lability of organic matter from varying depths, root and rhizome material (5 g wet weight) from depths corresponding to the sediment cores was placed in BOD bottles with seawater (297 ml) and a bacterial/sediment inoculum (3 ml), and respiration was measured. Dissolved o\\gen levels in all the ponds fluctuated greatly over a 24-h period, with values ranging from 0<7r to 200% of saturation (Fig. la). Along with high temperatures and salinity, this range of * Corresponding author: cnejl36@psu.edu dissolved oxygen emphasizes the extreme conditions of these ponds as habitat. These conditions seem paradoxical in view of the assumed value of these habitats to juvenile and larval organisms. The rates of gross primary production ranged from 2.75 to 6.98 g O-, m : d " ' in the ponds, and the rates of respiration from 3.6 1 to 6.9 1 g O, m 2 d~ ' . These rates are similar to those found in the Plum Island Sound estuary (J. Vallino. Marine Biological Laboratory, pers. comm.) and eutrophic estuaries in general (6). On average, rates of respiration exceeded gross primary production in the ponds such that net ecosystem production was negative 8 out of 12 days (Fig. Ib). A negative value for net ecosystem produc- tion indicates that more organic matter is consumed than produced internally (7). The most likely source of this organic matter is the highly organic marsh sediment. Our estimates of metabolism are likely to be conservative during this time of year to the extent that anaerobic products of sulfate reduction are stored temporarily in anoxic sediments (8). Decomposition rates of root and rhizome material decreased with depth, indicating that peat lability decreases with depth (Fig. Ic). Respiration rates in benthic cores were higher in the deep center of the pond than along the shallow edge (Fig. Id). On the basis of root and rhizome decomposition, however, we expected to see lower respiration rates in the benthic cores taken from the center of the pond than in those taken from the edge where the remains of recently dead macrophytes were evident. Higher res- piration rates in the pond center can be attributed to the presence of Emenimorpliii. This alga grows around the periphery of the ponds, and its remains tend to collect in the deeper, more central portions of the ponds. Benthic respiration decreased following removal of this highly labile, detrital Enteromoipha layer from the second center core (Fig. Id — labeled Center 2*). The importance of excess respiration as a mechanism contrib- uting to pond enlargement over time can be seen by comparing our measurements of net ecosystem production with our independent estimates of the rate of pond formation. Average depth-integrated carbon content of marsh peat surrounding ponds is approximately 38.446 g m"3. Assuming a maximum pond age of 50 y and an average pond depth of 20 cm (data not shown), we estimate a peat decomposition rate of 154 g C m~2 y"1 However, two mechanisms are responsible for the increase in pond depth: peat decomposition and accretion of organic and inorganic material in the marsh adjacent to the ponds. Because the areas of marsh surrounding the ponds are not yet inundated, we can assume that the surrounding marsh has been accreting sediment at a rate comparable to that of sea-level rise, which is 2.65 mm y locally (9). Accordingly, the adjacent marsh has accreted by at ECOLOGY AND POPULATION BIOLOGY 249 0:00 0:00 • DO Cone. DO Saturation Cone. Pond 1 Pond 2 Pond 3 Peat Respiration Rates (g 02 m 3 hr'1) 00 .0 Oe * .O OA *^\^ .4 00 .£. n r\ u.u 0 10 20 30 40 Depth (cm) C ^ O T "J "D A p=0.004 2 * '5. £ I I (0 CN 4 « O o °> 2 O *— ' t • !E <" = 1 0 fi £ edge n=6 cente n=6 Core I r D center n=2 2* Figure 1. Patterns nl metaholisni in three Milt inarsli /* mils, till Diurnal patterns of dissolved oxygen in Poml 3 over 11 3-day period, (b) Net ecosystem production rales in three ponds, (c) Peat respiration rates h\ depth, (dl Bentllic respiration rates in the cil.vc "nd center of H pond. Center 2* indicates that the respiration rates were measured after the removal <>t the delritul Enteromorpha layer. P-\-aliie refers to the left two columns of the graph only. least 8 cm over the past 50 years. Therefore, accretion of surround- ing marsh areas accounts for 40% of the depth of the ponds. The remaining 12 cm of depth increase can then be attributed to decomposition of the inundated peat. Decomposition of 12 cm of peat over 50 years is equivalent to 92 g C m"2 yr ' or an oxygen consumption of 0.67 g O, m~2 d~' (assuming a production-to- respiration ratio of 1) (7). The average rate of net ecosystem production in the ponds is -0.38 g O2 m"2 d~ '. Thus, on average, respiration can account for over half the required rate of decom- position: in fact, for the majority of the observations, it can account for nearly 100% of the required carbon loss (Fig. Ib). Although our study was conducted only during the summer season, our results do suggest that respiration is an important and actively functioning process in the development of marsh ponds. This work was supported by NSF-REU Site Grant (OCE- 0097498). the Boston University Marine Program. NSF Grant OCE 9726921. and EPA STAR Grant R828677. Literature Citt-d I. Redfield, A. 1972. Ecol. Monogr. 42: 201-237. 2 Friedrichs, C., and J. Perry. 2(101. ./. Coastal Res. 27: 7-37. 3. Kearney, M., R. Grace, and J. Stevenson. 1988. Geogr. Re\: 78(2): 205-220. 4. NOAA Coast & Geodetic Survey. 1953. Topographic Sheet, scale: 1:10.000. National Oceanic and Atmospheric Administration. 5. Emerson, S. 1975. l.imnol. Oceanonr. 20(5): 754-761. 6. Day, J., C. Hall, \V. Kemp, and A. Yanez-Arancibia. 1989. Estu- arine Ecology. John Wiley and Sons, New York. 558 pp. 7. Odum, H. 1956. Limnol. Oceanogr. 1: 102-117. 8. Howarth, R.. and J. Teal. 1979. Limnol. Uccanoxr. 24(6): 999- 1013. 9. Zervas, C. 2001. NOAA Technical Report. NOS CO-OPS 36, National Oceanic and Atmospheric Administration. Silver Spring. MD. 250 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull. 205: . 251. (October 2003) © 2003 Marine Biologic Transpb id Isotopic Evidence of the Relative Effects of Ambient and Internal Nutrient Supply on the Growth of Ulva lactuca A. B. Agniar1, J. A. Morgan2, M. Teichberg3'*, S. For*, and I. Valiela* 1 Lafavette College, Easton, PA 2 Yale University, New Haven, CT 3 Boston University Marine Program. Woods Hole, MA Growth of macroalgae in coastal environments is, to a large extent, limited by the available supply of nitrogen (1.2). Macroal- gal growth rates may be influenced by their internal pool of nitrogen, and also by the supply of new nitrogen provided by ambient water (3). Few studies have investigated the relative role of internal and external nitrogen supply on growth of macroalgae. In this study we investigate the net growth of Ulva lactuca, a widespread opportunistic species of macroalga found in the estu- aries of Waquoit Bay. Cape Cod. Massachusetts, in response to internal and external nitrogen supply by using a field transplanta- tion experiment and isotopic measurements (4). Fronds of U. lactuca were collected from Sage Lot Pond. Quashnet River, and Childs River, subestuaries in the Waquoit Bay estuarine system. The land-use patterns on the watershed of these estuaries differ enough to lead to substantially different nitrogen loads of 14, 350, and 601 kg N ha" ' y~ ', respectively (2). Fronds collected from these three estuaries will therefore have grown under different nitrogen regimes and have entered the experiment with different internal nitrogen contents. Two fronds from the same originating estuary were weighed (blotted wet weight) and placed inside cages constructed out of transparent disposable containers (GladWare) with two sides covered by mesh, allowing for water flow. To assess the effect of ambient nitrogen supply on growth of U. lactuca. the algae collected from each estuary and placed in the cages were then transplanted into either Sage Lot Pond, which has the lowest nitrogen load (and hence the lowest nitrogen supply for fronds (5)). or Childs River, which has the highest nitrogen load (and highest supply of nitrogen), with 30 cages per estuary. The cages were randomly set 1 m apart within the existing macroalgal canopy, and 0.2 m above the bottom in locations with salinities between 25 and 30 ppt. This experimental design was deployed from 18 to 27 June 2003 and was repeated from 17 to 24 July 2003. We measured the net growth response of U. lactuca by deter- mining the initial and final wet weights of each algal frond within each cage. Growth data from both runs of the experiment were pooled to span possible differences across the months. Fronds were dried at 60 °C, ground, and sent to the Stable Isotope Facility. University of California, Davis, for analysis of carbon and nitrogen content and stable isotope signatures. Net growth rate of U. lactuca depended on both the nitrogen pool within the fronds, obtained from the estuary from which the fronds were collected, and the nitrogen supply provided by the estuary to which the fronds were transplanted (Fig. 1A). Growth * Corresponding author: mirta«?'bu.edu rates of U. lactuca collected from Sage Lot Pond were significantly lower than those achieved by fronds collected from Childs and Quashnet rivers when transplanted into the Childs River (ANOVA, F = 13.66, P = 0.000017). An ad hoc Duncan's test showed no differences between growth rates of algae collected from the Childs and Quashnet rivers when transplanted into Childs River (ANOVA. F = 2.64. P = 0. 1 1 ). Growth rates of fronds from all three estuaries transplanted into Sage Lot Pond were not signifi- cantly different (ANOVA. F = 0.71. P = 0.40). These results suggest that, first, fronds of U. lactuca grown in an estuary with nutrient-poor water grew slowly if at all. even when transplanted in nutrient-rich estuaries. Second. U. lactuca fronds from nutrient- rich estuaries grew faster, and more so when transplanted in nutrient-rich estuaries. Third, fronds from nutrient-poor estuaries lag considerably, even when transplanted into nutrient-rich water; this is evidence of some other impairment of growth. To further examine the effects of internal and external nitrogen, we plotted net percent growth of U. lactuca in Childs River and Sage Lot Pond versus initial percent nitrogen in the fronds (Fig. IB). Some growth took place in all three estuaries (Fig. IB), which is not surprising because initial percent nitrogen in the fronds was above 0.71. the minimum <7r nitrogen content in the tissue of U. lactuca required for growth (3). The initial nitrogen content of fronds from the nutrient-rich estuary was 3-fold larger than that of fronds from the nutrient-poor estuary (Fig. IB). The steeper slope of the growth rate for fronds transplanted into Childs River (black points in the figure) suggests that net growth rates of U. lactuca were more affected by external nitrogen supply than by internal nitrogen content, as found in studies on other macroalgae (1. 6). The relative importance of external nitrogen supply and internal nitrogen content were corroborated by the isotopic signatures (Fig. 1C). Fronds initially differed markedly in carbon and nitrogen isotopic signatures; signatures in fronds from Childs River and transplanted into Childs River were notably different from those of Sage Lot Pond fronds transplanted into Sage Lot Pond (Fig. 1C). Transplanted fronds soon reflected the signature of the nitrogen in the estuaries in which they were incubated, and showed less influence from the estuary of origin. Although the initial percent nitrogen may have slowed growth of Sage Lot Pond fronds (Fig. 1A. IB), the nitrogen pools of the fronds from Childs River and from Quashnet River showed fast responses to the ambient nitro- gen supply (Fig. 1C). Some factor other than internal pool size must be responsible for the lag in growth of Sage Lot Pond fronds. The range of fiKSN between Childs River and Sage Lot Pond has been reported (7). and is associated with different land-derived nitrogen loads, bearing different SISN signatures, arriving from the ECOLOGY AND POPULATION BIOLOGY 251 Transplanted CR SLP SLP 12 Nitrate concentration (LiM) >,5°1 B CO ^40- / 0) }S A CL ./ 220- // O) "' ' o^ 10 - Qnr^" — ^^ ^ 0> Z n - 0123 Initial % N 8- . c 7- ,'A*»N, CCR ^* ' V »x' 2 6" •o A "» A " 0 A o O -Z. 5 g in 4 - ,'' D\ I „ 1 0 IP ^ bSLP Q v '—Lx -12 -10 -8 -6 S 13^ /o/ \ O U l/oo) Figure 1. Growth rales and isotopic evidence fur cl^iil fronds collected from Child', River (CR). Qiiaslmet River (QRl. tuul Sage Lot Pond (SLPl and trans- planted into CR aiul SLP. (A) Net <7c gnnvth per da\ (imwi - \.e.) o/U. lactuca transplanted into SLP and CR versus the average nitrate concentration tin jiMj tin-June 2002 (data from G. Toinasln; Boston Uim-ersin Marine Program > in the estuaries of origin of the fronds. SLP. QR. and CR. iBlMeun net 'i gn mill per das nt algae transplanted into CR, QR, and SLP in reliitmn In initial N content (%) in fronds of algae collected from each estuary. Symbols as in A. (Cl Coin/ninson of S''Nand 8"C isotupic i u/iio an',,) qfi}. lactuca p-nnil\ tnmsplanieJ to CR and SLP from the three estuaries of origin, CR. QR. and SLP- S\mhol\ as in A. Dulled outlines eneompass isotopic rallies of algae from SLP and mcithaled in SLP (Ssu>) and from CR and inctihated in CR (CCR). watershed to Childs River and Sage Lot Pond. The range of 513C values measured for (/. Im-titca in our study ( — 12%c to —T7cc) fall within the heavier part of the range ( — 359£c to -5%c) reported in a compilation of such values for macroalgae (8). Various mecha- nisms, such as the use of HCO, in photosynthesis (8, 9), have been proposed to explain the range of values, but further work is needed to understand the processes that control the values found in U. lactuca. The isotopic conversion evident in Figure 1C suggests that there is relatively rapid turnover of the internal N pools of U. lactuca. For the 8-day duration of the transplant, and assuming linear growth, we roughly calculate that the nitrogen pool turns over completely every 12-15 d. This relatively rapid turnover seems consistent with our conclusion that external nitrogen supply plays a larger role than initial nitrogen content in the growth of this macroalgal species. This research was supported by internships to A.B.A. from the Woods Hole Marine Science Consortium and to J.A.M. from a grant from the National Science Foundation's Research Experi- ence for Undergraduates (# OCE-0097498). This work was also supported by a grant from the National Oceanic and Atmo- spheric Association/NOS (ECOHAB award # NA16OP2728), and is ECOHAB publication #76. Literature Cited 1. Duarte, C. M. 1995. Oplu-lia 41: S7-112. 2. Valiela, I., J. McClelland, .!. Hauxwell, P. J. Behr, D. Hersh, and K. Foreman. 1997. Lininol. ()ceuiioKi: 42: 1105-1118. 3. Pederson, M. F., and J. Borum. 1997. Mar. Ecol. Prog. Ser. 161: 155-163. 4. McClelland, J. \V., and I. Valiela. 1998. Limmn. Oceanogr. 43: 577-585. 5 Valiela. I., K. Foreman, M. LaMontagne, D. Hersh, J. Costa, P. Peckol, B. DeMeo-Andreson, C. D'Avanzo, M. Babione. C. Sham, J. Brawlev. and K. Lajtha. 1992. Estuaries 15: 443-457 6. Peckol, P., B. DeMeo-Anderson, J. Rivers, I. Valiela, M. Maldo- nado, and J. Yates. 1994. Mai: Biol. 121: 175-185. 7. McClelland, J. \V., I. Valiela, and R. H. Michener. 1997. Linwcl. Oceanogr. 42: 93(1-937. 8. Fry, B., and E. B. Sherr. 1984. Mar. Sri. 27: 13-47. 9. Raven, J. A., A. M. Johnston, J. E. Kuebler, R. Korb, S. G. Mclnroy, L. L. Handley, C. M. Scrimgeour, D. I. Walker, J. Beardall, M. Vanderklift. S. Fredriksen, and K. H. Dunton. 2(102. Fimrr. P/«iif Biol. 29: 355-378. 252 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull. 205: -253. i October 2003) © 2003 Marine Bioloi'i >l .iratory Relative : ,• of Grazing and Nutrient Supply on Growth of the Green Macroalga Viva lactuca in Estuaries of Waquoit Bay, Massachusetts J. A. Morgan', A. B. Aguiar2, S. Far1, M. Teichberg~\ and I. Valielci" Yale University, New Haven, CT ~ Lafayette College, Easton, PA -1 Boston University Marine Program, Woods Hole, MA Nitrogen supply is a major control on growth of coastal mac- roalgae ( 1, 2. 3 1. Top-down effects in which grazing significantly affects macroalgae (4, 5). and nutrient-grazer interactions (3) have also been described. In this paper we describe an experiment in which we measured net growth of a common macroalga, Ulva lactuca, in treatments that allowed different numbers of grazers to access fronds as well as incubation of fronds in estuaries with demonstrably different nutrient supplies. These treatments were intended to assess the relative influence of grazer and nitrogen supply on net growth rates of a coastal producer. To examine the effect of grazing on growth of U. lactuca. we constructed acrylic plastic cages with sides of 1-mm, 4-mm, or 1 8-mm mesh. The different mesh openings were intended to allow entry to different numbers of grazers, which we took as a proxy for grazing pressure. The cage design also allowed for light penetra- tion and horizontal water flow. The 18-mm mesh permitted larger size classes and a greater number of grazers to enter the cages, while the 1-mm mesh excluded larger size classes and allowed fewer grazers. The 4-mm mesh was intended to furnish an inter- mediate grazer treatment. To evaluate the effect of nitrogen supply and grazing on algal growth, cages with the three mesh sides were placed in three estuaries in Waquoit Bay. Massachusetts. These three estuaries experience different nitrogen loads — Sage Lot Pond. 14 kg ha y ; Quashnet River. 350 kg ha y ; and Childs River. 601 kg ha~'y~' — from their watershed (6). These nitrogen loads led to different mean nitrate concentrations measured in the estuaries during July 2002. one year prior to the time of our experiments: 0.04, 6.1, and 1 1.75 /nM for Sage Lot Pond. Quashnet River, and Childs River, respectively (G. Tomasky. Boston University Marine Program, unpubl. data). To minimize effects of differences be- tween estuaries other than our treatments, we chose sites similar in salinity, depth, and algal composition. In each estuary we placed four replicates of each of the three grazing pressure treatments, for a total of 36 cages. Three fronds of U. lactuca, each approximately 300 mg (blotted wet weight), were suspended inside each cage. To measure the effect of top-down versus bottom-up factors, we measured net growth as the dependent variable. Net growth was the growth achieved by the fronds minus the biomass consumed by grazers. To determine net growth, the U. lactuca fronds were weighed initially (blotted wet weight) and again after 10 days of field incubation. First, we assess the successed of the treatments. To roughly measure the grazing pressure, we sorted and counted the potential grazers found in the cages for two replicates of each treatment at the end of the incubation. The grazers were sorted into four groups, amphipods. shrimp, crabs, and isopods. The total number of graz- ers in the 1-mm mesh cages was significantly lower in all three estuaries than the number in the 4-mm cages (Fig. la; ANOVA F = 31.0. P = 0.0014). The number of grazers found in the 18-mm mesh cages was also significantly different, although they contained lower grazer abundances than the 1 -mm and 4-mm mesh cages (Fig. la). Predatory fish and large shrimp entered the cages with IS mm mesh and likely fed on the smaller grazers, thus decreasing grazer abundances. This possible effect of predators on grazers suggests that there might be important top-down cascade effects in this system waiting to be studied. The difference in nitrogen load in the three estuaries provided quantitatively different nutrient supplies, as evident in the nitrate concentrations cited above. Bottom-up effects from these different nitrogen supplies on net growth of U. lactuca were dominant factors. Rates of net growth were higher in estuaries receiving larger external nitrogen loads (Fig. Ib; ANOVA F = 61.8, P < 0.001). Percent net growth of U. lactucu in Childs River was almost three times that in Sage Lot Pond (Fig. Ib). This response of macroalgae to nitrogen supply is similar to that reported by others (2). Top-down effects caused by grazers were small compared to the effects of nutrients (Fig. Ib). Across all estuaries, net growth in the 1-mm (fewer grazers) cages was higher than growth in 4-mm (more grazers) cages, but these differences were statistically in- significant. In contrast, there was more than a 200% increase in percent net growth caused by the nutrient effects. Taken together, these results suggest that the direct and indirect bottom-up effects associated with nitrogen loading were much larger than the top- down effects of grazing on the net growth of U. lactuca. Increased nutrient inputs might affect the composition of the grazers found in the three estuaries (Fig. Ic). Shrimp (Palaemon- etes sp.) and amphipods (Gammaridea) accounted for over 85% of grazers in all cages. Shrimp were far more abundant in high nitrogen load conditions (Childs River), while amphipods were more plentiful in lower nitrogen load conditions (Sage Lot Pond; Fig. Ic). Shrimp are predators as well as grazers (7). so they could have fed upon amphipods. Similar shifts in species composition have been reported elsewhere (3. 8). Increased anthropogenic supplies of nitrogen might therefore not only change the growth rates of U. lactuca directly, but also alter the relative abundances of consumers and lower abundances of grazers. This effect is possibly linked to more frequent anoxic and hypoxic conditions in the more nitrogen-loaded estuaries such as Childs River (9). To further assess possible grazer effects, we plotted percent net ECOLOGY AND POPULATION BIOLOGY 253 Mesh opening (mm) No. of grazers/ cage SLP QR CR 1 24±0.0 78.0±30.0 38.5±10.5 4 177.5±14.5 112.5±2.5 59.5±13.5 18 80.5±25.5 61.5±25.5 17.5±9.5 Mean 94.0±44.8 84.0±15.0 38.5±12.1 Mesh opening (mm) % net growth b SLP QR CR 1 69.8±10.0 118.1±12.8 247.1±7.5 4 61.3±5.8 104.2±19.2 223.0±36.3 18 76.6±15.3 92.1±14.7 285.5±31.6 Mean 69.2±4.4 104.8±7.5 251.9±18.2 60 -i 0) 0) to A •^ 40 - • SLP .c ^ . • QR d 20 - .'. '• ACR "Z. n . ^ — •• • m, * • •-• — A 40 80 120 No. amphipods/cage 400 -\ Q) c 200 - 80 160 No. amphipods/cage 400 o 200 0) c 0 40 No. shrimp/cage Fi|>ure 1. 77i<< effects of graying ami nitrogen load on the net growth of \J\\a lactuca HI i Barnstuble Harbor > Pleasant Bay (3, 4). Since increased urban development results in heavier nitrogen signatures of estuarine water and biota (2), we opted to study these estuaries with different land use in their watersheds to take advantage of potential resulting differ- ences in S'^N and 8I3C signatures (5, 6). We collected samples during July 2003 at a site in Barnstable Harbor and three sites in Stage Harbor in Cape Cod. We measured the size of horseshoe crabs as the width at the widest region of the prosoma (7). To obtain samples for isotope analysis, we sampled tissue from the last two segments of the second or third walking leg of adult horseshoe crabs ( 1 ). To identify some potential foods of horseshoe crabs, we also sampled quahogs (Mercenariti nwr- tenaria). polychaetes (Nereis sp., Nephtys sp., and Glvcera sp.), seston filtered from 1-1 water samples, and sediment from two 1 0-ml sediment cores taken 3 cm deep, which were pooled into a single sample. All samples were dried at 60CC, ground, and sent to the Stable Isotope Facility. University of California, Davis, for mass spectrometry. The 5'3C signatures of the horseshoe crabs suggest that their diet may have included a mix of polychaetes and quahogs (Fig. la). Quahogs. in turn, assimilated carbon from a mixture of seston and sediment, judging from their position relative to values on the 813C axis. The carbon in seston and sediment was likely initially derived from phytoplankton and macroalgal organic particulates (gray lines in Fig. la) (H). Polychaetes seemed to belong to a separate branch ut the food web. since they had relatively heavier signatures, and most likely incorporated a mix of carbon from macroalgae and Spartina grass (Fig. la). These S'3C values are consistent with carbon signatures found in Pleasant Bay ( 1 ). 8I5N signatures suggested that the horseshoe crabs consumed a combination of quahogs. polychaetes. and paniculate organic mat- ter, given the expected fractionation from food source to consumer of 2%c-4%P (Fig. la) (2). 815N values for bivalves and polychaetes were 2%o-5%c heavier than those of paniculate matter. In all cases, the 8I5N values for each taxon (shown by points within dashed ovals. Fig. la) seem to be heavier in Stage Harbor than in Barn- stable Harbor, suggesting, as suspected from the different land-use patterns, heavier land-derived nitrogen sources from freshwater inputs into Stage Harbor (5, 6). Isotopic signatures of individual crabs varied considerably. Part of the variation was associated with different crab size; larger crabs tended to have significantly heavier 8I5N signatures (F = 9.5, P = 0.004) and lighter 8I3C values (F == 13.94. P = 0.0005 ) (Fig. Ib. c). The increased 8I5N signatures may be related to increased prey size among larger crabs (9). The slopes of the regression lines in Figure Ib did not differ, but the intercepts were significantly different (ANOVA: F = 9.26, P = 0.004). The offset between the regressions shows that Stage Harbor crabs had a heavier 8l;iN signature than Barnstable Harbor crabs. In Figure Ic, we added a gray reference area that represents the prosomal width and 8I5N values for crabs from Pleasant Bay (1). the most pristine of the three estuaries. The Pleasant Bay values were appreciably lighter than those of either Stage Harbor or Barnstable Harbor (Fig. Ib). These isotopic comparisons suggested that the watershed-derived nitrogen loads (and contribution by wastewater) were greater in Stage Harbor than in Barnstable Harbor and that both these estuaries received greater anthropogenic nitrogen loads than Pleasant Bay. The significant difference in the isotopic signature of horseshoe crabs collected in the three estuaries can also be interpreted to mean that crabs tend to remain foraging in an estuary long enough to acquire 8I5N values characteristic of the estuary in which they were found. These results corroborate earlier observations ( 1 ) that in spite of the evident mobility of horseshoe crabs, they do tend to remain within relatively circumscribed areas for considerable pe- riods of time. The SI3C values provided additional information about the foraging and feeding behavior of horseshoe crabs (Fig. Ic). 8I3C became lighter as size of crabs increased (F =: 13.93. P - 0.0005) (Fig. Ic). As adult horseshoe crabs grew, signatures shifted from values near the 8' 3C for the Spartina-based side of the food web to values more closely associated with phytoplankton (dashed boxes in Fig. Ic. and Fig. la). This transition suggests that as adult crabs grew, a larger percentage of their diet consisted of ECOLOGY AND POPULATION BIOLOGY 255 a) Phytoplankton Macroalgae b) 14 - t ' r Horseshoe 12- Crabs ^*x - \ \ \' ~~*^ 10 - Quahogs >. 1 » 1 «, 1 / 8 • •^ _ x 1 / 6 - • / ^ Vx Polychaetes 4 • 2- Seston | B J^. • MS - Sediment A Or • MR 0- v BN 2 -20 -18 -16 -14 -12 513C %o 13 • 10 10- 60 100 140 180 220 260 300 OBN • SH O -16- -17 • -19 Polychaetes Quahogs 60 100 140 180 220 260 Prosomal width (mm) 300 Figure 1. laoiopic measurements of samples collected in Barnstahle Haihor (BN), Stage Harbor (SH). and Pleasant Bay (PB>. Cape Cod, Massachusetts, (a) S'*N and S"C signatures of horseshoe t rah and other components of the estuary' system of BN and SH. The dashed ovals include measurements taken in BN and the three other sites within SH (Main Bay, sources supported by the phytoplunkton-based portion of the food web. The results of this work lend support to earlier findings about the position of horseshoe crabs as generalist predators in the estuarine food webs of Cape Cod: demonstrate that horseshoe crabs are clearly linked to land-derived nitrogen sources; suggest a possible diet shift from S/wY/mi-based food sources to more phytoplank- ton-based sources as adult crabs grow: and show that the crabs exhibit considerable within-estuary loyalty in their foraging habits. This study was supported by an internship to C.W.O. from the Woods Hole Marine Sciences Consortium and funding from MIT Sea Grant. We are grateful to Ed Eichner of the Cape Cod Commission for sharing information on land use, and to Mirta Teichberg. Allyson Papa, and Rachel Allen for assistance in many ways. Literature Cited 1. Carmichael, R. H., D. Rutecki. B. Annett, E. Gaines. and I. Valiela. J. Exp. Mar. Biol. Ecol. (In press). 2. Peterson, B. J., and B. Fry. 1987. Alum. Re\: Ecol. Syst. 18: 293- 320. 3 Eichner, E. M., S. C. Michaud, T. C. Cambareri, B. Smith, G. Prahm. and M. Fenn. 20(12. Cape Cod Surface Water Nutrient Management Study. Unpublished report. Water Resource Office. Cape Cod Commission, Barnstuble, MA. 4. Eichner, E. M., K. Livingston, B. Smith, V. Morill, and A. Carbon- ell. 1998. Pleasant Bav Nitrogen Loading Analysis. Unpublished re- port. Water Resource Office. Cape Cod Commission. Barnstable. MA. 5. McClelland, J. W., I. Valiela, and R. H. Michener. 1997. Lunnol. Oceanogr. 42: 930-937. 6. McClelland. J. W., and I. Valiela. 1998. Limnol. Oceanogr. 43: 577-585. 7. Shuster, C. N. 1955. On morphometric and serological relationships within the Limulidae, with particular reference to Linmlus polyphemus (L.) Ph.D thesis. New York University. 8. McClelland, J. W., and I. Valiela. 1998. Mar. Ecol. Prog. Ser. 168: 259-271. 9. Valiela, I. 1995. Marine Ecological Processes. Springer- Verlag, New York. MS: Oyster Pond. OP: and Mill Pond. MP). The vertical lines show approximate mean value of o"C fin phytoplankton, macroalgae. and Spartina as found in similar Cape Cod estuaries (2, 8). (b) 8I5N value vs. prosomal width of horseshoe crabs collected in BN and SH. Regression information for BN: y = 0.01. 17\ + 8.9. F = 10.81. P = 0.002; for SH: y = 0.014\ + 8.41. F = 4.48. P = 0.05. Mean of horseshoe crab d'^Nfrom SH. BN. and PB /data from ( I )] are shown on \-a.\is. The shaded reference area covers range of PB from ( I ). (c) o"C values vs. prosomal width of horsecrabs collected in BN and SH. Svmho/s a.s in Fig. Ib. Dashed boxes include range of values for polychaetes and for quahogs for Fig. la. 256 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Binl. Bull. 2 :;•' 7. (October 2003) © 2003 Marine Bin!. .atory Incuh- •: ( onditions of Forest Soil Yielding Maximum Dissolved Organic Nitrogen Concentrations and Minimal Residual Nitrate C. Walker, E. Davidson,* W. Kingerlee, and K. Savage Woods Hole Research Center, Woods Hole, MA Soil is a dominant long-term sink for N in forest ecosystems ( 1 ). With increasing atmospheric N deposition in forests caused by combustion of fossil fuels (2. 3). concern over effects of N loading on ecosystem sustainability is substantial. Net primary productivity is thought to be limited by N in most temperate forests (4). but much of the N from deposition appears to be immobilized in the organic matter of the soil rather than being taken up by plants ( 1 ). However, the mechanisms of N immo- bilization as well as the bioavailability and ultimate fate of organic N remain poorly understood. One hypothesized path- way of immobilization is abiotic reduction of nitrate to nitrite and reaction of nitrite with dissolved organic matter to produce dissolved organic nitrogen (DON) (5). Studying the fate of DON would be facilitated by the development of a I5N label in a realistic soil DON product. In the present study, we tested incubation conditions that would permit nitrate immobilization to DON during 24 h, followed by conditions that would mini- mize the remaining nitrate. Water content and N concentrations were varied in 1-8-day incubations of organic horizon to study the dynamic of nitrate reduction and DON formation. Soil for these incubations was collected from the humus (O,) layer of a forest stand dominated by oak at the Harvard Forest. Massachusetts. The stand developed after agricultural abandon- ment at the end of the 19th century and after a devastating hurricane in 1938 (6). The soil samples were passed through a 2-mm sieve to remove large roots. Subsamples of 10 g (n = 64) were placed in 60-ml serum bottles, which then received a combination of two treatments in a factorial design. First. 3.33 ml of either deionized water or nitrate solution (50 /xg N/g dry soil as KNO3) was added. Second. 30 ml of deionized water was added to half of the samples to create saturated conditions, whereas those not receiving additional water were considered unsaturated. The bottles were sealed using rubber stoppers and tear-away metal crimping seals. Each treatment combination was replicated 16 times so that four replicates could be destruc- tively sampled by extraction in water at 0. 1. 4. and 8 days after the start of the incubation. The "day zero" extraction was done immediately after sample treatment (addition of nitrogen or water) to measure background nitrate and DON and to measure initial recovery of added nitrate. Ambient aerobic conditions prevailed during the first day of incubation to allow for aerobic immobilization processes. After the first day. air was flushed from the serum bottles with dinitrogen gas to create anaerobic conditions in an effort to increase denitrification. thus reducing *Corresponding author: edaudsonls'wrirc.org residual nitrate. At the end of each incubation period. 60 ml of water was added to the saturated samples and 30 ml to the unsaturated. All samples were shaken and immediately filtered through No. 1 Whatman filter paper. Filtrate from each incu- bation was analyzed for nitrate, ammonium, and total N using a Lachat 8000 flow-injection autoanalyzer with in-line persulfate digestion (7. 8). DON was calculated by subtracting the sum of nitrate and ammonium from total N. Nitrate concentrations in soil extracts remained at low values throughout the experiment in incubations under saturated and unsaturated conditions with no added nitrate (Fig. 1 ). In incuba- tions with added nitrate, concentrations increased slightly and similarly under saturated and unsaturated conditions during the first day. after which they declined for the remainder of the experiment. Incubation time, addition of nitrate, and the interaction of time by nitrate were significant (P < 0.01 ) in an analysis of variance. Saturation was also found to be significant (P < 0.05), as nitrate concentrations were lower in saturated incubations com- pared to those remaining unsaturated. Ammonium concentrations in soil extracts increased in a linear fashion, with day 8 values higher (close to 74 jug N/g dry soil with or without added nitrate) in incubations under saturated conditions than those remaining unsaturated (53 fig N/g dry soil with and 46 ^.g N/g dry soil without added nitrate). In summary, water satu- ration apparently enhanced denitrification and either stimulated mineralization or inhibited nitrification, thus yielding lower nitrate and higher ammonium than did unsaturated conditions. DON concentrations in soil extracts increased over time in incubations with and without nitrate addition under saturated and unsaturated conditions. An analysis of variance shows incubation time to be the only significant (P < 0.01 ) factor. Also, there was not a stoichiometric conversion of nitrate to DON, suggesting that DON was produced primarily from sources other than nitrate immobilization. However, only a small amount of nitrate immo- bilization to DON would be necessary to create a labeled DON pool when '^N-labeled nitrate is used. This experiment will be repeated with a '-"'N label in the added nitrate to determine the recovery of labeled N in the extracted DON, ammonium, and in the small remaining nitrate pool. These preliminary results using unlabeled N demonstrate that it may be possible to create a series of incubation conditions that would permit nitrate immobilization into DON while minimizing remain- ing nitrate. Hence, it may be possible to obtain an extract with labeled DON that is created naturally within the soil and that can be used to study DON bioavailability in subsequent experiments. This research was funded by the NSF Research Experience for Undergraduates site Grant (OCE-0097498). ECOLOGY AND POPULATION BIOLOGY 257 200 246 Days of Incubation 8 • DON with added N —A —Nitrate with added N — • - DON without added N - A - Nitrate without added N Figure 1. Mean concentrations (n = 4) of N in saturated (left) and unsammted (right) soil incubations. Solid lines represent DON concentrations of soil extract with added N. and dashed dotted lines represent concentrations without added N. Long dashed lines represent nitrate concentrations of soil extract with added N, and short dashed lines represent concentrations without added N. Literature Cited 1 . Nadelhofler, K. J., M. R. Downs, and B. fry. 1999a. Ecol. Appl. 9: 72-86. 2. Gunderson, P., B. A. Emmet, and O. J. Kjonaas. 1998. Forest Ecol. Manage. 101: 37-55. 3. Galloway, J. N., W. H. Schleisinger, and H. Levy II. 1995. Global Biogeochem Cycles 9: 235-252. 4. Vitousek, P. M., and R. VV. Howarth. 1991. Biogeochemistiy 13: 87-115. 5. Davidson, E. A.. J. Chorover, and D. B. Dail. 2003. Global Change Biol. 9: 228-236. 6. Foster, D. 1992. J. Ecol. 80: 753-772. 7. Ranger, C. B. 1981. Anal. Client. 20a: 53-59. 8. Ruzica, J. 1983. Amil. Client. 55( 1 1 ): 1040a-1053a. Reference: Biol. Bull. 205: 257-258. (October 20031 © 2003 Marine Biological Laboratory Building a Database of Historic Land Cover to Detect Landscape Change M. T. Holden.* C. Lippitt, R. G. Pontius. Jr., ami C. Williams Clark Universit\, Worcester, MA Since 1999, researchers at Clark University have collaborated with scientists at the Marine Biological Laboratory in Woods Hole, Massachusetts, to examine the relationship between land cover and nutrient concentrations in the Ipswich River watershed. The wa- tershed covers 404 km2, involves 22 towns, and drains into the Plum Island Ecosystem — Long Term Ecological Research site of the National Science Foundation. Previous research has associated increases in nitrogen concentrations with conversion from forest in 1971 to built land in 1999 (1). Land-cover before 1971 is also important in understanding nutrient cycling: however, maps of the pre-1971 landscape exist only in paper form. The first part of this paper describes the transformation of 1951 paper maps to digital land-cover maps (2). The second part describes a novel technique for comparing maps from two points in time and attributing the differences to either map error or true landscape change. To begin the digitizing process, the 14'/4 in. X 19 in. 1951 paper maps (1:31.680) were scanned in grayscale at 300 dpi using a large-format scanner. We used the GIS software ERDAS Imagine 8.5 to georeference (spatially orient) the scanned images. In order to do this, a digital data layer that matches features on the paper *Corresponding author: mholden@clarku.edu maps is needed. We used the Massachusetts 2000 Census roads layer (3) because roads are the only abundant features on the 1951 maps. We then imported the georeferenced images into the soft- ware R2V, which automatically vectorized (traced) the various land-cover areas. We manually edited this vector file for quality control and labeled each land category in the ESRI software ArcMap. Finally, we imported the vector land-cover data into the GIS software Idrisi at a resolution of 30-m pixels in order to make it compatible with the digital land-cover map for 1999 and to perform analyses. We reclassified the land of the 1951 and 1999 maps into the seven categories of the Anderson Level I classifi- cation system (4): Built. Agriculture. Range, Forest, Water, Wet- land, and Barren. The most common technique for assessing landscape change is to compare maps of two times, Tl and T2. A naive interpretation of this map comparison would lead to the conclusion that the differences between the maps of Tl and T2 indicate landscape change. However, even if there were no landscape changes be- tween Tl and T2, possible errors in both maps would result in differences between the two maps. To overcome this problem, we created a method to distinguish between the difference due to map error and difference due to true 258 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 1UU 0) a 98 •.• :- if a , . ^. ercent of Lands 00 U3 IO to U oo o to £> a 0 difference D agreement a. oc 1971-1999 1971-1971 1999-1999 Map Comparison Years Figure 1. Method lo estimate change from 1971 to 1999 among seven land categories for 22 towns of the Ipswich River watershed. change. The method estimates the expected difference between the maps assuming no change on the landscape between Tl and T2. based on accuracy assessments of the original maps. The true landscape change is thus calculated as the observed difference minus the expected difference. We have applied this technique using existing digital maps, where Tl = 1971 and T2 = 1999. We will also apply this technique to the 1951 maps when we are finished digitizing. Systematic accuracy assessment does not exist for the maps of 1971 and 1999. The map producer estimates the combined accu- racy of both maps to be approximately 98% because the categories seemed obvious from the high-quality photography used in the map-making process; the quality of the 1999 photographs was higher than those from 1971 (David Goodwin, University of Mas- sachusetts, Amherst, pers. comm.). Check plots were done on the 1971 and 1999 data to compare the final digitized land-cover map to the photo-created base map. In our analytic technique, the producer of the 1971 map is called A, and the producer of the 1999 map is called B. Based on the accuracies reported above, we assume that producer A makes maps with an accuracy of 97% for each category, and that producer B makes maps with an accuracy of 99% for each category. The left bar of Fisure 1 shows that the observed agreement is 88% in the direct comparison between the map of 1971 and 1999. The middle bar of Figure 1 shows that if producers A and B were each to make a map of the 1971 landscape, then the expected agreement between their two maps would be 96%. Since the observed agreement is 88%. the computed true landscape change is 8%. The right bar of Figure 1 shows that if producers A and B were each to make a map of the 1999 landscape, then the expected agreement between their two maps would be 97%, thus the com- puted true landscape change is 9%. Consequently, the technique estimates the true change to be between 8% and 9%. Our technique may underestimate the amount of true change because we assume that some of the observed differences are due to map error. Therefore, we should interpret the calculation of the tnie change as a lower bound on the estimate of landscape change. Our method is conservative because it recognizes that differences in maps do not always indicate true landscape change. The National Science Foundation (NSF) supported this work through the Research Experience for Undergraduates Summer Fellowship Program viu the Marine Biological Laboratory (Award OCE-9726921). Research was conducted at Clark University's Human Environment Regional Observatory, which is also sup- ported by NSF (Award SES-0243772, SubAward 2500-CU-NSF- 3772). David Goodwin of the University of Massachusetts Am- herst provided scanning equipment and expertise. Joseph Spencer and Louis Paladino provided their georeferencing knowledge. We thank reviewers for helpful comments. Literature Cited I Pontius, R. G., Jr., L. Claessens, C. Hopkinson. Jr., A. Marzouk, E. Rastetter, L. Schneider, and J. Vallino. 2000. Proceedings of the 4th International Conference on Integrating GIS and Environmental Modeling. CIRES. University of Colorado. Boulder. Online. Available: http://www.colorado.edu/research/cires/banff/pubpapers/ 1 65/ ( accessed June 2003). 2. MacConnell, W. 1951. Massachusetts Land-Use, 1: 31680. Univer- sity of Massachusetts. Amherst: U.S. Geol. Survey. 3 Massachusetts Geographic Information System. 2000. Online. Available: http://www.state.ma.us/mgis/cen2000_tiger.ntm (accessed June 2003). 4. Anderson, J. R., E. E. Hardy. J. T. Roach, and R. E. Witmer. 1976. U.S. Geol. Sun-ey Prof. Paper <->f>4. Kddvnce: Biol. Bull. 205: 259. (October 2003) . http://www.biolbull.org CIENCE HIITY EHICLE, MOTORIZED INVERTED MICROSCOPE. microscope will position your research lab at the very vanguard of multi-wavelength advanced fluorescence, DIG and deconvolution techniques. It is totally motorized from the built-in Z-Drive, 6-position nosepiece, 6-position condenser, 6-position fluorescence filter turret, and transmitted and ' shutters. Nine access ports allow you to keep dedicated cameras and lasers in place and still have plenty of ports available for new devices. The 1X81 demonstrates Olympus' leadership in Total Internal Reflection Fluorescence Microscopy (TIRFM) with exceptional e of operation and features such as the fully integrated TIRFM illuminator, a 1 .45 NA TIRFM objective and the exclusive 1 .65 NA objective. The Olympus 1X81 provides extraordinary system flexibility that will satisfy the most demanding research applications. OLYMPUS EOLYM ROCKET SCIENCE-. icroscopes :l 2003 OLYMPUS AMERICA INC. OLYMPUS Cover Sabellids, or feather-duster worms, are a diverse group of polychaete annelids common in many benthic marine habitats. Each adult sabellid secretes and inhabits an organic tube, from which it extends a crown of tentacles used for both suspension feed- ing and respiration. The image on the cover shows three individuals of a sabellid common in shallow waters of the northeastern Pacific, Schizobranchia insignis; its generic name reflects its distinctive, dichotomously branching tentacles (which, because of their respiratory function, are sometimes called branchiae). In life, the crown of the largest individ- ual shown here is about 4 cm in diameter. Like the planktonic larvae of other sabellids, those of Schizobranchia insignis do not ingest particulate food; instead, development to metamorphosis is fueled by lipids, proteins, and carbohydrates stored in the eggs. Phylogenetic evidence suggests that the nonfeeding mode of development common to sa- beiiids is a specific example of a frequent evolu- tioiK.r; transition among marine invertebrates — the loss of ihe requirement for particulate food during the larval stage. In most known cases, this transition in larval nulrition is soon followed by dramatic reduction or outright loss of the larval structures involved in feeding. But as reported by Bruno Per- net in this issue of The Biological Bulletin (p. 295), the nonfeeding larvae of S. insignis (and some other sabellids) possess ciliary bands that resemble, in form and behavior, bands that are used by the feeding larvae of closely related annelids to capture food particles (see Figures 3 and 4, pp. 300 and 301). In fact, larvae of S. insignis can use these vestigial feeding structures to capture food particles and transport them to the mouth; but because the mouth is not connected to the midgut, these parti- cles cannot be ingested. A functional digestive sys- tem does not develop in these larvae until well after metamorphosis. Why the larvae of some sabellids retain functional particle capture systems despite loss of both the need for food and the ability to ingest it is an interesting puzzle that remains unresolved. More generally, however, these observations suggest new, testable hypotheses about the developmental processes underlying the evolutionary loss of larval feeding in annelids and other animals with embryos that undergo spiral cleavage. The image on the cover was photographed by Bruno Pernet (Friday Harbor Laboratories, Univer- sity of Washington). The cover was designed by Beth Liles (Marine Biological Laboratory. Woods Hole. Massachusetts). Section Editor Online Editors THE BIOLOGICAL BULLETIN DECEMBER 2003 Iditor Associate Editors Editorial Board Editorial Office MICHAEL J. GREENBERG Louis E. BURNETT R. ANDREW CAMERON CHARLES D. DERBY MICHAEL LABARBERA SHINYA INOUE, Imaging and Microscopy JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG JOAN CERDA ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL KENNETH M. HALANYCH GREGORY HINKLE 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 Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center. Woods Hole Hunter College, City University of New York University of California. Davis Center of Aquaculture-IRTA. Spain Bodega Marine Lab.. University of California, Davis Louisiana State University Oregon Institute of Marine Biology. Univ. of Oregon Hopkins Marine Station, Stanford University Auburn University. Alabama Dana Farber Cancer Institute, Boston 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 Published by MARINE BIOLOGICAL LABORATORY Woons HOLE, MASSACHUSETTS http://www.biolbull.org 2004 SUMMER RESEARCH FELLOWSHIPS FUNDF AVAILABLE FOR SUMMER RESEARCH IN NEUROSCIENCE AT IE MARINE BIOLOGICAL LABORATORY IN WOODS HOLE The Marine Biological Laboratory is pleased to announce the availability of funding for the following summer research programs in Neuroscience in 2004. These programs will provide up to 550,000/year/award with a possibility for renewal for 3 years. Scholars in these programs will benefit from the rich intellectual and interactive environment of the scientific community at the MBL. APPLICATION DEADLINE: JANUARY 30, 2004 FOR NEUROSCIENCE FELLOWSHIPS Applications are encouraged from women and members of underrepresented minorities. Albert and Ellen Grass Faculty Grants Program Proposals must describe collaborative research in any area of neuroscience by teams of two or more investigators, with a minimum stay of six weeks at the Marine Biological Laboratory in Woods Hole. Collaborative teams must consist of a minimum of two Assistant/Associate Professor level neuroscientists. Collaborative units may be formed with senior investigators but only the more junior neuroscientists will receive direct funding. Awards provide costs for research and laboratory rental, plus housing and travel costs for the Pis. Dart Foundation Scholars Program in Learning & Memory Proposals must be devoted to the study of learning and memory with a minimum stay of six weeks at the Marine Biological Laboratory in Woods Hole. Applications are encouraged from junior or senior level neuroscientists holding a Ph.D., M.D. or equivalent degree. Awards provide costs for research and laboratory rental, plus housing and travel costs. FUNDING AVAILABLE FOR SUMMER RESEARCH The Marine Biological Laboratory is pleased to announce the availability of funding for the following summer research programs in 2004 for junior or senior investigators holding a Ph.D., M.D., or equivalent degree. These prestigious awards provide costs for research and housing. Proposals for Fellowship support will be considered in, but are not limited to, the following fields of investigation: Cellular & Molecular Physiology Developmental Biology Ecology Microbiology Molecular Biology Neurobiology Parasitology APPLICATION DEADLINE: JANUARY 15, 2004 FOR SUMMER RESEARCH FELLOWSHIPS Applications are encouraged from women and members oj underrepresented minorities. FOR APPLICATION FORMS AND ADDITIONAL INFORMATION, PLEASE CONTACT: Sandra Kaufmann, Fellowship Coordinator 508-289-7441; skaufman@mbl.edu Visit our web site at http://www.mbl.edu/fellowships Tlie MBL is an Equal Opportunity/ Affirmative Action Employer. Marine Biological Laboratory-? MBL Street~Woods Hole~Massachusetts~02543 CONTENTS VOI.UMK 205, No. 3: DECEMBER 2003 PHYSIOLOGY AND BIOMECHANICS Motokawa, Tatsuo, and Akifumi Tsuchi l)\namic mechanical properties of body-wall dermis in various mechanical states and their implications for the behavior of sea cucumbers 261 Tomanek. Lars, and Eric Sanford Heat-shock protein 70 (HspTO) as a biochemical stress indicator: an experimental field test in two congeneric intertidal gastropods (Genus: Tegula). . . 276 DEVELOPMENT AND REPRODUCTION Byrne, Maria, Michael W. Hart, Anna Cerra, and Paula Cisternas Reproduciion and larval morphology of broadcasting and viviparous species in the Cryptasterina species complex 285 Peniet, Bmno Persistent ancestral feeding structures in nonfeeding annelid lanae 295 Ruddell, Carolyn J., Geoffrey Wainwright, Audrey Gef- fen, Michael R. H. \VTiite, Simon G. Webster, and Huw H. Rees Cloning, characterization, and developmental ex- pression of a putative farnesoic acid O-methvl trans- ferase in the female edible crab Cancer pagimis .... 308 Lasker, Howard R., Michael L. Boiler, John Castanaro, and Juan Armando Sanchez Determinate growth and modularity in a gorgonian octocoral 319 SYMBIOSIS AND PARASITOLOGY Joyner, Joanna L., Suzanne M. Peyer, and Raymond W. Lee Possible roles of sulfur-containing amino acids in a chemoautotrophic bacterium-mollusc symbiosis . . . 331 Schwarz, J. A., and V. M. Weis Localization of a symbiosis-related protein. Sym32, in the Anthopleura ekgantissima-Symbiodinium musculnn-i associadon 339 ECOLOGY AND EVOLUTION Price, Rebecca M. Columellar muscle of neogastropods: muscle attach- ment and the function of columellar folds 351 Frick, Kinsey Response in nematocyst uptake bv the nuclibranch Flabellina vemicosn to the presence of various preda- tors in the southern Gulf of Maine 367 Index for Volume 205 377 ERRATUM On page 175 of the October issue (Volume 205. Number 2). we erred in listing the winner of the Senior In\ estimator category of the awards for a Short Report presented at the 2003 General Scientific Meetings of the Marine Biological Laboratory. Karen Crawford, who was listed as receiving honorable mention, should have been named as the winner of the award; Paul Gallant received honorable mention. 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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 vears of hindin 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 Afilysia ... 16 Bergman, Daniel A., and Paul A. Moore Field observations of intraspecific agonistic behavior of two crayfish species, Orconectes rusticus and <>r- conertes virilis, in different habitats 26 PHYSIOLOGY AND BIOMECHANICS Etnier, Shelley A. Twisting and bending of biological beams: distri- bution of biological beams in a stiffness mechano- space 36 Eyster, L. S., and L. M. van Camp Extracellular lipid droplets in Idiosepius notoides. the Southern pvgmv 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- gata 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 Anthnpleura brillii (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 Pecti-n maximus L 83 Annual Report of the Marine Biological Laboratory . . . Rl No. 2: OCTOBER 2003 RESEARCH NOTE Seibel, Brad A., and Heidi M. Dierssen Cascading trophic impacts of reduced biomass in the Ross Se.t, Antarctica: Just the tip of the iceberg? . . . Lee, Raymond W. Thermal tolerances of deep-sea hydrothermal vent animals from the Northeast Pacific. 93 98 ECOLOGY OF PARASITES Fingerut, Jonathan T., Cheryl Ann Zimmer, and Rich- ard K. Zimmer Patterns and processes of larval emergence in an estuarine parasite svslem Ill) 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 Vampyroteuthis iii/rninlis 102 DEVELOPMENT AND REPRODUCTION Gibson, Glenys D. Larval development and metamorphosis in Plfuro- branchaea maculata, with a review of development in the Notaspidea (Opisthobranchia) 121 CONTENTS: VOLUME 205 ECOLOGY AND EVOLUTION Chadwick-Fumv Nanette E., and Irving L. Weissman Effects nl neic contact on life-history traits of the coir.; •< : ascidian Botiyllus srhlossm in Monterey Bav 133 Bell, J. J., and D. K. A. Barnes Effect of disturbance on assemblages: an example using Porifera 144 PHYSIOLOGY AND BIOMECHANICS Hamdoun, Amro M., Daniel P. Cheney, and Gary N. Cherr Phenotypic 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 SHORT REPORTS FROM THE 2003 GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY The Editor The MBL Awards for 2003 175 DEVELOPMENTAL BIOLOGY Gilland, Edwin, Robert Baker, and Winfried Denk Long duration three-dimensional imaging of calcium waves in zebrafish using multiphoton fluorescence microscopy ................................. 176 < .ili-. nh. 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 HNK-1/N-CAM immunoreactivity correlates with cil- iary patterns during development of the polychaete Capitella sp. I ............................... 182 CELI Heck, D. i ;nd J. D. Laskin Ryanod \< < !-,hive calcium flux regulates motility of Arh/u in , - r, ^perm ...................... 185 Gallant, P. E. Axotomy inhibits the slow axonal transport of tubulin in the squid giant axon ....................... 1S7 Delacniz, John, Jeremiah R. Brown, and George M. Langford Interactions between recombinant conventional squid kinesin and native myosin-V 188 DeSelm, Carl J., Jeremiah R. Brown, Renne Lu, and George M. Langford Rab-GDI inhibits myosin V-dependent vesicle trans- port in squid giant axon 190 Pielak, R. M., V. A. Gaysinskaya, and W. D. Cohen Cytoskeletal events preceding polar body formation in activated Sp/sula 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-11-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 Micrurinna prolifera 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 Limit/us blood clot 201 Isakova, Victoria, and Peter B. Armstrong Imprisonment in a death-row cell: the fates of mi- crobes entrapped in the Limulu* 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, Limit/in polyphemui 205 NEUROBIOLOGY AND BEHAVIOR Bogorff, Daniel J., Mark A. Messerli, Robert P. Mai- chow, and Peter J. S. Smidi 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 Xenapus 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- lini has no residual effects on physiological record- ings of supramedullary/dorsal neurons of the cun- ner, Tautogolabrus adspersus 211 Redenti, S., and R. L. Chappell Zinc chelation enhances the sensitivity of the ERG b-wave in dark-adapted skate retina 213 CONTENTS: VOLUME 205 Molina, Anthony J. A., Katharine Hanimar, Richard Sanger, Peter J. S. Smith, and Robert P. Malchow [ntracellular release ol caged calcium in skate hori- zontal cells using line optical fibers -15 Palmer, L. M., B. A. Ginffrida, and A. F. Mensinger Neural recordings from the lateral line in free-swim- ming (oadfish, ()/><,nin<\ Ian 216 Child, F. M.. H. T. Epstein, A. M. Kuzirian, and D. L. Alkon Memon reconsolidation in Hermissenda -IS Kuzirian, A. M., F. M. Child, H. T. Epstein, M. E. Motta, C. E. Oldenburg, and D. L. Alkon Training alone, not the tripeptide RGI), modulates calexcitin in liiriin\\i-nda 220 Savage, Anna, and Jelle Atema Neuroclieinical modulation of behavioral response to chemical stimuli in Homarus nmmiinni* 222 Mann, K. D., E. R. Turnell, J. Atema, and G. Gerlach Kin recognition in juvenile zebrafish (Danio reria) based on olfactory cues 224 Turnell, E. R., K. D. Mann, G. G. Rosenthal, and G. Gerlach Mate choice in zebrafish (l)nnin rerio) analyzed with video-stimulus techniques 225 Mm n 1 1 \n BIOLOC,}, P.viiioi.iH-.Y. A\I> MICROBIOLOGY Roberts, S. B., and F. W. Goetz Expressed sequence tag analysis of genes expressed in the bav scallop. Argopecten irradians 227 Hsu, A. C., and R. M. Smolowitz Scanning electron microscopy investigation of epizootic lobster shell disease in Homanis ameri- canu\ 228 Orchard, Elizabeth, Eric Webb, and Sonya Dyhrman Characterization of phosphorus-regulated genes in Trichodesmiiim spp 230 Galac, Madeline, Deana Erdner, Donald M. Anderson, and Sonya Dyhrman Molecular quantification of toxic Alexandrium jundy- ense in the Gulf of Maine 231 Sangster, C. R., and R. M. Smolowitz Description of Vibrio algiTiolyticui infection in cul- tured Sepia offirintili\, Si'ji/f/ ri/if/iiui, and Si'/nn />/i/irn- ,»>is 233 Baird, Krystal D., Hemant M. Chikarmane, Roxanna Smolowitz, and Kevin R. Uhlinger Detection of Edit\n>im l/iu bv polvmerase chain reaction 235 Weidner, Earl, and Ann Findley Catalase in microsporidian spores before and during disc barge 236 Eioini.'i \\l> I'nri I \ll(>.\ BlOLoi,} 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 Thorns, T., A. E. Giblin, K. H. Foreman Multiple approaches to tracing nitrogen loss in the West Falmouth wastewaler 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 Radiochemical 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 Aguiar. 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 ilva lactura 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 Ulvn 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, Limnlm /M>l\/>li<'»ni\. 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 <>i; \i /'/,'/ s/ \ / i/vo.y.s Published bv title onl\ . . 259 CONTENTS: VOLUME 205 No. 3: DECEMBER 2003 PHT ;OGY AND BIOMECHANICS Motokatv: ; atsuo, and Akifumi Tsuchi |) mk mechanical properties of body-wall dermis in \.triotis mechanical states and their implications for the behavior of sea cucumbers 261 Tomanek, Lars, and Eric Sanford Heat-shock protein 70 (Hsp70) as a biochemical stress indicator: an experimental field test in two congeneric intertidal gastropods (Genus: Tegula). . . 276 DEVELOPMENT AND REPRODUCTION Byrne, Maria, Michael Hart, Anna Cerra, and Paula Cistern as Reproduction and larval morphology of broadcasting and viviparous species in the Crypla\lmna species complex 285 Fernet, Bruno Persistent ancestral feeding structures in nonfeeding annelid larvae 295 Ruddell, Carolyn J., Geoffrey Wainwright, Audrey Gef- fen, Michael R. H. White, Simon G. Webster, and Huw H. Rees Cloning, characterization, and developmental ex- pression of a putative farnesoic acid O-methyl trans- ferase in the female edible crab Cancer pagurus .... 308 Lasker, Howard R., Michael L. Boiler, John Castanaro, and Juan Armando Sanchez Determinate growth and modularity in a gorgonian octocoral 319 SYMBIOSIS AND PARASITOLOGY Joyner, Joanna L., Suzanne M. Peyer, and Raymond W. Lee Possible roles of sulfur-containing amino acids in a chemoautotrophic bacterium-mollusc symbiosis . . . 331 Schwarz, J. A., and V. M. Weis Localization of a symbiosis-related protein, Sym32, in the Anthopleura elegantissima—Symbiodinium muscatinei association 339 ECOLOGY AND EVOLUTION Price, Rebecca M. Columellar muscle of neogastropods: muscle attach- ment and the function of columellar folds 351 Frick, Kinsey Response in nematocyst uptake by the nudibranch Flabellina verrucosa to the presence of various preda- tors in the southern Gulf of Maine 367 Index for Volume 205 377 Reference: Bio/. Bull. 205: 261-275. (December 2003) © 2003 Marine Biological Laboratory Dynamic Mechanical Properties of Body-Wall Dermis in Various Mechanical States and Their Implications for the Behavior of Sea Cucumbers TATSUO MOTOKAWA* AND AKIFUMI TSUCHI Dcpanment of Biological Sciences, Graduate School of Bioscience and Biotechnology. Tokyo Institute of Technology, Meguro, Tokyo, 152-8551 Japan Abstract. The dermis of the sea cucumber body wall is a typical catch connective tissue that rapidly changes its me- chanical properties in response to various stimuli. Dynamic mechanical properties were measured in stiff, standard, and soft states of the sea cucumber Actinopyga mciuritiana. Sinusoidal deformations were applied, either at a constant frequency of 0.1 Hz with varying maximum strain of 2%- 20% or at a fixed maximum strain of 1.8% with varying frequency of 0.0005-50 Hz. The dermis showed viscoelas- ticity with both strain and strain-rate dependence. The der- mis in the standard state showed a J-shaped stress-strain curve with a stiffness of 1 MPa and a dissipation ratio of 60%; the curve of the stiff dermis was linear with high stiffness (3 MPa) and a low dissipation ratio (30%). Soft dermis showed a J-shaped curve with low stiffness (0.3 MPa) and a high dissipation ratio (80%). The strain-induced softening was observed in the soft state. Stiff samples had a higher storage modulus and a lower tangent 8 than soft ones, implying a larger contribution of the elastic component in the stiff state. A simple molecular model was proposed that accounted for the mechanical behavior of the dermis. The model suggested that stiffening stimulation increased inter- molecular bonds, whereas softening stimulation affected intra-molecular bonds. The adaptive significance of each mechanical state in the behavior of sea cucumbers is dis- cussed. Introduction Echinoderms have collagenous connective tissues that can alter their mechanical properties rapidly under nervous Received 1 2 May 2003; accepted 5 September 2003. * To whom correspondence should be addressed. E-mail: tmotokaw® bio.titech.ac.jp control (Motokawa. 1984a; Wilkie, 1996). Connective tis- sue with such mutability is called catch connective tissue or mutable connective tissue. The mutability has been attrib- uted to the changes in the mechanical properties of the extracellular materials in the tissue (Motokawa, 1984a; Wilkie, 2002; Tipper ct ai, 2003). The dermis in the body wall of sea cucumbers is a favored material for studies of connective tissue catch because of its large size. The me- chanical properties of the dermis have been described by various mechanical methods such as creep tests (Motokawa, 1981; Eylers, 1982), stress-strain tests (Motokawa. 1982), stress-relaxation tests (Motokawa, 1984b; Greenberg and Eylers, 1984), tensile tests (Motokawa. 1982), and dynamic tests (Shibayama et at.. 1994; Szulgit and Shadwick, 2000). These studies revealed the viscoelastic nature of the dermis. They drew, however, contradictory conclusions about which component, elastic or viscous, changed during alter- ations in mechanical properties. Motokawa (1984b) con- cluded from the results of creep tests and stress-relaxation tests that the changes in the mechanical properties mainly occurred in the viscous component, whereas Szulgit and Shadwick (2000) concluded that it was the elastic compo- nent that changed. The former used a fixed strain and the latter a fixed strain and strain rate. The apparently contra- dictory conclusions of these two studies are not surprising, because viscoelastic materials exhibit different properties when tested under different conditions of strain and strain rate (Wainwright et ai, 1976). Therefore, description of the mechanical properties of viscoelastic materials is not satis- factory in a limited range of strain and strain rate. The present study was undertaken to describe the dynamic me- chanical properties of the holothurian dermis under wide ranges of both strain and strain rate. The information ob- tained establishes a basis for understanding the mechanism of connective tissue catch and how sea cucumbers adapt to 261 262 T. MOTOKAWA AND A. TSUCHI various mechanic;' 1 environments by changing the mechan- ical propertie: •>' the Jermis. Materials and Methods Tissu Specimens of the aspidochirotid sea cucumber Acti- nopvgu mauritiuna (Quay and Gaimard) were collected from the lagoon in front of Sesoko Marine Science Center, University of the Ryukyus, Okinawa. They were shipped to Tokyo Institute of Technology and kept in an aquarium in our laboratory. This sea cucumber has a thick body wall (about 1-1.5 cm). The connective tissue dermis occupies most of the thickness. The outer side of the dermis is covered with a thin epidermis, and the inner side is lined with body wall muscles. Unlike some dendrochirotid spe- cies such as Cuciunaria frondosa, the dermis of this aspi- dochirotid sea cucumber looks uniform, showing no differ- entiation into layers. A dermis sample was dissected from the lateral interambulacrum of the body wall. Both the epidermis and muscles were removed and the middle por- tion of the dermis was cut out for experiments. The dimen- sions of the samples were measured with a caliper to a precision of 0.05 mm. The mean length of the samples from oral to aboral end was 2.3 mm (±0.89 mm SD; n = 79), and the mean cross-sectional area was 5.4 mm2 (±1.9 mnr SD; ;; = 79). The samples were rested in artificial seawater of normal composition (ASW) for 17-24 h before being subjected to mechanical tests. The temperature of the sea- water was 18-25 °C, which roughly corresponded to the temperature of the water from which the sea cucumbers were collected. Mechanical tests were performed at a con- stant temperature of 20 °C. Samples that were rested in ASW for 1 7-24 h and tested in ASW are described here as being in a standard state. Soft-state samples were prepared by removal of calcium ions (Hayashi and Motokawa. 1986); they were rested in calcium-free artificial seawater (CaFASW) and tested in CaFASW. Stiff-state samples are those that were tested in ASW without a resting period. Rough physical handling makes the dermis stiffen reversibly (Motokawa, 1984c). Soon after the dissection, the dermis experienced quite rough physical handling, so these are termed the physically stimulated samples. This state probably corresponds to the state the sea cucumbers are in after being stimulated phys- ically. Two other kinds of stimulation were employed to invoke the stiff state (possibly through stimulating the stiff- ening iM-'-hanism in vivo) — chemical stimulation by the neurotrniismitter acetylcholine (ACh) (Motokawa, 1987) and chemi'." simulation using artificial seawater with an elevated pot: Vm concentration (KASW). The latter likely stimulates a a - mechanism that controls stiffness, such as nerves, through depolarization (Motokawa, 1981). The samples for chemical stimulation were rested for 17-24 h in ASW, and chemicals were applied 20 min before the mechanical testing. The composition of ASW was as follows (in mmol/1): NaCl. 433.7; KC1, 10.0; CaCk 10.1: MgCU. 52.5; NaHCO,, 2.5. In KASW, the concentration of potassium was raised to 100 mmol/1, and CaFASW con- tained 5 mmol/1 EGTA (ethylene glycol bis(j3-aminoethyl- ether)-/V, N, N' yV'-tetraacetic acid). In both cases, the so- dium concentration was adjusted to keep the osmotic concentration constant. ACh concentration was 10~4 mol/1 in ASW. The pH of all the solutions was adjusted to 8.2. Experimental apparatus A dermis sample was subject to forced vibrations using sinusoidal displacements. The sample was stretched and compressed cyclically, and the resulting forces were re- corded. The experimental apparatus (Fig. 1) included a vibrator (511-A, EMIC, Japan) driven by sinusoidal cur- rents that were generated by a function generator (SG-4101, Iwatsu, Japan). The force developed in the dermis was measured by a micro load cell (LTS-200GA, Kyowa, Ja- pan). The compliance of the load cell was 0.3 jum/g, which contributed at most 4% to the measured value of strain in the present experiments. The deformation of the dermis was monitored by an eddy-current displacement sensor (502-F, EMIC, Japan). Force signals were amplified by a strain amplifier (DPM-602A, Kyowa, Japan). Both force and dis- placement signals were displayed on an oscilloscope and simultaneously recorded by a computer through a data- acquisition unit (Lab Stack, Keisoku Giken, Japan). The dermis sample was glued with cyanoacrylate glue to the holders, one attached to the vibrator and the other to the load Figure 1. Schematic of the experimental apparatus tor dynamic tests. VISCOELASTICITY OF SEA CUCUMBER 263 cell, and an experimental solution was introduced to the trough. The sample was usually rested for 20 min in the trough; however, the physically stimulated sample was tested immediately. The trough was water-jacketed to keep the temperature constant at 20 °C. We performed two series of experiments. In the constant- frequency experiments, the frequency of vibration was kept constant at 0.1 Hz and the maximum strain in a cycle was varied between 2% and 20%. A frequency of 0.1 Hz was chosen because the differences in the mechanical properties of the three states were most prominent at this frequency (see Results). In the constant-maximum-strain experiments, the maximum strain in a cycle was kept at 1.8% and the frequency was varied between 0.0005 and 50 Hz. A maxi- mum strain of 1 .8% was chosen because the dermis behaved as a linear viscoelastic material at this strain (see Results). The samples were preconditioned by applying oscilla- tions for 10 min prior to the test; the amplitude and fre- quency were the same as those in the test. A steady-state response was reached by the preconditioning. Constant-frequency experiment In experiments that examined the effects of strain, a dermis sample was subjected to successive oscillatory tests with different levels of maximum strain. The hysteresis loop of the stress-strain relationship showed almost point sym- metry. The point of symmetry was brought to the origin of the coordinates by adjusting the length of the sample. Data were then collected, and a typical hysteresis loop was gen- erated for that maximum strain. When the data acquisition was finished at a certain maximum strain, the maximum strain in an oscillatory cycle was increased by about 4% (range 2%— 7%), the sample was preconditioned, and data were collected with the new maximum strain. Strain (e) was defined as the length change divided by the original length of the sample, and stress (cr) as the force divided by the cross-sectional area at the original length. When stress was plotted against strain, a closed hysteresis loop was obtained (Fig. 2). Because the dermis behaved quite similarly in tension and in compression, the values given in this paper are the averages of the absolute values at equal strains in tension and in compression. The maximum strain imposed in a loop was denoted as the maximum strain (emax), and the maximum stress observed in a loop was denoted as the maximum stress (crmax). A quarter of the loop had the shape of the letter "J" with a flat "toe" region and a more vertical "pole" region. We introduced an index (the J index) to quantify the degree of concavity of the stress- strain curves in the loading and unloading phases in tensile strain. We defined the J index as the area enclosed by the stress-strain curve and the line connecting the peak point with the intersecting point at 0 strain in the curve, divided by the area of a right triangle whose hypotenuse was the same line as before, one side was a horizontal extending stress strain Figure 2. Schematic of a hysteresis loop of the dermis in standard state under a sinusoidal deformation of 10% maximum strain at 0. 1 Hz. The loop showed clockwise rotation (arrows). The stiffness was defined as the slope of the dotted line. The darker hatched area represents the dissipated energy, the lighter hatched areas the conserved energy, and the total hatched area the deformation energy. The inset shows how the J index was measured: it was the percent ratio of the hatched area to the area of the triangle shown by dotted lines. from the intersecting point, and the other side was a vertical extending from the peak point (Fig. 2 inset). The J index was equivalent to the difference between the energy fed into the specimen and the deformation energy of an elastic body with a linear stress-strain curve; the J index is thus a measure of how concave the stress-strain curve was. The J index was defined as 0 when the curve was convex. The stiffness was defined as the slope between the two peaks of a hysteresis loop. The deformation energy (£,) was defined as the hatched area in Figure 2. It corresponds to the energy required to deform the dermis of a unit volume for one cycle. In each cycle of deformation, the deformation energy is partly conserved and reused as elastic recoil to restore the geometry of the sample; the rest is dissipated and lost, primarily as thermal energy. The energy dissipated (£(/) corresponds to the area enclosed by the hysteresis loop. The dissipation ratio D was defined as D = EJE, and was expressed as a percentage. The limit strain was estimated as the average of the smallest maximum strain that caused the strain-induced softening and a maximum strain one step smaller (where strain-induced softening was not observed). Reversibility of the response was determined by succes- sive mechanical tests using the same sample. The sample in the stiff state caused by physical stimulation was tested, then rested for 22 h and tested again. In the chemically 264 T. MOTOKAWA AND A. TSUCHI stimulated stiff samples, the preparations were first tested in ASW and then trea-.'.-d with stimulation media for 20 mm before the second mechanical tests. Samples were washed thoroughlv in ASW for 24 h. and the third mechanical test was performed. We also determined whether softening induced by stretching the samples in CaFASW beyond the limit strain was recoverable when the maximum strain was reduced to less than the limit strain. The sample was tested at smax = 2% in CaFASW. Then oscillations of emax of about 30% were given for 10 min, and the sample was tested again at Smax = 2%. Constant-maximum-strain experiment In this experiment, the maximum strain was kept constant at 1.8% and the frequency was varied in a dermis sample. A sample was tested first at 0. 1 Hz. Then the test frequency was either increased stepwise to 0.5. 1, 5. 10. and 50 Hz or decreased to 0.05. 0.01. 0.005. 0.001. and 0.0005 Hz. The data from 10 cycles at each frequency were averaged in all experiments except at the lowest two frequencies, where data for two cycles were averaged. In a viscoelastic material, stress and strain are not in phase — rather, strain lags behind stress by a phase angle 8. For a linear viscoelastic material, the complex modulus E* , the storage modulus £". and the loss modulus £"' are defined as follows (Oka. 1974). E* = ale = E' + IE" E' = \E* cos 8 E" = |E* | sing tan 8 = E"/E' The complex modulus represents the conventional "stiff- ness." The storage modulus is equivalent to the elastic modulus in phase with the stress and is a measure of the energy elastically stored in each cycle. The loss modulus is the out-of-phase component and is a measure of the energy dissipated. Tangent 8 is the ratio of the energy lost to the energy stored. The soft state was induced by a vibration of 0. 1 Hz with ±20% maximum strain applied for 30 min in CaFASW. Such a treatment caused strain-induced softening (see Re- sults). The softened sample was subsequently tested in CaFASW. Results Constant-f ret/i. < Hvsteresis loop at maximum strain -• 70% and fre- quency = 0.1 Hz. When stress was plotted against strain, a closed hysteresis loop of clockwise rotation was generated. The loop could be divided into four phases: a tensile phase in which the dermis was gradually stretched, a tension- unloading phase in which the tensile strain decreased, a compressing phase in which the dermis was gradually com- pressed, and a compression-unloading phase in which the compressive strain decreased. The hysteresis loop of a sample in the standard state at 10% maximum strain is shown in Figure 3a. When stretched from zero strain, the dermis could be deformed quite easily: thus the slope of the curve was flat at first, corresponding to the toe region of the J. When strain exceeded about 5%, the slope became progressively steeper; thus the tensile stress- strain relation followed a typical J-shaped curve (Wain- wright etui.. 1976). The J index averaged 43% (±9.1% SD, n ---- 17). After the tensile stress peaked, the unloading phase started and stress decreased rapidly. The slope was steeper than that in the loading phase at each strain. As the strain progressively decreased, the slope declined and the curve became almost flat. The J index of the unloading phase averaged 71% (±13% SD. n == 17). much greater than that of the loading phase (significant difference by paired t test, P < 0.01). In the loading and unloading phases of the compression half of the cycle, the curve followed almost the same course as that of the tensile half with the sign of stress and strain reversed. Thus the dermis behaved similarly in tension and compression. The four phases of the hysteresis loop all exhibited a J shape, and the whole loop showed approximate point symmetry. The loops of stiff samples differed from those of samples in the standard state both in shape and in the stress devel- oped (Fig. 3b, c. d). The J shape became less prominent: the toe region became short (restricted to strains under 2%-4%) and was not flat but had a steep slope so that there was only a small kink between toe and pole. Thus each phase ot the hysteresis loop in stiff samples was rather straight, which made the J index small. The J indices of the loading phase in samples stiffened by physical stimulation. KASW stim- ulation, and ACh stimulation were 9.0%- ± 8.2% (n = 4). 19% ± 1 1% (n = 4), and 26 ± 6.0 (n = 5), respectively (average ± SD). The average J indices were significantly smaller than that of the standard state (Scheffe test. P < 0.01 ). In stiff samples, the shape of the stress-strain curve during the unloading phase showed no great difference from that of the loading phase. The J indices during the unloading phase in samples stiffened by physical stimulation. KASW stimulation, and ACh stimulation were 14% ± 8.6% (n = 4). 23% ± 12% (// = 4), and 33 ± 8.9 (/; == 5), respectively (average ± SD). The J index of the unloading phase in a hysteresis curve was slightly larger than that of the loading phase, but the statistical comparison of the averages showed no significant difference. In stiff samples, the maximum stress was much larger and the area enclosed by a loop was much smaller than in samples in standard state. These features implied that the stiffened dermis be- haved like a stiff, resilient spring. The hysteresis loop of soft samples was rather flat in VISCOELASTICITY OF SEA CUCUMBER 265 0.5 o o -0.5 -1 I 0 -2 -10 0 strain (%) 10 -2 -4 -2 -10 10 -10 10 -10 10 0.6 0.3 2 0 -0.3 -0.6 -10 0 10 Figure 3. Typical hysteresis loops at 10% maximum strain and 0.1 Hz frequency, (a) Standard state, (b-d) Stiff state induced by physical stimulation (b), by KASW (c), and by 10~4 mol/1 acetylcholme (d). (e) Soft state. loading (Fig. 3e). This is because the toe region was similar, both in length (strain) and in height (stress) to that of the standard, but the pole was shorter: for example, the maxi- mum stress was much smaller in the soft state than in the standard state. The J index in loading phase averaged 27% ± 8.9% (average ± SD, n = 9) which was significantly 266 T. MOTOKAWA AND A. TSUCHI smaller than that of the standard state (Scheffe test, P < 0.01). The shape if the stress-strain curve during the un- loading phas ! ed marked differences from that of the loading ph;< ; 11^ unloading phase, stress sharply de- creased fff .he peak to give a J index as high as 86% ± 7.1% (average ± SD, n -- 9). which was statistically different from that of the loading phase (paired / test. P < 0.01 ). This made the area enclosed by the hysteresis loop larger. These features implied that the dermis in the softened state became more compliant and less resilient. Influence of maximum strain on the hysteresis loop. At emax = 0.5%-3%, the hysteresis loop followed a skewed ellipse regardless of the state of the samples, showing no signs of the J-shaped curve seen at larger strains. Both E* and tan 5 were constant in this strain range. Thus the dermis behaved as a linear viscoelastic material. The maximum strain was increased stepwise by incre- ments of 2%-4% starting from emax = 2%-3%. In standard samples, the hysteresis loops of emax -= 5%-25% were composed of prominent J curves. The peak stress became higher as emax increased (Fig. 4a). The J index increased as emax increased from 5% to 15%, and thus the stress-strain curve showed a more pronounced J shape as emax increased up to 15%. In stiff samples, the shape of the loop was quite similar for emax between 5% and 15% (Fig. 4b) with a constant J index. The maximum stress (/ MH cucumber dermis Maximum strain Mechanical properties 3% 10% 18% Stiffness (MPa) Stiff physical 3.1 ± 0.37 (5)** 3.4 ± 0.61 (4)** KASV\ 3.1 ± 0.95 (5)** 2.7 ± 0.53 (4)** ACh 2.3 ± 0.60(6)** 2.4 ± 0.85 (5)** Standard 0.77 ± 0.30(14) 1.0 ± 0.34(17) 1.1 ± 0.32(9) Soft 0.28 ±0.11 (4) 0.29 ±0.25(9)** 0.12 ±0.070(5)** Deformation energy (kPa) Stiff physical 5.0 ± 0.85 (5)** 38 ± 6.9(4)** KASW 5.1 ±1.1 (5)** 28 ± 9.1 (4)** ACh 3.7 ±0.61 (6)** 22 ± 7.6(5)** Standard 1.4 ±0.53(14) 9.4 ±3.9(17) 25 ±9.8 (9) Soft 0.48 ±0.31 (4) 3.0 ±2.8(9) 3.1 ±2.3 (5)** Dissipation ratio (%) Stiff physical 14 ± 1 1 (5)** 29 ± 7.0(4)' KASW 20 ± 11 (5)** 31 ± 5.1 (4)* ACh 32 ± 8.3 (6) 32 ± 11(5)* Standard 48 ± 10(14) 59 ± 18(17) 54 ± 12(9) Soft 61 ± 14(4) 78 ± 11 (9)* 77 ± 16(5)" A series of data at different maximum strain were obtained from the same sample. Values are averages ± SD (»). The average is that of samples that were taken from different individuals. For 3% and 10% maximum strains, the Schefte test was used for /mw hue statistical analysis after the analysis of \anance. An unpaired / test was used for 18% maximum strain. * The mean is significantly different from that of the standard (P < 0.05 ). '* The mean is significantly different from that of the standard (P < 0.0 I ). — No data were obtained because the samples detached from the holders when maximum strain exceeded about 15%. than 107r, the stiffness of soft samples was one-tenth that of the stiff state and one-third of the standard state. Because the peak stress (and thus the stiffness) decreased above the limit strain, the differences in the stiffness were more marked at emax = 18%, where stiffness was one-tenth of the standard (Table I ). Deformation energy increased with emax in all three states except for the region above the limit strain in soft samples (Fig. 5b). When compared with standard samples, stiff samples required 2-4 times more energy to deform, whereas soft samples required less energy, especially when em.,x exceeded the limit strain (Table 1 ). & S 0.1 0.01 5 10 15 20 maximum strain (%) 100 60 20 5 10 15 20 25 maximum strain (%) 5 10 15 20 maximum strain (%) 25 Figure 5. Effects ol maximum strain on mechanical properties measured at 0.1 Hz. A typical example was chosen for each state from 5 to 6 dermis samples from different individuals: (a) stiffness; (b) deformation energy; (c) dissipation ratio. Curves with the same symbol are from the same preparation. The stiff sample (cross) was produced by physical stimulation. Filled circle, standard sample; diamond, soft sample. 268 T. MOTOKAWA AND A. TSUCHI The dissipation ratio increased as emax increased from 2% to 5%, but it reimirvd almost constant for larger emax (Fig. 5c). About 601 deformation energy was dissipated in standard samp .s at smax = 10% (Table 1). The dissipation ratio was hah ed in stiff samples but increased to as much as 80% in soft samples. The average values at a maximum strain of 10% in the stiff and soft states were significantly different from that of the standard state (P < 0.05). Reversibility of responses. Whether the stiffened dermis, which was stimulated from the standard state, resumed the standard state after removal of the stimuli was examined by successive measurements on the same sample. The stiffen- ing response was reversible — stiffened samples resumed the standard state after resting in ASW for 1 day (Fig. 6). Although it took 2 days from the time of preparation to the completion of measurements in the chemically stimulated samples, the samples did not show any sign of decay, and the mechanical parameters in ASW after stimulation were similar to those in ASW before stimulation. Constant-maximmn-stniin experiments No increase in stress was observed during the precondi- tioning period, which implied that the imposed vibration of 1.8% strain did not act as a mechanical stimulus for the dermis. Stiffening of the dermis by forced vibration has been reported in other sea cucumbers but at much larger strains (Shibayama et til.. 1994). The frequency dependence of £*, £", £", and tan 8 in the standard state is given in Figure 7. The complex modulus £* gave a sigmoid curve (Fig. 7a). £* took a low and rather constant value of about 20 kPa at frequencies lower than 0.005 Hz. At 1 Hz and higher it showed a rather constant value of about 3 MPa. Tan 6 was more or less constant in the lower frequencies with a maximum value of about 1. and for frequencies exceeding 0.01 Hz it decreased with fre- quency to 0.06 at 50 Hz (Fig. 7b). £' exhibited a curve similar to that of £*, with the value for each frequency a little smaller than that of £* (Fig. 7c). £" also showed a sigmoid curve (Fig. 7d) with values similar to that of £' for frequencies lower than 0.01 Hz. However. £" was about one-tenth of £' in the high-frequency range, reflecting the fact that tan 5 was less than 1 at higher frequencies. The frequency dependence of stiff and soft states is given in Figure 8. All curves of stiff states induced by different stimuli coincided well, suggesting that the three stimuli invoked an identical mechanical state. The curve of £* in the stiff state was different in shape and position from that in the standard state (Fig. 8a). The curve was not sigmoid. It sharply increased with frequency in the low frequency range (5 X 10~4- 1 X 10~3 Hz): the rate of increase diminished as frequency increased, reaching a constant value of around 3 MPa. In the stiff state, £* was higher than E* of the standard at every frequency — by as much as two orders of magnitude at the lower frequencies. At frequencies higher than 0.5 Hz, however, the difference decreased by a factor of 2. In the soft state, £* was different in shape and value from both those in the standard state and those in the stiff state (Fig. 8a). £* was rather constant at a low value (about 10 kPa) for frequencies lower than 0.5 Hz but sharply increased at higher frequencies to about 1 MPa. £* was 1/100-1/10 the value of £* of the standard state seen in the middle frequency region (0.05-5 Hz). Tan 8 in the stiff states decreased with frequency to reach rather a constant value at 5 Hz and higher. The values were similar to those in the standard state at low frequencies (less than 0.005 Hz), but at higher frequencies tan d was much less. In the soft state, tan 8 was about 1 . with a little decrease with increasing frequency in the range higher than 0.005 Hz. but a sharp drop between 0.0005 and 0.005 Hz. The values of tan 5 were similar to those of the standard in the frequency range 0.005-0.05 Hz. but at in other frequen- cies— higher or lower — the values were much higher than those in either the standard or stiff states. The curve of £' in the stiff state appeared on the top, that 0.1 b) before stimulation after 10 0.1 before stimulation after c) 50 r 10 before stimulation after Figure 6. Reversibility of stiffening responses induced by ACh (triangle), KASW (square), and physical stimulation (cross). For chemical stimulation, measurements in ASW were made both before and after the stimulation, (a) Stiffness; (b) deformation energy; (c) dissipation ratio. Identical symbols are from the same sample. 107 106 10= 10" 103 102 VISCOELASTICITY OF SEA CUCUMBER 10" 269 10'" 10'3 10'2 10"1 10° 101 102 frequency (Hz) 1CT4 10'3 10'2 10'1 10° 10' 102 frequency (Hz) 10' 10° 10'1 10- b) 106 — 105 =- 10" , 103 =- 10" 10- 10'2 ID'1 10° frequency (Hz) 10' 102 102 10" 10- 10- 10' 10° 10' 102 frequency (Hz) P'igurc 7. Frequency dependence of mechanical properties of samples in the standard state at 1.8% strain. Circles give the average of 8 samples from 4 different individuals; bars are ± SD. (a) Complex modulus £*; (b) tangent 8; (c) storage modulus £"; (d) loss modulus E". in the soft state on the bottom, and that in the standard in-between (Fig. 8c). As for £*, the difference in the values of £' became smaller with increasing frequency in the frequency range higher than 1 Hz, especially the difference between standard and stiff states. E' for the stiff state gave a curve similar to that of E*. The curve of E' in the soft state was similar to that of E* except at low frequencies (less than 0.005 Hz), where E' showed a sharp increase with frequency, reflecting the sharp decrease of tan 8 in this frequency range. The curve of E" in the stiff state was rather flat with a value on the order of 100 kPa. The curve of E" in the soft state was sigmoid. as was that in the standard. E" in- creased with frequency, but plateaued above 0.1 Hz in standard state, whereas in the soft state it plateaued at a higher frequency (5 Hz). The saturated E" value of about 200 kPa was the same in all three states (frequency range 5-50 H/i. Comparison of curves in different states (Fig. 8) showed that both the viscous component and the elastic component changed with tissue states; the relative contributions of the two components to the changes in E* appeared to be dif- ferent at different frequencies. For example, a fairly large increase in E* occurred at 0.005 Hz without a change in tan 8 when the dermis stiffened from standard state. At this frequency, both increases in the elastic component (£" ) and increases in the viscous component (£"') contributed equally to the increase in £*. At 5-50 Hz, however, a decrease in £' without changes in £" caused a decrease in £* when the dermis softened from the standard state. Discussion Strain and strain-rate dependence The present work studied the mechanical properties of the catch connective tissue in the holothurian dermis and their changes upon stimulation. Dynamic sinusoidal strain of 0.5%-25% over a frequency range covering 5 logarithmic decades was applied and mechanical parameters were ex- amined. This is the first description of the dynamic visco- elasticity of mutable connective tissues that systematically varied both strain and strain-rate over wide ranges. The study revealed that the viscoelastic properties of the ho- lothurian dermis is both strain and strain-rate dependent. 270 107 T. MOTOKAWA AND A. TSUCH1 10" io6 L • 104 103 102 10'" 10'3 10'2 10'1 10° frequency (Hz) 101 102 10'' TO'2 b) 10' 10'3 10' 10'1 10° frequency (Hz) 101 102 10'4 10'3 10' 10'1 10° frequency (Hz) 107 id) - 106 |r no5 V 103 102 10'4 10"' 10'1 10° frequency (Hz) 101 102 Figure 8. Frequency dependence of mechanical properties of the stiff state and of the soft state (strain l.S^c). (a) Complex modulus E*: (b) tangent 5; (c) storage modulus E'\ (d) loss modulus E". Values are averages of 6 samples from 3 individuals. Hollow circle, stiff state (dissection); hollow diamond, stiff state (KASW); cross, stiff state (ACh); filled circle, soft state. Broken lines are the curves of the standard state. We observed several kinds of strain dependence. The dermis behaved as a linear viscoelastic material at small strains (less than 3%), whereas it showed nonlinearity at larger strains. Two kinds of notable nonlinear strain depen- dence were observed. One was the J-shaped stress-strain relationship, which is a common feature of soft biological materials (Wainwright eluL, 1976). The J-shaped curve was apparent in the standard and soft states but not in the stiff state. The introduction of the ./ index enabled us to quanti- tatively describe the difference in the shape of the curves. The other nonlinearity. which was observed only in the soft state, was strain-induced softening — the stiffness decreased when strain exceeded the limit strain of about 10%. These nonlinear strain dependences were thus tissue-state depen- dent. e obtained a modulus-frequency curve by varying the ! nc\. The curves of £*, £", and E" in the stan- dard s ere sigmoid, which clearly showed that the mech:i iperties of the dermis were strain-rate de- pendent. Thi rurves and thus the dependence were quite different in different tissue states. The curves of £" in stiff, standard, and soft states were different from each other, which implied that changes in elasticity accompa- nied the alteration in tissue states. The curves of E" in the three states were also different, which implied that the changes in viscosity also occurred at tissue-state changes. Thus, both elasticity and viscosity changed with the changes in tissue states. The curves of tan 8 for the three states gave different values at most frequencies. This clearly showed that the ratio of the contribution of the viscous component to the contribution of the elastic component changed with tissue states. The change in elasticity and the change in viscosity appeared either simultaneously or independently at a given frequency. Previous studies based on experiments with a fixed, ar- bitrarily selected strain rate drew contradictory conclu- sions on which components (elastic or viscous) mainly changed at alteration in tissue states (Motokawa, 1984b; Szulgit and Shadwick, 2000). The present study clearly showed that both components change. VISCOELASTICITY OF SEA CUCUMBER 271 Stiff state The present study employed three different stimuli that possibly acted through stimulating the stiffening mechanism active in the intact dermis. Although the stiff state was induced by different methods, all produced almost identical parameter-frequency curves. Therefore we concluded that the same mechanical state was induced by these methods. Acetylcholine is a ubiquitous neurotransmitter that func- tions in the control of dermal stiffness in sea cucumbers (Motokawa, 1987; Birenheide el ai. 1998). The potassium- rich media very likely worked through stimulating cellular elements controlling the dermal stiffness by membrane de- polarization (Motokawa, 1994). The stiffening caused by the handling at preparation likely corresponds to the dermal stiffening that occurs when the organism is mechanically disturbed in nature. We thus regard the stiff state seen in the present study as representative of the stiff state occurring naturally in the intact dermis. The stiff state was character- ized, in the constant-frequency experiments, by high stiff- ness, high deformation energy, low dissipation ratio, and low J index. In the constant-maximum-strain experiments, this state was characterized by high moduli and low tan 5. The low values in tan 5 and dissipation ratio implied that elasticity prevailed over viscosity. Tan 8 was less than 0.1 when the frequency exceeded 0.1 Hz, which implies that the contribution of the viscous component was quite small. This and the low J index imply that the tissue behaved rather like a linear elastic solid at frequencies higher than 0.1 Hz. The high values of stiffness, moduli, and deformation energy are the features associated with the increase in the elastic mod- ulus. Thus, in short, the dermis in the stiff state behaved like a stiff spring. This state has been believed to function in posture maintenance and mechanical defense (Motokawa, 1985). The stiff, spring-like properties seem to be adaptive features for such functions. The high stiffness is helpful in defense and body support, and the springy feature helps restore the original posture after the imposed force is re- moved. The dominance of elasticity over viscosity also helps by minimizing plastic flow. Soft state The soft state is characterized in the constant-frequency experiments by low stiffness, low deformation energy, high dissipation ratio, high J index in unloading, and strain- induced softening. In the constant-maximum-strain experi- ments, the soft state was characterized by low moduli and high tan 8. Tan 8 was about 1 over a wide frequency range, which implies that the contribution of the viscous compo- nent was as large as that of the elastic component. At the lowest frequency, tan 8 exceeded 1 . indicating the domi- nance of the viscous component. A significant contribution of the viscous component was also found in the constant- frequency experiments that showed a high dissipation ratio in the soft state. When the maximum strain exceeded a limit strain of about 10%, stiffness decreased as the maximum strain in- creased. This phenomenon was never observed in other states. Although strain-induced softening has not been re- ported previously, we could explain the rather contradictory results reported for other sea cucumbers by this strain- induced softening phenomenon. The soft state in the present study was induced by calcium chelation. This procedure was found to cause drastic softening in creep tests (Hayashi and Motokawa. 1986), whereas in dynamic tests it caused much less softening (Szulgit and Shad wick, 2000) or no detectable changes (Shibayama el al.. 1994). Although Szulgit and Shadwick (2000) measured shear modulus, not elastic modulus, and thus strict comparison is not possible, the difference in the extent of softening seems to be inter- pretable, at least in part, by the difference in the strain used. In creep tests, the dermis was usually subjected to fairly large strain, while in dynamic tests, the strain imposed was much smaller than the limit strain of the present sea cucum- ber. In creep tests, the dermis is very likely in the strain- induced soft state, but in the previous dynamic tests it probably was not in that state. Strain-induced softening of the dermis was observed in an intact sea cucumber that was subjected to large repetitive deformations (Motokawa. 1988), and thus it seems very likely that the state in CaFASW mimicked the soft state of intact animals. The dermis in CaFASW showed a drastic decrease in stiffness when the deformation exceeded the limit strain. This unique behavior, together with the quite high energy dissipation ratio in the soft state, seems to have adaptive significance in autotomy and fission. Sea cucum- bers show evisceration, a kind of autotomy. They contract the body to increase the pressure in the coelomic cavity, causing a rupture in the body wall; they eject their viscera through that hole. Because the dermis in the ruptured por- tion is very soft to the touch, that part is no doubt in a soft state. The scenario for how the strain-induced softening works in the evisceration process is as follows. The animal would first make a small portion of the dermis — that to be ruptured — soft. At this stage, the softened part still contrib- utes to the integrity of the body wall because the stiffness of the soft state at low strain is not as low as that after having exceeded the limit strain. The animal would then increase the coelomic pressure, causing larger deformations in the softened part. Once the deformation exceeds the limit strain, the stiffness drastically decreases and so the dissipation ratio increases, which allows the dermis to continue deform- ing at the same pressure (or even under lower pressure) until rupture. This is positive feedback: the more deformed the dermis, the more easily the dermis is deformed. Such me- chanical properties allow the animal to eviscerate with only a transient increase in the coelomic pressure, and thus to confine the rupture to the small portion initially softened, leaving the rest of the dermis intact. 272 T. MOTOKAWA AND A. TSUCHI Standard state We emplo d the convention used in previous studies that the , .sied in ASW, was taken as the standard state (M .ukuwa, 1984a). We tested the dermis after a resting period of 1 day in ASW. Such a lengthy resting period was chosen because the dermis, when rested for less than 15 h, showed stiffness values that were between those of the stiff state and the standard state. Thus, recovery from the effects of handling at preparation took quite a long time in this species. The long resting period seems not to ad- versely affect the dermis, because the sample rested for 1 day showed clear responses both to KASW and to ACh. Previous studies did not employ such a long resting period, which may be one reason for the notoriously large varia- tions in the reported mechanical properties of non-stimu- lated dermis in ASW (Motokawa, 1984c; Hayashi and Mo- tokawa, 1986; Szulgit and Shadwick, 2000). By touching the living sea cucumber, we can feel the stiffening of the body wall. If such a stiffened body wall is then vigorously squeezed, it becomes very soft — soft enough to show a viscous flow (Motokawa, 1988). The isolated dermis in the standard state also showed both stiffening and softening responses. Thus it seems reasonable to suppose that the dermis of the intact animal at rest is in a state that corresponds to the present standard state; in this state, the animal is likely to change its body shape for movement. The standard state showed a J-shaped stress- strain relationship with a prominent flat toe region followed by a steep pole region. The toe region allows animals to change their posture easily, with little energy expenditure, by using their body wall muscles. In contrast, to protect the animal from damage, the steep region resists large, exter- nally imposed forces. Thus the standard state with its J- shaped stress-strain curve seems to have adaptive signifi- cance. The standard state showed mechanical properties inter- mediate between the stiff and soft states, and thus the standard state appears to be just an intermediate between two extremes. Close inspection of the stress-strain relation- ship, however, suggests that the mechanisms of stiffening and of softening from the standard state are probably dif- ferent (see next section), and thus we conclude that the dermis of the sea cucumber can assume three distinct me- chanical states — stiff, standard, and soft. Simple polymer model and implications for mechanism of catch The derniv of the sea cucumber is composed mainly of extracellulm -i aerials whose mechanical properties have been thought lo i.Mermine those of the whole dermis. The main componem ; (he extracellular materials are macro- molecules such as collagen and proteoglycans (Matsumura. 1974; Kariya et ai, 1990). The dermis shows continuous creep to final breakage under even a small load. This be- havior is observed not only in the standard state (Motokawa, 1981 ) but also in soft and stiff states (unpubl. obs.), which suggests that the main components of the dermis do not make covalent cross-links with each other. It seems to be possible to regard dermis as a blend of non-cross-linked polymers, and thus it is tempting to interpret the present results in terms of polymer science. Let us suppose that the force-bearing structure in the dermis is a meshwork of polymers. In the meshwork, poly- mer molecules make a noncovalent bond with adjacent molecules at each crossing point of the molecular mesh. The polymer chain between adjacent crossing points is here called a segment. Two kinds of bonds are postulated in the meshwork. One is the intermolecular bond that forms the crossing point of the meshwork, and the other is the seg- mental bond found within the molecular chain that com- prises the segment (Fig. 9). A part of the molecule in the segment is presumed to take a folded structure in which the folds were maintained by intra-molecular or segmental bonds. Such bonds make the segment less flexible and resistant to stretch. The introduction of inter-molecular bonds implies that more molecules in the dermis are re- cruited into the force-bearing meshwork. When the dermis in the standard state is stretched, each molecular segment freely rotates around the crossing points of the mesh until all the segments become parallel to the direction of stretch. Further stretch directly stretches the segment and thus stretches the segmental bonds (central column of Fig. 9). The free rotation of segments corre- sponds to the toe region of the J curve, and the direct stretching of segments corresponds to the pole region. The unloading curve shows a higher J index than that of the loading curve. This implies that stretching segments induces some plastic deformation. Such plastic deformation must be temporary rather than permanent, because the next hyster- esis loop exhibited exactly the same shape. To explain this behavior, we postulate that some segmental bonds are stretched plastically, but they recover their original state in the unloading and compressing phases that follow. Tempo- rarily plastic behavior explains the rather high dissipation ratio seen in the standard state. Stiffening stimulation induces inter-molecular bonds, which reduces the segment length of the mesh (upper right of Fig. 9). A reduced segment length accounts for the short toe region. It also increases the resistance to rotation around crossing points because a smaller mesh size increases the resistance to displacement of water from the mesh, and thus increases the slope of the toe region. An increase in inter- molecular bonds recruits more molecules into the mesh- work, and thus increases the resistance to stretch of the meshwork. This explains the higher stiffness of the pole region. The newly recruited molecules are presumed not to contain plastic segmental bonds given the small difference between loading and unloading curves and the low dissipa- tion ratio in the stiff state. VISCOELAST1CITY OF SEA CUCUMBER W 273 Figure 9. Schematic of a simple polymer model for the dermis. A single mesh is drawn. Top row portrays. from left to right, the soft state, the standard state, and the stiff state. Hollow circle, intermolecular bond; rilled circle, segmental bond that deforms elastically when stretched; stippled circle, segmental bond that deforms plastically when stretched. The folded structure of the segment is drawn only for one side of a mesh. Central column is the mesh of the standard state under increasing strain from top to bottom when stretched horizontally. The mesh is free to deform (middle): the segmental bonds experience deformation after the segments become oriented parallel to the direction of stretch (bottom). In the soft state, the length of the toe region remained the same as that in the standard state. From this result we postulate that the segment length remains the same, and thus the number of inter-molecular bonds remained unchanged at the transition from standard to soft states (upper left of Fig. 9). The segmental bonds are, however, postulated to de- crease in number. Removal of segmental bonds makes the segment more flexible and stretchy, which accounts for the low stiffness of the pole region in the soft state. A fair proportion of the remaining segmental bonds show plastic deformation on stretch, producing a fairly large J index at unloading and a large dissipation ratio in the soft state. In the soft state, segmental bonds break when the strain ex- ceeds some limit. This endows the model with strain-soft- ening behavior. The loss of segmental bonds that had held the folded structure of the chain results in elongation of the segment. This explains the longer toe region in samples that experienced strain-induced softening. The breakage of bonds also accounts for the decrease in the maximum stress. This meshwork model simulates well the mechanical behavior of the dermis with only a small number of assump- tions. The model suggests that the molecular mechanism involved in stiffening is different from that involved in softening. Inter-molecular bonds are associated with changes between the standard state and the stiff state, whereas segmental bonds or intra-molecular bonds are as- sociated with changes between the standard state and the soft state. An increase in E' associated with the formation of bonds between molecules has been reported in the collage- nous mesogloea of a sea anemone (Gosline, 1971). The stiffening mechanism and the softening mechanism seem to have their own cross-bridging molecules. Tensilin, a protein that stiffens the dermis by binding to collagen (Tipper et ai, 2003), is a candidate for the inter-molecular bonding agent. The presence of a softening molecule has also been shown (Koob et ai, 1999; Szulgit and Shadwick, 2000). The stiff- ening mechanism and the softening mechanism also seem to have their own neural pathways (Motokawa. 1987; Biren- heide et al., 1998). In the present model, calcium ions are involved in segmental bonds. Calcium ions have a number of possible sites that they affect in both polymer systems and in biological systems. They seem to have some roles in a "polymer system" of the dermis because detergent-treated dermis is quite sensitive to calcium ions (Motokawa, 1994). They are also probably involved in the "biological system" of the dermis, affecting neuronal activities, secretion pro- cesses, or both (Motokawa and Hayashi, 1987; Trotter and Koob, 1995). Calcium translocation in the holothurian der- mis has been suggested (Matsuno and Motokawa, 1992). Thus calcium ions no doubt play one or more important roles in the mechanism of connective tissue catch. A meshwork of noncovalently cross-linked polymers gives an £" -frequency curve with four characteristic regions (Ferry. 1980). They are, from the high-frequency end to the low-frequency end, the glass region with a high value of £" , the transition region with decreasing E' as frequency de- creases, the rubbery or plateau region of constant E' , and the flow region of decreasing E' as the frequency decreases. 274 T. MOTOKAWA AND A. TSUCHI These four regions are all apparent only when measure- ments are per.orrned over a very wide range of frequencies, which is often not practicable. In the present study, the dermis in the suit state showed an £' -frequency curve with decreasing E' as frequency decreases in the lowest fre- quency region. It is reasonable to regard this region as the flow region because a tan 8 value of 10 implies that the dermis behaves like a liquid, and because the softened intact dermis does exhibit flow (Motokawa, 1988). The stiff state showed an increase in £" with increasing frequency to reach a constant value of £" of about 5 MPa. Because £" in the glass state is 2 orders of magnitude higher than this (Fuka- hori, 2000), the high-frequency region of the stiff state is very probably the plateau region, not the glass region. In polymer science, it is possible to construct a single E' -frequency curve from experimental data of limited fre- quency ranges by using the time-temperature superposition principle. For linear viscoelastic materials such as amor- phous polymers, the effects of time and temperature on mechanical properties are equivalent; thus the curve at one temperature can be superimposed upon those at different temperatures by shifting the curves from lower temperatures to the left and those from higher temperatures to the right along the frequency axis to generate a smooth master curve (Ferry, 1980). This method is convenient because it gener- ates a single curve that gives an overview of the frequency dependence of mechanical properties. A master curve is usually made by varying the temperature. It is, however, sometimes composed from curves derived from different concentrations of polymers or in solutions of different ionic strength (Gibbs et al., 1968). We attempted to generate an "apparent master curve" in order to get a single curve that gives an overview of the frequency dependence of mechan- ical properties of the holothurian dermis. Temperature ma- nipulation was not practicable because temperature greatly affected the mechanical properties, acting not only directly on the polymer meshwork but also indirectly through af- fecting the activities of nerves and secretory cells control- ling mechanical properties. Instead we shifted the curve in the stiff state to the right and that in the soft state to the left, leaving that of the standard state as a reference. We could construct a smooth curve of £" with four different phases quite similar to the usual master curve of polymers (Fig. 10), although there is no physicochemical theory at hand that supports the present procedure. Therefore, the similar- ity is just an apparent one. A smooth curve could also be generated on E" by lateral shifting (data not shown). The fact that we could construct a smooth curve suggests that some physicochemical processes corresponding to fre- quency shifts occurred at state changes. » Acknowledgments We thank Drs. Makoto Kaibara and Akio Sakanishi for discussion, and the staff of Sesoko Station. Tropical Bio- 107 10" - o5 w 104 103 102 10-' 10" iry2 10° frequency (Hz) 102 Figure HI. "Apparent master curve" of storage modulus E' . Hollow circle, the stiff state (dissection}; hollow diamond, the stiff state (KASW); .-'. the stiff state (ACh); +, the standard state; filled circle, the soft state. The logarithm of the shift factor was 1 .7 for the stiff states; for the soft state it was — 2. meaning that the curve shifted to the left by 2 orders of magnitude in frequency. sphere Research Center, University of the Ryukyus, for the supply of sea cucumbers. Supported by Grant-in-aid for scientific research on priority area (A) "Molecular synchro- nization for design of new materials system." Literature Cited Birrnheide, R., M. Tamori, T. Motokawa, M. Ohtani, E. hvakushi, V. Muneoka, T. Fujita, H. Minakata, and K. Nomoto. 1998. Peptides controlling stiffness of connective tissue in sea cucumbers. Biol. 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(December 2003) © 2003 Marine Biological Laboratory Heat-Shock Protein 70 (Hsp70) as a Biochemical Stress Indicator: an Experimental Field Test in Two Congeneric Intertidal Gastropods (Genus: Tegula) LARS TOMANEK1 AND ERIC SANFORD2 Hopkins Marine Station, Stanford University, Pacific Grove, California 93950-3094 Abstract. Although previous studies have demonstrated that heat-shock protein 70 (Hsp70) can be induced by en- vironmental stress, little is known about natural variation in this response over short time scales. We examined how Hsp70 levels varied over days to weeks in two intertidal snail species of the genus Tegula. Sampling was conducted both under naturally changing environmental conditions and in different vertical zones on a rocky shore. The subtidal to low-intertidal T. brunnea was transplanted into shaded and unshaded mid-intertidal cages to assess temporal variation in Hsps under conditions of increased stress. For compari- son, the low to mid-intertidal T. funebralis was transplanted into mid-intertidal cages, within this species' natural zone of occurrence. Snails were sampled every 3 to 4 days for one month, and endogenous levels of two Hsp70-kDa family members (Hsp72 and Hsp74) were quantified using solid- phase immunochemistry. Following periods of midday low tides, levels of Hsps increased greatly in transplanted T. brunnea but not in T. funebralis. Levels of Hsps increased less in T. brunnea transplanted to shaded cages than to unshaded cages, suggesting that prolonged emersion and reduction in feeding time per se are factors that are only mildly stressful. Upregulated levels of Hsps returned to base levels within days. In unmanipulated snails collected from their natural zones, Hsp levels showed little change with thermal variation, indicating that these species did not ex- perience thermally stressful conditions during this study. However under common conditions in the mid-intertidal Received 28 May, 2003; accepted 24 September 2003. 1 To whom correspondence should he addressed. Present address: Dept. of Animal Science. University of California, One Shields Avenue, Davis. CA 95616. E-mail: ltomanek@ ucdavis.edu " Present address: Eric Sanford. Dept. of Ecology and Evolutionary Biology, Brown University, Providence, Rl 02412. zone, Hsp70 levels reflected the different thermal sensitiv- ities of the physiological systems of these two species. Introduction The synthesis of heat-shock proteins (Hsps) is induced when environmental variation perturbs an organism's phys- iological system to the extent that its proteins denature. Under such environmental conditions Hsps and other mo- lecular chaperones stabilize denaturing proteins, refold re- versibly denatured proteins, and facilitate the degradation of irreversibly denatured proteins (Lindquist, 1986; Lindquist and Craig, 1988; Parsell and Lindquist, 1994; Feige et <;/.. 1996; Frydman, 2001; Haiti and Hayer-Hartl, 2002). Nu- merous studies have investigated the relationship between Hsp synthesis and various potential stress factors; however, very few studies have investigated the variation of Hsp levels under varying natural conditions (for review see Feder and Hofmann, 1999). Even fewer studies have inves- tigated short-term variation in Hsp levels (e.g., hours, days to weeks) in response to variable physical conditions in the field (Hofmann and Somero, 1995; Nakano and Iwama, 2002). A comprehensive understanding of such a time course of variation in Hsp levels under natural conditions is needed to interpret Hsp levels from field-collected organ- isms and therefore to evaluate stress under natural condi- tions. Furthermore, predictions about the ecological role of the heat-shock response that are made from laboratory com- parisons of species that occupy widely varying thermal environments have not been tested under natural conditions. Our study focuses on Hsp variation in response to phys- ical stress in intertidal organisms. Physical factors, in par- ticular temperature, play an important role in setting the upper limits to the vertical distribution range of intertidal organisms and confine them to distinct bands on the shore 276 HSP70 AS STRESS INDICATOR 277 with species-specific upper and lower vertical limits (see reviews in Benson, 2(102; Tomunek and Helmuth, 2002). This view is supported by numerous laboratory studies demonstrating that physiological resistance to physical con- ditions is greater in species that live at higher tidal heights (Newell. 1979; Somero. 2002). Intraspecific variation in the expression of Hsps has been found in intertidal species in association with seasonal ac- climatization (Dietz and Somero. 1992; Hofmann and Som- ero. I99v Roberts ct al.. 1997; Chappie et al., 1998: Buck- ley et al., 2001). laboratory acclimation (Hofmann and Somero. 1996a; Roberts et al., 1997; Tomanek and Somero. 1999. 2000. 2002). competition for space (Rossi and Sny- der. 2001). food availability and wave exposure (Dahlhoff et cil.. 2001). and microhabitat (Helmuth and Hofmann. 2001). Interspecific differences in the heat-shock response, especially among congeneric species, often correlate posi- tively with thermal extremes in the environment (Sanders el ul., 1991; Dietz and Somero, 1993; Hofmann and Somero. 1996a: Tomanek and Somero. 1999. 2000. 2002: Nakano and Iwama. 2002: Tomanek, 2002). Some of these studies have shown how Hsp levels vary over hours in response to thermal stress under natural (Hofmann and Somero, 1995. 1996b; Nakano and Iwama, 2002) as well as laboratory conditions (Tomanek and Somero. 2000). To our best knowledge, nothing is known about the variation of Hsp levels in response to changing natural conditions over days to weeks. Furthermore, whether interspecific differences in the heat-shock response limit a species' thermal niche and contribute to setting its vertical distribution range within the intertidal zone has not been tested under natural conditions. This study focuses on two herbivorous gastropod species of the genus Tegula that occupy distinct vertical zones on the shore: T. bninnea (Philippi. 1848), common in the subtidal to low-intertidal zone, and T. funebralis (Adams. 1855), common in the low- to mid-intertidal zone (Riedman et al.. 1981: Watanabe. 1984). Our previous laboratory work suggested that heat stress might prevent the low- intertidal T. bninnea from occupying the mid-intertidal zone (Tomanek and Somero. 1999, 2000, 2002). Thus, we predicted that T. bninnea transplanted upward into the mid- intertidal zone would express increased levels of Hsp70 in its new thermal environment. Laboratory results (Tomanek and Somero. 1999) also suggested that the mid-intertidal T. funebralis would activate the heat-shock response fre- quently in its natural zone of occurrence due to the temper- ature extremes and fluctuations that characterize the mid- intertidal zone. In this study we test these predictions by following the time course of expression of two Hsp70 isoforms (Hsp72 and Hsp74) over a month-long sampling period in specimens transplanted from the low-intertidal into the mid-intertidal zone, and in control individuals col- lected from their natural vertical zones. This combination of ecological and molecular approaches thus attempts to comprehensively test how Hsp levels vary over time in response to the changing physical conditions within and beyond a species' natural thermal zone. Materials and Methods Stiu/y organisms and distribution patterns The two Tegula congeners used in this study differ in their biogeographic and vertical distributions. Tegula briin- nea inhabits the subtidal to low-intertidal zones of the eastern Pacific Ocean from Cape Arago, Oregon (43° 25 'N) to the Channel Islands. California (34° OO'N) (Abbott and Haderlie, 1980: Riedman et al., 1981: Watanabe. 19X4). Tegula funebralis is found in the low- to mid-intertidal zone and has a wider latitudinal range, from Vancouver Island. British Columbia, Canada (48° 25'N), to central Baja Cal- ifornia. Mexico (28° OO'N) (Abbott and Haderlie. 1980; Riedman et al.. 1981). Experimental design Field experiments were conducted at Hopkins Marine Station of Stanford University in Pacific Grove. California (36° 36'N. 121° 54'W). To test the role of temperature in setting the upper vertical limit of the subtidal to low-inter- tidal T. bninnen, we transplanted this species above its natural zone of occurrence. Snails (shell diameter = 20-25 mm. marked with yellow nail polish) were placed into stainless steel cages (20 x 20 cm wide and 5 cm high; 316 stainless steel wire cloth with a 3-mm opening size) that were positioned at similar heights in the mid-intertidal zone (+0.64 ± 0.12 m above mean lower low water). These enclosures had no bottom and thus allowed snails to move over the rock surface and feed on the algal species present naturally. Cages were either unshaded (sun-exposed treat- ment) or shaded by covering them with plastic mesh (two lav ers of a black polyethylene mesh with a 3.8-mm opening size). Each treatment included seven cages, with 10 snails per cage. In addition, we transplanted T. funebralis from the mid- intertidal zone into unshaded mid-intertidal cages (;; = 7) positioned at the same tidal height. This treatment served two purposes. First, it allowed us to compare the thermal stress of T. bninnea transplanted into the mid-intertidal zone (above its natural upper limit) to that of similarly caged T. funebralis. the natural inhabitant of the mid-inter- tidal zone. Secondly, it allowed us to test for caging artifacts by comparing caged mid-intertidal T. funebralis to unma- nipulated snails found naturally in that zone. Single snails were collected from each cage and replaced with an unmarked specimen (to avoid changes in snail density during the experiment) every 3rd or 4th day over a month-long sampling period (31 March to 1 May 2000; see Figs. 1 and 2). To evaluate the response of snails in their 278 L. TOMANEK AND E. SANFORD native thermal environment, we also collected unrestricted snails from i n aid intertidal (T. fiinehralis) and the shal- low subtid;; one (T. bntnnea; specimens were always submen.'. o i'ore and during collection). All collections were mack- within 45 min of low tide to minimize any physiological variation that might be related to endogenous tidal rhythms. Snails were frozen immediately on dry ice following collection and kept at -70 C until further pro- cessing. To obtain a record of Tegula body temperature, temperatures in gelatin-filled snail shells were recorded inside mid-intertidal cages and outside (on adjacent rocks) by a Stow A way XTI temperature data logger (Onset Com- puter Corp, Pocasset, MA). For further details on this method, see Tomanek and Somero (1999). Because of equipment failure, only one complete record, from a T. fiinebralis shell attached to open rock adjacent to a mid- intertidal cage, was obtained from the six data loggers installed. Temperatures shown (Figs. I and 2) therefore represent the thermal variation of field-acclimatized T. fii- nebralis from the mid-intertidal zone. Immediately follow- ing the experiment (and discovery of the equipment failure), additional data loggers were deployed to characterize the difference among our treatments. During midday low tides, temperatures within unshaded cages were typically 3-6 °C cooler than on the open rock outside the cage, whereas temperatures in shaded cages were an additional 2-5 °C cooler than in cages without shades. Tissue preparation Gill tissue was dissected from whole snails that were thawed under conditions that do not induce heat shock ( 1 3 °C) and immediately placed in 200 /xl (T. fiinebralis, 15.0 to 25.0 mg wet weight) or 300 /j,l (T. hrunneci, 30.0 to 45.0 mg wet weight) of homogenization buffer (32 mmol 1~' Tris- HC1, pH 7.5 at 4 °C, 2% (w/v) SDS. 1 mmol T1 EDTA, 1 mmol I"1 Pefabloc (Boehringer Mannheim), 10 ^g ml"1 pepstatin, and 10 /n,g ml"1 leupeptin). Tissues were incu- bated for 5 min at 100 °C and homogenized. The procedure was repeated and homogenates were centrifuged at 15,800 X g for 15 min. The supernatant was removed and stored at -70 "C. Protein concentrations were determined using the Micro-BCA assay (Pierce) according to the man- ufacturer's instructions. Gel electrophoresis and immunodetection (Western) protocol In general, we followed the procedure described in To- manek and Somero (2002). Briefly, proteins were separated electrophoretically and subsequently transferred onto nitro- cellulose membranes (Nitrobind, Schleicher and Schuell) in transfer buffer (25 mmol 1~' Tris-base, 0.193 mol 1"' glycine, 20% methanol (v/v), pH 8.3 at 20 °C). After mem- branes were dried overnight they were treated with blocking buffer (25 mmol 1~' Tris-HCl, pH 7.5 at 20 °C, 150 mmol 1 ' NaCl, 0.1% (v/v) Tween. 0.02% (w/v) Thimerosol, 5% (w/v) nonfat dried milk) for 1 h, subsequently washed with Tris-buffered saline (TBS; 25 mmol 1~' Tris-HCl, pH 7.5 at 20 °C. 150 mmol 1~' NaCl), and then incubated with a solution of a monoclonal rat antibody (IgG) against Hsp70 (clone 7.10; Affinity BioReagent, MA3-001; 1:2500 dilu- tion of Hsp70 antibody in buffer A (BA): TBS, 2.5% (w/v) bovine serum albumin in TBS) for 1 h. After washing the membranes, we incubated them for 30 min with a rabbit- anti-rat bridging antibody (IgG) solution ( 1 :2000 dilution in BA; Vector. AI-4000). followed again by several washing steps. Finally, we incubated membranes with a horseradish- peroxidase protein A solution (1:5000 dilution in BA; Bio- Rad) for 30 min. Membranes were washed and overlaid with a solution of enhanced chemiluminescent (ECL) re- agent (Amersham Pharmacia) according to the manufac- turer's instructions for 1 min. Under dark room conditions, we exposed membranes onto pre-flashed Hyperfilm (Amer- sham Pharmacia) for 5. 10. 20. 30. and 50 min after ECL treatment to obtain various exposures that were in the linear range of detection. All samples were run at least twice. linage analysis and quantification of expression of heat - shock proteins Film images were scanned on a densitometer (Sharp JX-330) and the digitized images were analyzed with image analysis software (ImageMaster ID. ver. 2.01, Pharmacia) to quantify band intensities of the two Hsp70 isoforms, one with a molecular mass of about 72 kDa (Hsp72), the other of about 74 kDa (Hsp74). We express band intensities relative to a known amount of a bovine heat-shock cognate 70 (80 ng: StressGen, SPP-750) to account for variation amonc Western blots. Statistical anahsis Variation in Hsp72 and Hsp74 was compared using a two-factor analysis of variance (ANOVA) with experimen- tal treatments and sampling days as the main effects. We conducted post hoc comparisons of all five treatment groups (Student-Newman-Keuls test) within each sampling day separately. To calculate the critical value, we used the appropriate Studentized range statistic (a = 0.95; m = number of means: df = degrees of freedom of the error term from the ANOVA) and an adjusted H value (/;,,) to account for unequal sample sizes among the means using the fol- lowing equation: «o = a - \ HM-70 AS STRESS INDICATOR 279 with "a" the total number of means compared (a = 50) and "/;," the sample size for each mean. Variances were not heterogeneous (Cochrane's test, P > 0.05). and therefore there was no need to transform the data. A cross-correlation analysis (MatLab Software) com- pared average, minimum, maximum daily temperatures as well as daily temperature range with endogenous levels of Hsp72 and Hsp74 over the entire sampling time for field- acclimatized mid-intertidal T. funebralis only. Results Data logger records indicate that the body temperature of mid-intertidal Tegula varied dramatically with tidal cycle and date over the course of this experiment (Figs. 1 A and 2 A). The three panels B, C, and D (Figs. 1 and 2) show the endogenous levels of Hsp72 and Hsp74 over the month of sampling. Variation of Hsp70 in Tegula brunnea transplanted above its natural limit Specimens of T. bninneti transplanted into unshaded cages in the mid-zone often showed dramatically elevated levels of both Hsp72 and Hsp74 relative to control individ- uals collected from the shallow subtidal zone (for statistical results, see Figs. IB and 2B). The overall response of both Hsps was very similar. Moreover, these elevated levels of Hsps seen in transplanted individuals were correlated with environmental factors: 3 days on which Hsp72 and Hsp74 levels were 2 to 4 times higher in sun-exposed mid-inter- tidal snails than in control snails from the shallow subtidal were preceded by periods of 2 or more days of midday low tides that greatly raised body temperatures for 1-4 h (3 and 13 April and 1 May 01). However, on 21 April, sun-exposed specimens of T. brunnea showed 6 times higher endogenous levels of Hsp72 and Hsp74 than control animals, but max- imal daily temperatures during the preceding 4 days were relatively low compared to other time periods. In addition, individuals of T. brunnea that were trans- planted into shaded mid-zone cages showed elevated levels of Hsps only slightly more often than control snails from the shallow subtidal, and to a greater degree in the case of Hsp74 (e.g.. 10 April and 24 April) than in Hsp72. In contrast, sun-exposed individuals often showed greater Hsp levels relative to their shaded conspecifics that were trans- planted into the mid-zone, with Hsp72 levels almost always being higher in the sun-exposed T. brunnea (Fig. IB). Time course of Hsps in transplanted and field- acclimatized Tegula funebralis We quantified the time course of Hsp levels in specimens of T. funebralis from the mid-intertidal zone that were either experimentally caged (sun-exposed) or unrestricted (field- acclimatized) to address three issues. First, we tested for caging artifacts by comparing caged and uncaged snails within the same zone. Second, we tested the prediction that the mid-intertidal Tegula congener would activate the heat- shock response more frequently than the shallow subtidal congener due to their differing thermal environments and heat-shock responses (Tomanek and Somero. 1999, 2000). Third, we compared the response to thermal stress in the two temperate Tegula congeners that occupy different tidal heights under "common garden" conditions (see below). Unrestricted individuals of T. funebralis were collected in crevices next to the unshaded cages. The two groups did not differ until the last week in April, when Hsp72 levels were higher in sun-exposed caged snails than in field-acclima- tized snails (Fig. 1C). Preceding this time period, peak temperatures from data loggers were relatively low, but they increased greatly with the onset of a 10-day period of early to midday low tides. Hsp74 levels showed a similar pattern, but in addition, field-acclimatized individuals showed higher levels than caged snails from 10 to 13 April (Fig. 2C). Thus, caged specimens of T. funebralis were appar- ently more thermally stressed than their unrestricted con- specifics during the last week of the study, perhaps because caging limited the access of snails to shaded and moist microhabitats. With time and changing temperatures, Hsp72 levels var- ied little until they increased in caged but not in unrestricted individuals (Fig. 1C). Although Hsp74 levels showed greater temporal variability, the resulting changes were still within the range of variation observed for shallow subtidal T. brunnea (Fig. 2B, C — field samples). Neither Hsp72 nor Hsp74 showed any correlations with any of the temperature variables (cross-correlation analysis, P > 0.05). These results suggest that mid-intertidal T. funebralis does not elevate levels of Hsps more often than T. brunnea does under the less thermally variable conditions of the shallow subtidal. Interspecific comparisons of Tegula in the mid-zone Transplanting both species to unshaded cages in the mid- intertidal zone allowed us to compare their responses to thermal stress under common garden conditions. Whereas transplanted specimens of T. brunnea responded to thermal stress, specimens of 7". funebralis caged in the mid-zone (their natural zone of occurrence) changed little (Figs. ID and 2D). Elevated levels of both Hsps indicate a response to thermal variation during time periods of midday low tides in T. brunnea (see above for details). Although base levels of Hsp72 in sun-exposed T. brunnea were close to levels found for T. funebralis. Hsp74 levels were, regardless of the tidal 280 L. TOMANEK AND E. SANFORD CM 200 150 100 50 T. brunnea - sun-exposed T. brunnea - shaded T. brunnea - field B 200 - J 150 O g 100 S> 50 200 150 100 50 T. funebralis - sun-exposed T. funebralis - field T. brunnea - sun-exposed r. funebralis - sun-exposed D 03-Apr-OO 10-Apr-OO 17-Apr-OO 24-Apr-OO 01-May-OO Time (days) Figure 1. Environmental variation and time course of beat-shock protein expression (Hsp72) in experimen- tal and control snails. (Al Tidal heights (m above mean lower low water) for Monterey Bay, day and night (gray) cycles, and temperatures recorded in a gelatin-filled snail shell (Tegula funebralis attached in the mid-intertidul zone). Snails within unshaded (sun-exposed) and shaded cages experienced lower temperatures. (B) Time course of endogenous levels of Hsp72 for specimens of Tegula briinneu transplanted to unshaded and shaded mid-intertidal cages versus field-acclimatized conspecifics (shallow subtidal zone). (C) Hsp72 levels for restricted (caged) and unrestricted (field-acclimatized) individuals of T. funebralis (mid-intertidal zone) and (D) for T. funebralis and T. brunnea individuals transplanted into unshaded mid-intertidal cages. Levels are expressed relative to an internal control (a bovine heat-shock cognate 70). Values are mean ± 1 SEM; * indicates significant differences (P £ 0.05) among treatments; n = 5-7 snails for all data points (except n = 4 for sun-exposed T. brunnea on 21 April). regime, consistently elevated in T. hninnea (with one ex- ception on 7 April), supporting our previous results from laboratory acclimation experiments (Tomanek and Somero, 2002). Discussion Although temporal variation in levels of heat-shock pro- tein (Hsp) has been suggested to closely track sublethal Hsp70 AS STRESS INDICATOR 281 200 - 150 100 - 50 - T brunnea - sun-exposed T. brunnea - shaded T. brunnea - field B 200 150 Q. I •5 50 0> T. funebralis - sun-exposed 7" funebralis - field 200 -, 150 100 50 - 7. brunnea - sun-exposed T. funebralis - sun-exposed D 03-Apr-OO 10-Apr-OO 17-Apr-OO 24-Apr-OO 01-May-OO Time (days) Figure 2. Environmental variation and time course of heat-shock protein expression (Hsp74) in experimen- tal and control snails. (A) Tide heights, day and night cycles, and mid-intertidal temperatures (see Fig. 1 legend for details). (B) Time course of endogenous levels of Hsp74 for specimens of Tegiila brunnea transplanted to unshaded (sun-exposed) and shaded mid-intertidal cages versus field-acclimatized conspecifics (shallow subtidal zone). (C) Hsp74 levels for restricted (caged) and unrestricted (field-acclimatized) individuals of Tegula funebralis (mid-intertidal zone) and (D) for T. funebralis and T. brunnea transplanted into unshaded mid- intertidal cages (see Fig. 1 legend for details). stress, there have been few tests of this hypothesis under natural conditions. Furthermore, the ecological importance of interspecific variation in the heat-shock response deduced from laboratory- studies has not been tested in the field. In this study we show that the subtidal to low-intertidal Tegula brunnea transplanted above its natural zone of occurrence experienced sublethal thermal stress, as reflected by ele- vated levels of two isoforms of the 70-kDa family of heat- shock proteins (Hsp72 and Hsp74). In contrast, levels of Hsp72 and Hsp74 varied little in control specimens of Tegula brunnea collected from the shallow subtidal zone, and less in T. brunnea individuals transplanted to shaded mid-intertidal cages. In addition, T. funebralis transplanted within its natural zone of occurrence, to mid-intertidal cages, but not field-acclimatized individuals, showed slightly elevated Hsp levels during a prolonged midday 282 L. TOMANEK AND E. SANFORD low-tide period mly. Here we discuss (1) how the time course of Hsr ion correlates with an organism's thermal hiv' and (2) how interspecific variation in the heat-sh*' Mise may limit the vertical distribution range L rtidal invertebrates. Time course of Hsp levels One of the objectives of this study was to examine the correlation between thermal events and the organism's re- sponse through time to better interpret the role of Hsps as biochemical indicators of sublethal thermal stress. The time course of levels of Hsp70 shows that periods of extreme thermal conditions upregulate endogenous levels of Hsp70 isoforms in transplanted T. hnmnea, but not in individuals of both species that were collected from their natural ther- mal environment (Figs. 1B-C and 2B-C). One exception to the association between increased Hsp levels in transplanted T. brunnea and peak temperatures occurred on 21 April. However, changes in the daily temperature range (To- manek. 2002) during the preceding days, from 16 to 17 April, were of similar magnitude as at the onset of a midday low-tide series (e.g.. 8 to 9 April), even if maximal temper- atures were relatively low. Hsp levels were upregulated in response to physiological stress for not more than 3 to 4 days. It is still unclear what thermal signals elicit an increase in Hsp levels. Levels in field-acclimatized mid-intertidal T. fiinebralis did not con-elate with any of the thermal vari- ables tested. Hsp levels will respond differently to chronic and acute thermal stress (Helmuth and Hofmann, 2001), but further studies are needed to test the response of Hsps to more complex thermal signals. We predicted increased Hsp levels in transplanted T. hnmnea on the basis of its long recovery (> 50 h) in the laboratory from a heat-shock-inducing thermal exposure typical of the mid-intertidal zone (30 °C; Tomanek and Somero, 2000). These studies also indicated that T. fune- hralis would activate Hsp synthesis for at least several hours (< 6 h for Hsp70) in response to temperatures of 30 °C or above — exposures that were reached at least several times during this month-long field experiment (Fig. 1A). In addi- tion, the activation temperature of Hsp synthesis is 27 °C in laboratory-acclimated (constant temperature) specimens of T. fiinehrtilis that were exposed to a wide range of incuba- tion temperatures following rapid heating in seawater (To- manek and Somero, 1999). Although these laboratory con- ditions do not match field conditions completely, the activation temperature should be within a few degrees of 27 °C, certainly below 35 °C — one of the highest temperatures that field-acclimatized specimens of T. fiinebralis experi- enced during our month-long sampling period. Addition- ally, Hsp synthesis js upregulated for several hours in re- sponse to an acute thc/mal stress in mussels collected from the field after low tide, presumably in response to protein denaturation due to thermal stress (Hofmann and Somero, 1996b). This discrepancy between our field results and our labo- ratory-based predictions could be due to several factors: first, most of our laboratory incubations were done in water, leading to a high rate of heating. However, heat stress in the intertidal zone is typically experienced under aerial condi- tions, when heating is slower (Tomanek and Somero, 2000). Second, our collection interval of 3 to 4 days may have missed an increase in levels of Hsp70 isoforms in response to thermal stress on some collecting days that followed several midday low tides after which individuals may show an attenuated response. Yet some of those collecting days were preceded by the "first" extreme low tide following several days of minor tides (e.g., 10 and 24 April). Although the recovery time of elevated Hsp synthesis in response to heat stress in T. fiinebralis is short (6 h; Tomanek and Somero. 2000), we should have detected elevated endoge- nous Hsp levels over a much longer time period. Other studies have observed elevated Hsp levels in response to short-term acute severe heat stress (elevation lasting up to 2 weeks; Clegg et ai, 1998) and to long-term chronic mild heat stress (elevation as long as 4 days; Nakano and Iwama, 2002). Alternatively, Hsp70 is closely regulated and quickly eliminated in granules following heat shock (Morimoto, 1998), and we may have therefore missed briefly elevated levels of Hsp70. A final factor may be that the moderation of temperatures generated by the cages themselves (even those cages without additional shading), may have kept the temperatures of caged snails below 30 °C. Interspecific variation in the heat-shock response and vertical distribution limits in intertidal invertebrates By transplanting T. brunnea above its natural zone of occurrence and by following the time course of changes in endogenous levels of Hsps over a month of thermal varia- tion, we were able to directly evaluate the importance of interspecific variation in the heat-shock response in relation to vertical distribution limits. Transplanted specimens of T. brunnea increased their endogenous levels of both Hsp72 and Hsp74 in response to midday low-tide periods and therefore reached, as pre- dicted, temperatures above the activation threshold for their stress response (Tomanek and Somero. 1999, 2000). Mor- tality in transplanted T. hnmnea is also indicative of severe stress (8.5% mortality in sun-exposed individuals over the entire month; no individuals of T. fiinebralis died). Endog- enous levels of Hsp72 and Hsp74 changed little in T. fiinebralis in comparison to T. brunnea, and this could be mainly due to this species' higher activation temperature. In the laboratory, T. fiinebralis activates the heat-shock re- sponse at 27 CIC versus 24 °C in T. hnmnea (following H.SP70 AS STRESS INDICATOR 283 acclimation to 13 C and after rapid heating in seawater to a wide range of incubation temperatures). Thus the relative differences between activation temperatures of the stress response are good predictors of the relative levels of sub- lethal thermal stress and therefore the relative increases in endogenous levels of Hsps under common garden condi- tions, although the actual activation of the stress response depends on the heating rate and the medium (air versus water: Tomanek and Somero, 2000). An increase in endogenous levels of Hsps in T. hnmnea could also have been caused by the reduction in feeding time that accompanied the transplantation from the low- into the mid-intertidal zone. A reduction in feeding time is likely to lower metabolic rates (Shick. 1981: Branch et ai, 1988) and cellular energy levels (e.g.. ATP), which may disrupt protein homeostasis. Individuals of T. hninneu transplanted to shaded mid-intertidal cages differed from unmanipulated snails collected from the shallow subtidal zone in experiencing slightly higher body temperatures and much longer emersion times. Yet levels of Hsp72 and Hsp74 did not differ consistently between shaded mid- intertidal transplants and shallow subtidal controls, suggest- ing that longer emersion times per se were not activating increased Hsp levels. Other stress factors, e.g., osmotic and desiccation stress, may also contribute to changes in Hsp70 levels, but these were not addressed in this study. These results suggest that thermal conditions in the mid- intertidal zone are stressful for the subtidal to low-intertidal T. bnmnea. but not for the low- to mid-intertidal T. fnne- bralis, and thus may contribute to preventing T. hrunnea from inhabiting the mid-intertidal zone. This is in large part due to the lower activation temperature (7",,,,) of the stress response, the 6 °C lower temperature of maximal Hsp synthesis (T/>CII^. and the lower temperature at which the synthesis of proteins (including Hsps) ceases in T. brunnea (7",,,,: Tomanek and Somero, 1999). In addition, levels of the heat-shock transcription factor! (HSF1 ) are lower in T. hnmnea than in T.funebrulis (Tomanek and Somero. 2002). T. finiehralis is therefore better adapted to the physical conditions of the mid-intertidal zone, but such adaptations in the heat-shock response may be costly. For example, higher Hsp70 levels due to experimentally higher gene copy numbers of Hsp70 can impact life-history traits (e.g.. mor- tality and developmental time) that determine fecundity in Drosophila (Krebs and Feder. 1997a. b). and yeast strains with lower levels of Hspl04 grow faster (Sanchez et al., 1992). Furthermore. Hsps can interact in detrimental ways with native proteins under nonstressful conditions and are therefore rapidly sequestered from the cytoplasm (Feder et al.. 1992). Thus, higher costs due to adaptations in the stress response to the mid-intertidal environment may in part explain why T. fimebralis shows slower growth rates than its low-intertidal to subtidal congeners T. hnmnea and T. montereyi (Frank, 1965: Paine. 1969: Watanabe, 1982). However costly elevated levels of Hsps are. the transient upregulation of endogenous levels in subtidal to low-inter- tidal gastropods in the mid-intertidal zone shows that Hsps are good indicators of the thermal sensitivities of physio- logical systems under common field conditions. Our results also confirm our prediction that interspecific variation in the heat-shock response of Tegula congeners is adaptive to life in the thermally variable mid-intertidal zone (Tomanek. 2002). 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(December 2003) © 2003 Marine Biological Laboratory Reproduction and Larval Morphology of Broadcasting and Viviparous Species in the Cryptasterina Species Complex MARIA BYRNE1'*, MICHAEL W. HART2, ANNA CERRA1. AND PAULA CISTERNAS1 1 Department of Anatomy and Histology. F13. University of Sydney, NSW 2006, Australia; and 2 Department of Biology. Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada Abstract. The Cryptasterina group of asterinid sea stars in Australasia comprises cryptic species with derived life histories. C. pentagona and C. hystera have planktonic and intragonadal larvae, respectively. C. pentagona has the gonochoric. free-spawning mode of reproduction with a planktonic lecithotrophic brachiolaria larva. C. hyxtera is hermaphroditic with an intragonadal lecithotrophic brachio- laria. and the juveniles emerge through the gonopore. Both species have large lipid-rich buoyant eggs and well-devel- oped brachiolariae. Early juveniles are sustained by mater- nal nutrients for several weeks while the digestive tract develops. C. hystera was reared in vitro through metamor- phosis. Its brachiolariae exhibited the benthic exploration and settlement behavior typical of planktonic larvae, and they attached to the substratum with their brachiolar com- plex. These behaviors are unlikely to be used in the intrago- nadal environment. The presence of a buoyant egg and functional brachiolaria larva would not be expected in an intragonadal brooder and indicate the potential for life- history reversal to a planktonic existence. Life-history traits of species in the Cryptasterina group are compared with those of other asterinids in the genus Patiriella with vivip- arous development. Modifications of life-history traits and pathways associated with evolution of viviparity in the Asterinidae are assessed, and the presence of convergent adaptations and clade-specific features associated with this unusual mode of parental care are examined. Received 28 March 2003: accepted 23 July 2003. * To whom correspondence should be addressed. rnbyme@anatomy.usyd.edu.au E-mail: Introduction Speciation in marine invertebrate taxa is strongly influ- enced by the evolution of life-history traits. Evolutionary changes that influence speciation include modifications to gamete-binding proteins, oogenesis, larval nutrition (plank- totrophic, lecithotrophic). and location (planktonic, benthic) of development (Strathmann, 1985; Reid, 1990; Palumbi. 1992; Vacquier et ai. 1995; Byrne et al., 1999, 2003; Duda and Palumbi, 1999: OFoighil and Taylor, 2000; Villinski et al.. 2002). In a large number of these cases, the combination of rapid and diverse evolution of larval forms and stasis in adult stages has resulted in congeneric species with mark- edly different larval phenotypes and habitats but similar adult phenotypes and ecologies. This decoupling of larval and adult morphological evolution suggests that critical examination of suspected morphospecies will reveal undis- covered marine biodiversity. Molecular and developmental studies have shown that many problematic taxa include a suite of cryptic species (Reid. 1990; Knowlton. 1993; Deg- nan and Lavin. 1995: OFoighil and Smith, 1995; Arndt et al.. 1996; Huber et al., 2000). Application of the compara- tive approach has made many of these taxa important mod- els for the investigation of processes underlying evolution and development, and speciation in the sea (Hart et al., 1997; Degnan and Lavin. 1995; Huber et al.. 2000). The potential for species divergence through life-history evolution is common in some marine invertebrate taxa but rare in other, even closely related, taxa. Genera in which speciation is associated with evolution of development are found among gastropods (Littorina, Conns), clams (La- saea), soft corals (A/cyoniiinn. asteroids (Asterina. Putiri- ella). echinoids (Heliocidaris) and ascidians (Molgulu) (Reid. 1990; OFoighil and Smith. 1995; Raff. 1996: Hart et 285 286 M. BYRNE ET AL. til., 1997; Duda ;ind Palumbi, 1999; Huber et ai, 2000; McFadden et u!.. 2001). Why some taxa are prone to de- velopment! 'ion and not others is not known. In the Asteroidea. th. Asterinidae is a species-rich family com- prisini that share derived forms of reproduction. larval 'ij.irphology, and brood protection (Byrne and Cerra. 1996: Byrne et til., 1999b). Molecular phylogenetic analy- ses of these sea stars revealed many examples of conver- gence in which derived life-history traits have evolved through independent pathways (Hart et til., 1997. 2003). In temperate Australia, the Patiriella exigua species group includes three species: one benthic egg layer and two viviparous lineages (Dartnall. 1969. 1971; Keough and Dartnall. 1978). Until the observation of live birth, the viviparous species were considered to be morphs of P. exigua. Detailed analysis of mtDNA sequences revealed the presence of a second cryptic group of asterinids in the genus Cryptasterinu, which occurs throughout Australasia (Hart et ai, 2003; Dartnall et at.. 2003). These sea stars, formerly in the Patirielln pseudoexigua species complex (Dartnall. 1971: Rowe and Gates, 1995), have been reassigned to Cryptasteriiw in a recent taxonomic review (Dartnall et al., 2003). One lineage. C. pentagona, (formerly P. pseudoex- igua) occurs in Queensland. A second lineage, Cryptast- erina n. sp. (also formerly known as P. pseudoexigua) occurs in Wanlitung, Taiwan, and its planktonic lecithotro- phic life history is well-documented (Chen and Chen. 1992). The viviparous species described by Hayashi ( 1977). Patiriella pseudoexigua pacifica, has been reassigned to C. pacifica (Dartnall et til.. 2003). On the basis of the position of C. pentagona in the phylogenetic tree, nested between broadcasting and brood- ing species (Hart et til., 1997), and in light of the very broad geographic range of this nominal species, it appeared likely that Australian morphs of C. pentagona would have inter- esting modes of development. A recent molecular study revealed the relationships among four lineages of this taxon recently called P. pseudoexigua (Hart et ai, 2003). In Queensland these lineages comprise two species with dif- ferent life histories (Hart et til.. 2003). One of these species, C. hystera, is a recently described intragonadal brooder (Dartnall et al., 2003). In this study we examined popula- tions of Cryptasterina from the original type locality in Queensland and elsewhere along the coast to document details of their reproduction and development. We com- pared the life history traits of C. liystera to those of other viviparous asterinids in the genus Patiriella and closely related broadcasting species to assess the changes associ- ated with the evolution of viviparity. This life history is at the extreme end of the broadcast-brooding modes of prop- agation in the Asteroidea. The pathways in the evolution of viviparity in the Asterinidae are assessed, and the potential for convergent Captations in species with this unusual mode of parental .are is examined. Materials and Methods Cryptasterina pentagona was collected from five loca- tions along the Queensland coast (Fig. 1) at irregular inter- vals between 1996 and 2002. This included several sites in North Queensland (10/00: 11/00; 10/02), including Airlie Beach (20°30'S; 148°45'E); Rowes Bay, Townsville (19°15'S; 146°50'E); and Bingil Bay, Mission Beach (17°50'S; 146°06'E). C. liystera was collected from Statue Bay (23°15'S; 150°45'E) in central Queensland (8/96; 2/96; 9/97; 10/99). The samples were used to assess the condition of the gonads and preserve samples for histology. The type locality for P. pseudoexigua is Airlie Beach (Dartnall, 1971). In October 2002. the gonads of specimens from Airlie Beach and several sites in Bowen (20°1'S; 148°16'E) — Dalrymple Point, Rose Bay, and Murrays Bay (Fig. 1) — were examined and processed for histology. Iso- lated ovaries of females from these sites and from Rowes Bay, Townsville, were induced to spawn through the use of Figure 1. Map of Queensland showing sample locations and current known distributions of Cr\piu\tcrinti and C. hystera. DEVELOPMENT IN CRYPTASTERINA 287 the ovulatory hormone 1 -methyl adenine in tillered seawater (10 5 M in tiltered seawater [FSW]). The eggs were fertil- ized with sperm removed from the testes. and the larvae were reared in FSW at 23-25 °C. Settlement substrata including glass slides aged in seawater and pieces of coral- line algae were introduced into some culture dishes contain- ing competent larvae. The gonads of the Statue Bay sea stars contained embryos. These were removed and reared in vitro in FSW (1.0 jLim) at 22 °C through metamorphosis. For histology, the gonads were fixed in Bouin's fluid for 24 h. rinsed in distilled water, dehydrated in graded eth- anols, and embedded in paraffin. Serial sections were stained with hematoxylin and eosin. For scanning electron microscopy, larvae and juveniles were fixed for 1 h in 2.5% glutaraldehyde in 0.45 ju,m FSW. Use of a secondary fixa- tive was found to be unnecessary (Byrne, pers. obs.). After fixation, the specimens were dehydrated, critical-point- dried, and viewed with a JEOL JSM-35C scanning electron microscope. Results Our field survey of life-history traits in six populations of Cryptasterina provided data on the distribution of two lin- eages, one broadcasting species and one intragonadal brooder. C. pentagona from the type locality Airlie Beach (Fig. 1 ) was found to be a free-spawner with a short-lived planktonic larva. This species was found from Airlie Beach to Mission Beach in north Queensland (Fig. 1 ). Mission Beach is near the northern limit of the distribution of C. pentagona in Queensland (A.J. Dartnall, James Cook Uni- versity, pers. comm.). This species was located under rocks high in the intertidal zone which dry out at low tide. Reports of C. pentugona south of Airlie Beach will have to be checked in light of the discovery of C. hystera in Statue Bay, Central Queensland (Fig. 1 ). C. hystera is an intrago- nadal brooder. Molecular data indicate that this new species also occurs in Yeppoon (Hart et al., 1997). It occurs high in the intertidal under small rocks in mangrove habitats. The distribution of the two species may overlap south of Airlie Beach. A thorough search of the sites used in this study indicated that the two species do not co-occur in north Queensland. Planktonic developer C. pentagona is a dioecious free-spawner with a plank- tonic lecithotrophic brachiolaria larva (Figs. 2A, B, D; 3A- C). Ovaries of specimens collected in October and Novem- ber 2000 and October 2002 were gravid (Fig. 2A). They contained large eggs dominated by large lipid droplets. Spawned eggs (413 /im diameter; SE = 6.4 /urn, n = 20) were an amber-gold color. They were positively buoyant and floated to the surface as they emerged from the gono- pore. Mature testes were typical of asteroids, having a layer of spermatogenic columns along the germinal epithelium and a lumen filled with sperm (Fig. 2B). Development of C. pentagona through the stages of ho- loblastic radial cleavage, early blastula, wrinkled blastula. and gastrula was typical of development in lecithotrophic sea stars (Byrne, 1995). At 23 °C the early larvae (2 days) had a large preoral lobe and developing larval arms (bra- chia) (Fig. 3A). The central brachium developed as a bulge that emerged from the preoral lobe, flanked posteriorly on either side by two lateral braehia (Figs. 2D, 3B). Advanced brachiolariae (5 days) had a prominent brachiolar complex comprising three braehia and a central adhesive disc (Fig. 3C). Early larvae swam at the surface propelled by their cover of cilia (Fig. 3A, B, F); but as the juvenile rudiment developed in the posterior region, they swam at the bottom of the culture dishes, anterior end up. An extracellular matrix with meshlike holes covered the surface of the larvae and juveniles (Fig. 3F). This feature was most evident over the braehia of the larvae and on the oral surface and tube feet of the juveniles. Advanced larvae adhered to surfaces using their braehia. As they explored the substratum, they flexed dorsally to bring the adhesive disc to the surface. Once they were committed to metamorphose, permanent benthic attachment was achieved by the adhesive disc as- sisted by the braehia. The larvae attached to a range of substrata including the walls of the culture dishes, although they appeared to favor coralline algae (Fig. 2F). Most larvae metamorphosed regardless of whether a specific settlement substratum was introduced into the culture dishes. Many of them settled on the walls of their containers. Newly meta- morphosed juveniles (620 /u,m diameter; SE = 11.8, H = 15) had two pairs of tube feet in each radius (Fig. 3D, E). They were a dark amber color, indicating the presence of extensive maternal nutritive reserves. The mouth did not open for 3 weeks after settlement (Fig. 3D). Development to the settled juvenile stage took 9 days at 23 °C, while at ambient temperatures (30 °C) in Queensland, development was completed in 6 days (Dartnall, pers. comm.). Intragonadal developer C. hystera had ovotestes that were a mosaic of oogenic and spermatogenic areas (Fig. 2C). Gravid specimens were present in all the samples obtained from September to November, indicating that the reproductive period lasts at least 3 months. In December 1999, the gonads contained juveniles and few gametes. The amount of spermatogenic tissue in the gonads varied among individuals (/; = 20). In some specimens, sperm was only detectable histologically; in others, white testieular regions of the gonad were evident by direct examination. Like those of C. pentagona, the eggs were large (440 jum diameter; SE = 6.0 /urn, /i = 8) and contained abundant lipid droplets. They floated to the sur- 2X8 M. BYRNE ET AL Figure 2. Histology and light microscopy. (A, B) Cryptasterina pentagona ovaries and testes. o, oocyte; s. spermatozoa: sc. spermatocyte columns. (C) C. /jv.v/t-™. The ovotestis contains lipid-rich eggs (o). sperm (s). and developing embryos (arrows); g, gastrula. (D, E) Brachiolaria larvae of O. pi'iiuigona and C. hyxlcnt respectively, hu, hrachia; h. hydropore. (F) Metamorphosing juvenile C. iJeniu^onu on coralline algae (ca). (G) Dissected C. liy\lciv showing juveniles (arrows) in gonad. (H) Newly released C hysteru. Scales: A-E = 100 ftm; F, H = 200 )j.m; G = 500 ju.ni. face when removed from the gonad and were gold-orange, with a dark vegetal pole. Developing embryos and larvae were interspersed with iKimetes in the gonud (Fit;. 2C. G). Embryos removed from the gonad at the early blustulu stage developed indepen- dently of the parent through the wrinkled blustulu and gastrula stages into a planktonic highly buoyant brachio- laria. The developing brachia appeared as three bulges (Fig. DEVELOPMENT IN CRYPTASTERINA 2S9 Figure 3 Scanning electron microscopy, Cryptasterina pentagonu. (A. B) Brachiolaria larvae have a uniform cover of cilia. The central brachium (arrow) develops as a protrusion of the preoral lohe. (C) Advanced larva with a well-developed adhesive disc (ad) at the hase of the brachia (ba). (D. E) Recently metamorphosed juvenile with two pairs of tube feel in each radius and with a mouth opening. (F) Detail of cilia (c) and meshlike matrix on larval surface. Scales: A. B = 100 fim; C, E = 50 /im; F = 4 jitm. 4A). and the adhesive disc developed between the arms (not illustrated). As in C. pennigonu. advanced larvae had a well-developed preoral lobe from which the central bra- chium emerged as a bulge-like protrusion (Fig. 4B. C). They swam anterior end up with their ciliary cover (Fig. 4F). Advanced larvae ( 10 days) had a well-developed brachiolar complex which was used for benthic attachment. They exhibited typical settlement behavior while exploring the 290 M. BYRNE ET AL Figure 4. Scanning electron microscopy. Cryptiuiciinii Imtera. (A. B) Early larvae with developing hiachia (ha I. (C) Advanced larva. The central brachium (arrow ) develops as a posterior protrusion of the preoral lobe. (D) Metamorphosing larva with resorbing larval body (arrow) and developing tube feet (t). (El Recently metamorphosed juvenile with two pairs of podia in each radius and with a mouth opening- (F) Detail of cilia on larval surface. Scales: A. B, E = 100 /Am: C. D = 51) jum; F = 12 /im. substratum and adhered to the surface of the culture dishes either with the tips of the brachia or by flexing the body to attach the adhesive disc. The juvenile rudiment developed in the posterior region as the larval body was resorbed (Fig. 4D). Development in vitro to the juvenile stage took 16 days. Newly settled juveniles had a dark amber pigment due to the presence of maternal nutritive reserves. It took 3 weeks for the mouth opening to develop, and by this time maternal reserves were no longer evident (Fig. 4E). Three- week-old juveniles had a well-developed skeleton. In aquaria, juveniles (800 ju,m diameter. SE = 6.3. n = 10) with two pairs of tube feet in each radius emerged from the gonopore on the aboral surface of the adults (Fig. 2H). These juveniles had a mouth opening, a functional digestive tract, and a well-developed skeleton. Newly released juve- niles were white due to the color of the skeleton, and they appeared to lack residual maternal nutrients. Discussion Recent discoveries of cryptic species in a range of taxa have been facilitated by investigation of developmental evolution and molecular phylogeny (Reid. 1990; Degnan and Lavin. 1995: OFoighil and Smith. 1995; Williams, 2000; Flowers and Foltz, 2001; McFadden et ai, 2001). Observations of juvenile birth resulted in the description of new viviparous PatirielUi in the P. exigiui group (Dartnall. 1969, 1971; Keough and Dartnall, 1978), and our investi- DEVELOPMENT IN CRYPTASTERINA 291 gallon of cryptic biodiversity in Cryptasterina was prompted by the results of molecular phytogeny ( Hart ct nu group are shown in Table 1 and Figure 5. The gonochoric. free-spawning mode of reproduction seen in C. pentagona and Cryptasterina n. sp. is ancestral for the Echinodermata, while acquisition of hermaphroditism, a derived character, is exhibited by most echinoderms that brood their young (Strathmann el ai, 1984; Byrne, 1991, 1999; Hendler. 1991). Like those of the other viviparous species (Table 1 ), the gonads of C. hystera were ovotestes. This indicates the potential for self-fertilization, as appears to be the case for P. vivipuru (Byrne. 1996). The amount of sperm in the gonads of C. h\stcra is more than sufficient to fertilize all the eggs produced, and so it is likely that some individuals release sperm. For out-crossing to occur, the sperm would have to gain access to eggs by swimming through the gonopore. A genetic study is required to deter- mine if progeny in the gonads of the viviparous Cryptast- crinii and Putiriellii are full siblings or half siblings. The diversity of the fertilization biology in these asterinids with complete out-crossing in the free-spawners, partial self- fertilization in the benthic egg layers, and potential for selling in viviparous forms provides a useful model in which to investigate the relationships between the evolution of mating systems and the genetic structure of sea star populations (Byrne. 1995. 1996). As characteristic of echinoderms, the evolution of leci- thotrophy in Cryptasterina species involved acquisition of a large egg (Table I ). The increase in egg size from what would have been an ancestral form with a small egg and planktotrophic development is considered to have been nec- essary to sustain development without feeding (Mortensen, 1921; Strathmann, 1978; Emlet ct na-Y\ke ancestor. This suggestion is supported by molecular phylogenetic data (Fig. 5; Hart ct al., 2003). The intragonadal brachiolariae of P. vivipara and P. parvivipara are vestigial, unlike those of C. hystera. Their minute larvae have a reduced brachiolar complex compris- ing three small nonsticky protrusions that cannot function in attachment, and some embryos do not develop brachia at all. Intragonadal development in P. vivipara and P. pan'ivipara is thought to have evolved through a P. e.\igua-\\ke ancestor that laid benthic egg masses and had highly modified benthic nondispersive larvae (Fig. 5; Byrne, 1995; Hart et al.. 1 997). Evolution of viviparity through retention of eggs by an ancestor with benthic egg masses is suggested to have been a likely pathway for the acquisition of this form of brooding in asterinids (Strathmann et al.. 1984; Byrne, 1996). Despite their intragonadal location, the larvae of C. hys- DEVELOPMENT IN CRYPTASTERINA 293 /era exhibited exploratory settlement behavior and attached to the substratum with their brachia and adhesive disc prior to metamorphosis, in a manner typical of planktonic aster- oid larvae. This behavior is unlikely to serve any function in the intragonadal environment. A reversal to a planktonic larva is readily envisaged for C. hystera and C. pacifica. a suggestion also made for Pteruxter tesselatus (McEdward, 1992). These species could potentially use two modes of development, releasing some progeny as dispersive larvae and others as juveniles. By contrast. P. ririptirci and P. parvivipani are committed to intragonadal development. A reversal to reacquire a large egg and functional larva ap- pears unlikely for P. vivipara and P. /Hirvivi/nini. Intragonadal development is rare in the Echinodermata. Among asteroids, intragonadal development and live birth is known for only three genera. Cryptasterina, Putiriella (Table 1 ). and the aberrant XylopUi\ medusiformis from the deep sea (Rowe et enet. Evol. 15: 301-313. O'Loughlin. P. M., J. M. Waters, and M. S. Roy. 2002. Description of a new species of Patiriella from New Zealand and review of Patirellti regularis (Echinodermata, Asteroidea) based on morphological and molecular data. J. R. Soc. NZ 32: 697-7 1 1 . Palumbi, S. R. 1992. Marine speciation on a small planet. Trends Ecol. Evol. 7: 114-118. Raff, R. A. 1996. The Shape of Life. University of Chicago Press, Chicago. Reid, D. G. 1990. A cladistic phylogeny of the genus Littorinu (Gas- tropoda): implications for evolution or reproductive strategies and for classification. Hydrohiologia 193: 1-19. Rowe, F. W. E., and J. Gates. 1995. Echinodermata. In Zoological Catalogue of Australia. Vol. 33, A. Wells, ed. CS1RO, Melbourne. Rowe, F. W. E., A. N. Baker, and H. E. S. Clark. 1987. The morphol- ogy, development and taxonomic status of Xylopla.\ Baker, Rowe and Clark (1986) (Echinodermata: Concentricycloidea), with the descrip- tion of a new species. Proc. R. Soc. Loud. B. 233: 431-459. Strathmann, R. R. 1978. The evolution and loss of feeding larval stages of marine invertebrates. Evolution 32: 894-906. Strathmann, R. R. 1985. Feeding and nonfeeding larval development and life history evolution in marine invertebrates. Annu. Rev. Ecol. Syst. 16: 339-361. Strathmann. R. R., M. F. Strathmann, and R. H. Emson. 1984. Does limited brood capacity link adult size, brooding and simultaneous hermaphroditism? A test with the starfish Asterina phylactica. Am. Nat. 123: 796-818. Vacquier, V. D., W. J. Swanson. and M. E. Hellberg. 1995. What have we learned about sea urchin bindin? Develop. Growth Differ. 37: 1-10. VandenSpiegel, D.. D. J. W. Lane, S. Stampanato, and M. Jangoux. 1998. The asteroid fauna (Echinodermata) of Singapore with a dis- tribution table and an illustrated identification to the species. Ruffles Bull. Zool. 46: 431-470. Villinski, J. T., J. L. Villinski, M. Byrne, and R. R. Raff. 2002. Convergent maternal provisioning and life history evolution in echino- derms. Evolution 56: 1764-1775. Williams, S. T. 2000. Species boundaries in the starfish genus Linckia. Mar. Biol. 136: 137-148. Wray. G. A. 1996. Parallel evolution of nonfeeding larvae in echinoids. S\st. Biol. 45: 308-322. Reference: Bio/. Bull. 205: 295-307. (December 2003) © 2003 Marine Biological Laboratory Persistent Ancestral Feeding Structures in Nonfeeding Annelid Larvae BRUNO FERNET Frida\ Harbor Laboratories, 620 University Road, Fridav Harbor. Washington 98250 Abstract. Evolutionary loss of the requirement for feed- ing in larvae of marine invertebrates is often followed by loss of structures involved in capturing and digesting food. Studies of echinoderms suggest that larval form evolves rapidly in response to loss of the requirement for feeding, but a lack of data from other taxa makes it difficult to assess the generality of this result. I show that many members of a large clade of annelids, the Sabellidae, retain ancestral sys- tems for particle capture despite loss of the need and ability to feed. In at least one species, Schizobranchia in.signis. an opposed-band system of prototrochal. food-groove, and metatrochal ciliary bands can concentrate suspended parti- cles and transport them to the mouth, but captured particles are invariably rejected because larvae lack a functional gut. The persistence of particle capture systems in larvae of sabellids suggests that they have lost larval feeding very recently, that opposed bands are inexpensive to construct and operate, or that opposed bands have some alternative function. These observations also suggest a hypothesis on how the ability to feed is lost in larvae of annelids and other spiralians following increases in egg size. Introduction Larvae of some marine invertebrates require paniculate food to complete development to the juvenile stage, but others cannot feed and instead rely on materials stored in the egg. These alternative nutritional modes are associated with differences in many other traits, including embryonic de- velopment (Wray and Bely, 1994), dispersal and population genetic structure (Palumbi, 1995), species duration (Jablon- ski, 1986). and perhaps most obviously, larval form. Feed- ing larvae bear structures that function in particle capture, ingestion. and assimilation, while nonfeeding larvae tend to lack such structures and are relatively simple in external Received 3 April 2003; accepted 22 September 2003. E-mail: pernetb@hotmail.com form (Emlet, 1991). Many species with nonfeeding larval development evolved independently from ancestors that had feeding larvae (Strathmann. 1978). This suggests a strong evolutionary association between the loss of the require- ment for feeding and the loss of larval feeding structures (Wray, 1996). How does this association arise? One model suggests that the first step in the evolution of nonfeeding development is an increase in the energy content of the egg (Jagersten, 1972; Strathmann, 1975: Raff, 1987; Kempf and Todd, 1989: Hart, 1996: Wray, 1996; McEdward and Janies, 1997). Increased egg energy content permits larvae to com- plete development without paniculate food. These derived larvae may feed facultatively. However, once larvae have lost the requirement for food, stabilizing selection on feed- ing performance is weakened, and mutations that affect the form or function of feeding structures may accumulate (Strathmann, 1975). Selection on other larval functions like swimming or developing rapidly and efficiently may also lead to changes in larval form (Wray and Raff, 1991; Emlet. 1994). Under these conditions, feeding structures eventually become nonfunctional, and the resulting larvae are obli- gately nonfeeding. Loss of the ability to feed may occur by any of many different changes in morphology, physiology, or behavior, but details of this process are mostly unknown (Kempf and Todd. 1989; Wray, 1996). Further reduction of ancestral feeding structures, which may occur rapidly after loss of the requirement for paniculate food (Wray and Raff, 1991; Hart, 1996; Wray, 1996), then leads to simplification of external form in nonfeeding larvae (Emlet. 1991; Byrne el al., 2001 ). This scenario is a specific example of a more general model of the reduction and loss of nonfunctional characters (Fong et al., 1995). Though larval feeding has been lost in many lineages of marine invertebrates (Strathmann. 1978). subsequent pat- terns of change in larval form have been studied primarily in members of only one phylum, the Echinodermata. 295 296 B. FERNET Examples from other phyla would be useful in assessing the generality of thev results. Here I provide such an example from a phylum only distantly related to the echinoderms, the Annelida Vjoellid annelids are sessile, tube-dwelling worms often known as "feather-duster worms" because of the crown of tentacles they extend from their organic, un- mineralized tubes for suspension feeding. The family in- cludes about 490 species that fall into two clades, the subfamilies Fabriciinae and Sabellinae (Rouse and Pleijel. 2001). All fabriciins whose reproduction has been studied (about 15 of 75 species) brood embryos that undergo direct development. Sabellins are more variable in reproductive biology, with some species brooding embryos and larvae through the juvenile stage; others brooding embryos and larvae for most of their development, releasing larvae for a brief planktonic phase before settlement; and still others releasing gametes directly into the sea where they are fer- tilized and develop into planktonic larvae. Though repro- duction and development has been studied in few sabellins (about 30 of 415 species), these are broadly distributed in phylogenies of the family (Rouse and Fitzhugh, 1994). No sabellid larvae are known to feed (Rouse and Fitzhugh, 1994). A look at the relationships of annelid worms suggests that nonfeeding development in sabellids represents a loss of larval feeding (Fig. 1 ). Sister clade to the Sabellidae is the Serpulidae, which includes both species with feeding larvae and species with nonfeeding larval development. Sister to the clade (Serpulidae, Sabellidae] is the Sabellariidae. All sabellariids whose development has been described have feeding larvae. These character states are shown in Figure 1, with inferences on larval nutritional mode in ancestral forms. As no extant sabellid is known to have feeding Sabellidae Sabellariidae Serpulidae Fabriciinae Sabellinae ffl BD D D • feeding larvae D nonfeeding larvae Figure 1. Relationships of sabellariid, serpulid, and sabellid annelids, as indicated by cladistic analyses of morphological and reproductive char- acters by Fitzhugh (1989) and Rouse and Fitzhugh (1994). This topology is also supported by analyses of DNA sequence data from the nuclear gene elongation factor- 1 alpha (D. McHugh, Colgate University, pers. comm.). Inferences on ancestral character slates are discussed in the text. larvae, nonfeeding development is likely plesiomorphic in the family (Rouse and Fitzhugh, 1994). The inference that the common ancestor of serpulids and sabellids had a feed- ing larval stage relies primarily on the observation that feeding larvae of sabellariids and serpulids capture particles using similar systems of three parallel ciliary bands, the prototroch, food groove, and metatroch (Fig. 2: Strathmann et nl.. 1972; Strathmann, 1987; Strathmann and Fernet, unpubl. data). The cilia of the prototroch, which form a transverse band anterior to the mouth, beat from anterior to posterior. These cilia create a swimming current as well as being involved in feeding. Suspended particles passing near them are overtaken and moved towards the surface of the larval body. Cilia of the metatroch. located posterior to the mouth, beat from posterior to anterior. Particles caught between these two ciliary bands are transported towards the mouth by cilia of the food groove. This is usually referred to as "opposed band" feeding. (Note that Rouse [1999. 200()a] argues that sabellariid larvae do not feed with op- posed bands of cilia. However, larvae of the only sabellari- ids in which larval feeding has been studied, Sabelluria alreolala and S. cementarium, do feed in this way [Strath- mann, 1987; Strathmann and Fernet, unpubl. data].) The presence of similar feeding structures in sabellariids and serpulids suggests that ancestors of the clades [Sabellariidae [Serpuiidae, Sabellidae]] and [Serpulidae, Sabellidae] had larvae that fed with opposed bands of cilia. Nonfeeding development in the sabellids thus represents a loss of larval feeding. Rouse (2000a) also concluded that sabellids were derived from ancestors that had feeding larvae. Here I show that despite loss of the requirement and ability to feed as larvae, members of at least eight genera of sabellids retain opposed bands of cilia. In larvae of at least one species, Schizobranchia insignia, these structures can concentrate particles from suspension and transport them to the mouth, where they are invariably rejected because the larvae lack functional digestive systems. The persistence of ancestral systems for particle capture in nonfeeding sabellid larvae is unexpected in light of data from echinoderms, which suggest that once the requirement for larval feeding is lost, feeding structures are rapidly reduced or lost. These observations suggest that sabellids lost larval feeding very recently, that opposed bands are inexpensive to construct and operate, or that opposed bands have alternative func- tions in these larvae. These observations also suggest a hypothesis on how development mediates the loss of feed- ing ability in larvae of annelids and related phyla after increases in egg size. Materials and Methods Collection, xf>nwnin}>, and lurval culture I studied larvae of the sabellin sabellid Schizobranchia insignis Bush, 1905, intensively, and made additional ob- servations on larvae of three other sabellins, Denwnax me- OPPOSED BANDS IN SABELLID LARVAH 297 ANTERIOR prototroch \ food groove metatroch neurotroch mouth POSTERIOR Figure 2. Diagrammatic venlral view of a larva of a serpulid annelid, showing structures used in the opposed-band feeding system and the path of a particle (arrowhead) captured from suspension. Cilia ot a preoral hand, the prototroch, beat from anterior to posterior. As they beat, panicles that pass within their reach may be moved into a perioral band of cilia, the food groove. Metatrochal cilia beat from posterior to anterior and may help trap particles in the food groove. Once in the food groove, particles are transported ventrally to the mouth. Rejected particles are moved posteriorly by cilia of the neurotroch. dins (Bush. 1905). Myxicola aesthetica (Claparede. 1870), and Pseudopotamilhi occelata (Moore, 1905). All of these species except D. medius are broadcast spawners; adults of D. medius brood embryos and larvae in a gelatinous mass around the opening of the adult tube, and larvae emerge from masses for a brief (~1 day) planktonic period (McEuen et ai, 1983). Adult S. insignis and P. occelata were collected from floating docks on San Juan Island, Washington, from January to April in 2002 and 2003. The tube of each animal was cleaned of fouling organisms and trimmed to the length of the worm it contained. To induce spawning. 10-20 conspecific individuals were placed in a large container through which fresh seawater flowed. About half of the worms so treated spawned within 48 h. usually at night. I collected fertilized eggs from the bottom of the container the following morning, rinsed them several times in coarsely filtered seawater (FSW, mesh size ~5 /nm). and cultured them in 1 -liter beakers of FSW at densities of ~5 per milliliter. Beakers were partially submerged in flowing seawater at temperatures of 8-10 °C (near field tempera- tures) and stirred with paddles (M. Strathmann, 1987). Wa- ter in larval cultures was changed every 3-6 days. No food was added, but cultures certainly contained bacteria and unicellular protists. Larvae of S. insignis were followed through metamorphosis. These larvae settled on small pieces of adult tube that were scored with a razor blade to provide crevices. Juveniles were fed the unicellular algae Isochrysis sp. and Rhodomonas sp. at high concentrations. I collected adult Mvxicoln aesthetica from floating docks in Anacortes. Washington, in July 2002. Adults spawned when removed from their tubes and subjected to gradual warming (to room temperature) and rapid cooling of the seawater they were incubated in. Embryos and larvae were raised as described above. I obtained two egg masses of Demonax medius from the intertidal zone on the west side of San Juan Island in February and April 2002, and incubated them in FSW at 8-10 °C in the laboratory. I examined larvae that had been removed from masses, as well as planktonic larvae that had emerged from masses naturally. Planktonic larvae were raised as described above. Lan'al morphology Developmental stages were examined with a light micro- scope equipped with differential interference contrast (DIG) optics. Larvae were relaxed in a 1:1 solution of 7.5% MgCK and seawater before examination. Body dimensions were estimated with a calibrated ocular micrometer. For scanning electron microscopy, relaxed larvae were killed by gradual addition of dilute formalin. They were then rinsed in Mil- lipore-filtered seawater (MPFSW, mesh size 0.45 jitin) and fixed for 2 h in 2% OsO4 in 1.25% sodium bicarbonate buffer (pH 7.2). Fixed larvae were rinsed in distilled water. then dehydrated in ascending concentrations of ethanol. They were critical-point-dried, mounted on stubs with car- bon adhesive disks, and sputter-coated with gold-palladium before being examined and photographed with a JEOL JSM-35 scanning electron microscope. Larvae of Schi-o- hnmchia insignis were also sectioned for study of internal anatomy. Relaxed larvae were fixed in 2.5% glutaraldehyde in 0.2 M Millonig's phosphate buffer for 1-3 h. rinsed in buffer, then post-fixed in 1% OsO4 in buffer for 1 h. After dehydration in ethanol, larvae were embedded in EMBed- 812 (Electron Microscopy Sciences, Inc.) with propylene oxide as an infiltration solvent. Sections -1 /am thick were cut with glass knives and stained with Richardson's stain for light microscopy (Richardson et ai, 1960). Functional morphology of ciliary bands Beat patterns of cilia were recorded with a high-speed video camera (Motionscope 1000-S. RedLake Imaging Inc.) mounted on a compound microscope with DIC optics. Lar- vae were restrained under a coverslip supported with plas- ticene modeling clay. Images were collected at 250 frames per second and replayed at 10 frames per second. Sequences were routed from the camera through a Sony DVMC-DA2 analog-digital converter to a Macintosh iBook and saved as iMovie files (Apple Computer, Inc.). Capture and transport of particles by larvae of Sclii:o- branchia insignis were visualized by videotaping larvae in a suspension of particles. Larvae were mounted on a slide under a coverslip supported with plasticene modeling clay. 298 B. FERNET The coverslip was pressed down just enough to prevent larvae from s miming rapidly. High concentrations of polystyrene ti; mvlbenzene beads (3-/Mm diameter, blue- dyed, Polvi ,-:v,ces. Inc.) were added and larvae were ob- served with a compound microscope. Beads had previously been incubated in a 2.5% solution of bovine serum albumin (BSA) in distilled water for 1-2 h, then rinsed and resus- pended in MPFSW. Such beads are readily captured and ingested by free-swimming or restrained feeding larvae of sabellariids and serpulids (Strathmann and Fernet, unpubl. data). Images were collected with a video camera (Sony CCD SSC-S20) and recorded on VHS tape, with a date-time generator providing a time signal. Tapes were played frame by frame for analysis. Possible functions of opposed ciliary bands in sabellid larvae I tested two hypotheses on functions of opposed ciliary bands in larvae of sabellids. Opposed ciliary bands are used for capturing paniculate food during the planktonic larval stage. I verified that larvae of Schizobranchia insignis and Demonax medius are non- feeding by offering them particulate "food" through devel- opment and later examining their guts for ingested particles. For each assay, 20 larvae were placed in 20 ml MPFSW in a vial. Two types of particles were added to each vial: blue-dyed polystyrene divinylbenzene beads 3 and 6 /u,m in diameter, previously incubated in 2.5% BSA as described above, and single-celled algae (Dunaliella sp.). Final con- centrations of beads were 5-10 per microliter of each di- ameter: I did not measure algal concentrations. I included several feeding larvae of the echinoid echinoderm Strongy- locentrotus droebacliiensis or the serpulid annelid Serpula coluinbiana in each vial as positive controls. Vials were slowly rotated on a plankton wheel in the dark at 10 °C for 1-2 h. Larvae of sabellariids and serpulids feed at high rates under these conditions (Fernet, unpubl. data). Larvae were preserved at the end of feeding assays by addition of buff- ered formalin. They were later cleared and mounted in glycerin, and examined with a compound microscope to determine whether they had ingested particles. Through processing, beads retained blue dye, and algae retained pigment; both types of particles were easily visible when present in larval guts. Ten sabellid larvae, as well as several larvae known to feed, were examined in each assay. For S. insignis. assays were conducted every 5 days, from the 5th day after fertilization until larvae were 30 days old. For D. nii'diiis. feeding assays were carried out with swimming larvae that had left egg masses on their own. Opposed ciliary bands are sites of uptake of dissolved proteins. Moran (1999) found that encapsulated larvae of several marine gastropods use velar cells to take up dis- solved proteins from the capsular fluid. In related species with planktonic feeding larvae, these cells bear cilia that function in an opposed-band feeding system. Using the methods of Rivest (1981) and Moran (1999), I tested the hypothesis that larvae of Schizobranchia insignis or De- monax medius take up proteins with the cells that bear the opposed ciliary bands. Ten larvae were placed in 1 ml of fluoresceinisothiocyanate-conjugated bovine serum albumin (FITC-BSA) dissolved in MPFSW (1 mg/ml). Negative controls were incubated in MPFSW only. Larvae of species known to take up large proteins (veligers of Littorina sit- kana [Moran, 1999] or Crepidula aditnca [Rivest, 1981]) were included in all vials as positive controls. Larvae were incubated in test solutions for 6 h at 10 °C in the dark. After incubation, they were rinsed in MPFSW and observed by epi fluorescence microscopy. At least five sabellid larvae, as well as several positive controls, were examined from each treatment. For S. insignis, assays were conducted every 5 days, from the 5th day after fertilization until larvae were 30 days old. For D. medius, assays were carried out with larvae that had hatched from their individual capsules but had not yet left egg masses on their own (these larvae were about 5-15 days old, and were removed from egg masses by agitation). Results Lan'al development and form Schizobranchia insignis. Except for notes on egg size and developmental mode (Lee, 1970. 1975; McEuen et ai. 1983), development of this species has not previously been described. A summary of its development can be found in Table 1. In the spawning events I observed, males always spawned before females. Fertilized eggs were spherical, opaque, neg- atively buoyant, gray in reflected light, and surrounded by an elevated envelope (Fig. 3 A). Mean diameters (± one standard deviation) of 20 eggs from each of three separate females were 154 (3.4), 156 (2.8), and 157 (2.8) ^m; the fertilization envelope brought the total diameter to 180-190 jam (Fig. 3A). First and second cleavages were both mark- edly unequal (without the formation of polar lobes), leading to a four-cell stage with one cell that was much larger than the others (Fig. 3B). Two days after fertilization, embryos had become pear- shaped trochophore larvae (Fig. 3C). The widest part of the body bore a transverse, preoral band of cilia, the prototroch. The prototroch was composed of three parallel tiers of cilia — an anterior tier of short cilia, a middle tier of long compound cilia, and a posterior tier of short compound cilia. Cilia of all three tiers protruded through the fertilization envelope. They could be identified as preoral because a slight depression had appeared just behind them, midven- trally at the site of the developing mouth. By the 3rd day after fertilization, two additional bands of transverse cilia had appeared (Fig. 3D). Immediately pos- terior to the prototroch, at the level of the larval mouth, was OPPOSED BANDS IN SABELLID LARVAE 299 Table 1 Si'lieilnle nj ilcreltipnicnt in Schizobranchia msignis raised at temperatures of 8-10 °C Time Stage or event Planktonic larval development (I Fertilization I d Prototrochal cilia protrude through egg envelope; weakly swimming - d Pear-shaped trochophores; prototroch well developed; ocelli, head and anal vesicles present ? d Metatroch and food-groove cilia present; neurotroch present; competent to settle •4 d Notochaetae in chaetiger 1 5 d Notochaetae in chaetiger 2 I 1 d Notochaetae in chaetiger 3; neurochaetae (uncim) in chaetigers 2 and 3 12-1? d Neurochaetae (uncini) in chaetiger 4 Post-settlement development II Settlement; mucus tube formed around body 1 d Neurotroch between peristomium and pygidium lost in some indmduals. cullar lobes present on peristomium 2 d Neurotroch between peristomium and pygidium lost in all individuals; prototroch lost middorsally; radiole buds present on prostomium 3 d Prostomial snout present; radiole buds each divided into 2 lobes; anus present 4 d Radiole buds each divided into 3 lobes, ciliated; all long cilia of larval prototroch gone; ingestion of snout begins? 5 d Radiole buds each divided into 4 lobes 6-7 d Juvenile feeding begins Timing data are summarized from observations of cohorts of offspring from six separate females. Though competent to settle 3 days after fertil- ization, larvae, if not offered a suitable settlement substrate, continue swimming and remain competent to settle until at least 30 days post- fertilization. For the schedule of post-settlement development, larvae were allowed to settle after a 15-d planktonic period. The timing of events in post-settlement development was similar in larvae that had 9-d and 30-d planktonic periods. a narrow band of short, simple cilia. The width of this perioral band was about 4-5 /xm in rive 10-day-old larvae. Just behind this perioral band was a postoral band of simple cilia. These ciliary bands are shown in greater detail in an illustration of an older larva (Fig. 3E). Positionally, these bands of cilia are identical to the perioral food groove and postoral metatroch known from annelid larvae that feed with opposed bands of cilia, and I will refer to them as food groove and metatroch in the rest of this paper. Both food groove and metatroch were incomplete dorsally; the dorsal gap in the food groove was substantially wider than that in the metatroch. Three-day-old larvae had also developed a midventral band of short cilia, the neurotroch, which stretched from the metatroch to the posterior end of the body. Three-day-old larvae were competent to settle and meta- morphose. However, if larvae were not offered settlement substrates and were maintained in stirred cultures in clean glass beakers, most remained planktonic until at least 30 days after fertilization. Changes in form in larvae from 3-30 days of age — mostly involving Ihe appearance of chaetae in developing segments (e.g.. Fig. 3F) — are summarized in Table 1 . Between the 25th and 30th day of development, many larvae began to show signs of metamorphosis while still in the plankton. In particular, they developed a pair of dorsolateral bulges on the prostomium. In normally meta- morphosing worms, these bulges go on to form the adult feeding tentacles, or radioles. Mortality of larvae appeared to increase greatly at around 30 days. Sections of larvae fixed at various ages allowed descrip- tion of changes in gut morphology during development. Transverse sections of 8-day-old larvae showed that the mouth was represented only by a shallow, ciliated midven- tral depression (Fig. 3G). This depression was not con- nected to the midgut. The midgut wall was composed of endodermal cells that were so large that they completely occluded its lumen. Eight-day-old larvae had neither an intestine nor an anus. In 20-day-old larvae, the mouth had increased in depth and led to a very narrow ciliated stomo- daeum (Fig. 3H). In serial sections of five 20-day-old larvae, I was unable to find a connection between the stomodaeum and midgut. The cells lining the midgut had shrunk slightly, and it now had a narrow central lumen at the level of the stomodaeum. This lumen disappeared posteriorly. I ob- served no intestine or anus in these larvae. Larvae of Schizobranchia insignis were competent to settle from 3 to at least 30 days after fertilization. When offered pieces of adult tube in still water, 20%-50% of larvae settled within 24 h. Larvae usually settled in crevices in the tubes, or on the glass bowl beneath pieces of tube. The events following settlement of 15-day-old larvae are summarized in Table 1. Note that prototrochal cilia had been resorbed or shed by 4 days after settlement, before juveniles began feeding. Demonax mediits. All larvae of D. mediits that I exam- ined— larvae extracted from gelatinous brood masses, and larvae that had escaped from masses for a brief planktonic period — had prototroch, food groove, and metatroch cilia similar to those seen in Schizobranchia insignis (Fig. 4A). The width of the food groove ranged from about 6-8 jam in five larvae that had left egg masses on their own. The metatrochal band was slightly broader than that of S. insig- ni.\. In most other respects my observations of development of two broods of D. mediits are in accord with those of McEuen et til. (1983). One addition to their description is that embryos and early larval stages in gelatinous egg masses were individually encapsulated in spherical capsules only slightly larger in diameter than the developing worms themselves. Capsules are present in addition to fertilization envelopes like those described above for S. insignis. In the broods I observed, larvae hatched from capsules about 5-7 300 B. FERNET B i» Figure 3. Embryonic and larval stages of Schizobranchia insignis. (A) Fertilized egg. (Bl Four-cell embryo. (C) Two-day-old larva. (D) Three-day-old larva. (E) Detail of opposed ciliary bands of fourteen-day-old larva. (F) Fourteen-day-old larva. (G) Transverse section of eight-day-old larva at the level of the mouth. |H) Transverse section of twenty-day-old larva at the level of the mouth. Scale bars = 50 pim, except for (E) where the scale bar = 15 /im. chl = first chaetiger, ch2 = second chaetiger. ch3 = third chaetiger. fe = fertilization envelope, fg = food groove, me = metatroch. mg = midgut wall, mgl = midgut lumen, mo = mouth, ne = neurotroch, po = pore of one of the paired anal vesicles, pr = prototroch, pr2 = second tier of prototrochal cilia, pr3 = third tier of prototrochal cilia. days after egg deposition, and they emerged from egg masses about 7-15 days after deposition. Myxicola aesthetica. Fertilized eggs of M. aestlietica were spherical, opaque, negatively buoyant, rose-colored in reflected light, and surrounded by an elevated envelope. The mean diameter (± one standard deviation) of 10 eggs from OPPOSED BANDS IN SABELLID LARVAE 301 Figure 4. Opposed ciliary bunds of Demonax metliiis. Myxicola aes- iltciHLi, and Psi-iul,ip<>kiiiiilla occelata. (A) Ciliary bands in the right ventral region of larva of D. mediiis. (B) Left lateral view of ciliary bands of 1 1 -day-old larva of M. aesthetica. (C) Left dorsal view of ciliary bands of 3-day-old larva of P. occelata. Scale bars = 25 /u.m. fg, food groove; me. metatroch; mo, mouth; pr, prototroch. a single female was 130 (4.9) /urn. First and second cleav- ages were distinctly unequal. At incubation temperatures of 12-13 °C. larvae began swimming by about 18 h after fertilization. By 2 days after fertilization, larvae bore pro- totrochal. food-groove, and metatrochal cilia similar to those seen in Schizobranchia insignis (Fig. 4B). The width of the food groove was about 6 jum in five larvae. As in Demonax medius, the metatrochal band of M. aesthetica was broader than that of S. insignis. Pseudopotamilla occelata. Fertilized eggs of P. occelata were spherical, opaque, negatively buoyant, gray in re- flected light, and surrounded by an elevated envelope. The mean diameter (± one standard deviation) of 15 eggs from a single female was 142 (2.9) /u,m. First and second cleav- ages were distinctly unequal. By 3 days after fertilization, larvae bore prototroch, food-groove, and metatroch cilia similar to those seen in Schizobranchia insignis (Fig. 4C). The width of the food groove was about 6 jurn in five larvae. Functional morphology of ciliary bands Food-groove and metatrochal ciliary bands in larvae of all four species behaved like similar ciliary bands in larvae that feed with opposed bands of cilia (e.g., Strathmann et «/., 1972). The directions of effective strokes of cilia were anterior to posterior (prototroch), posterior to anterior (metatroch). and laterally towards the mouth (food groove). All three bands of cilia could arrest their beat, apparently independently of the others. When metatrochal cilia were not beating, they lay flat along the larval body, pointed posteriorly. These ciliary beat patterns were confirmed by analyses of high-speed video footage, but were also clearly visible by inspection of living larvae at 40QX final magni- fication. I examined the ability of larvae of Schizobranchia insig- nis to concentrate particles from suspension and transport them to the mouth using opposed bands of cilia. I video- taped four 9-day-old larvae in suspensions of 3-/u,m beads for a total of 22 min. This footage included at least 30 particle captures. Analysis of these video sequences indi- cated that beads were caught in the current generated by the prototrochal cilia and moved into the food groove. Resolu- tion of images was not sufficient to determine if metatrochal cilia were actively beating during captures. Captured beads were transported in the food groove around the body to- wards the larval mouth (Fig. 5). Once at the mouth, beads were moved along the neurotroch until they fell off the posterior end of the body. Possible functions of opposed ciliary bands in sabellid larvae I tested the hypothesis that planktonic larvae of Schizo- branchia insignis or Demonax medius feed on paniculate food by offering larvae particles that are readily ingested by 302 B. FERNET A 0:00 D 0:51 Figure 5. Capture and transport of a particle by a 9-day-old larva of Schi-ohranchia insignis. in ventral view. ( A. B) Capture of a particle. (C, D) Transport to the mouth in the food groove. (E) Transport posteriorly on the neurotroch. Time (s) is marked on each frame. Scale bar = ?() /j.m; the asterisk is adjacent to the particle. (F) Composite diagram of particle positions in (A-E): me = metatroch. pr = prototroch. For clarity, only positions of the prototroch and metatroch are shown. Food-groove cilia lie between them, and the neurotrochal cilia run from the gap in the metatroch to the posterior tip of the body. The mouth is slightly anterior to the position of the particle shown in (D). related feeding larvae. Paniculate food was never observed in the guts of larvae of 5. insignis ranging from 5 to 30 days old, or in the guts of larvae of D. mediits that had hatched naturally from egg masses. Feeding larvae of echinoid echi- noderms or serpulid annelids included as positive controls always ingested many of both types of particles. I also tested the hypothesis that ciliated cells of the prototroch, food groove, or metatroch are involved in the uptake of dissolved proteins by endocytosis. Larvae of Schizobranchia insignis and Demonax inedins exposed to FITC-BSA never showed any differences in fluorescence relative to larvae incubated only in MPFSW. In both treat- ments, larval chaetae autofluoresced when excited with ul- traviolet light. Larvae known to be able to take up large dissolved proteins always showed fluorescence indicating uptake oi FITC-BSA in velar cells (Littorimi sitkana: Mo- ran. 194" or cells of the "larval kidney" (Crepidiilti adunca: RivvM. 1981) when incubated in FITC-BSA. but not when incubated in MPFSW. Discussion Evolutionary loss of the requirement for larval feeding has occurred repeatedly in many phyla, and is typically followed by the loss of structures formerly involved in feeding (Strathmann, 1978: Emlet, 1991; Wray. 1996). De- tailed hypotheses on how this association arises, however, have been tested mainly in the context of comparative data on members of only one phylum, the Echinodermata (Hart. 1996: Wray, 1996). The observations reported here on lar- vae of annelids permit an initial assessment of their gener- ality. These data are also useful in generating hypotheses on other topics, including the specific sequence of events that lead to loss of feeding ability after increases in egg size and energy content in animals that develop via spiral cleavage. Ancestral feeding structures in nonfeeding annelid lan'ae I interpret the transverse ciliary bands of the sabellid larvae described here as homologs of the prototroch, food OPPOSED BANDS IN SABELLID LARVAE 303 groove, and metatroch of closely related feeding larvae (i.e.. those of serpulids and sahellariids). Almost all annelid lar- vae bear a prototroch. and in all cases where detailed observations have been made, these arc homologous by developmental criteria (Rouse. 1999). The lineages of the cells that bear food-groove and metatrochal cilia are not known in any detail, so other criteria must be used to infer homology of these ciliary bands. Two criteria that support my interpretation are those of position and behavior. The perioral and postoral cilia of larvae of Schizohranchhi i/i- xignis, Dcmoiutx nicdins. Myxicola aesthetica, and Pseudo- /><>t(iinil/ii occe/- hmnchia insignis. where the two new rows of cilia form the food groove and metatroch, and where the metatroch, when arrested, points posteriorly. Finally, a text description and drawings of larvae of Chime teres also suggest the presence of opposed bands. Okuda (1946) states that the "prototroch" of larvae of C. terex is composed of one band of long cilia followed posteriorly by two adjacent bands of short cilia. Unfortunately, he does not specify the spatial relationships of these cilia to the larval mouth. His drawings show ciliary bands similar to those observed here in four genera of sabellin sabellids. Published illustrations of larvae of sev- eral other sabellins (e.g., Anipliigleiui mitluie [Rouse and Gambi. 1998] and Pcrkinsiana riwo [Rouse. 1996|) may indicate the presence of only a single band of cilia behind the prototroch, but more observations of these larvae are needed. I found no descriptions of sabellin larvae that un- equivocally indicate the absence of food groove and metatroch. Thus, members of at least eight genera of sabellins likely possess opposed bands of cilia. According to recent phy- logenies (Rouse and Fitzhugh. 1994; Fitzhugh and Rouse. 1999). these genera are not clustered in any particular sub- clade of the Sabellinae: some (e.g.. Amphicorina, Myxicola) arise from deep nodes in the tree, and others (e.g., Laonome, Schiyhnincliiii) from shallow nodes. All known sabellin reproductive strategies (brooding to the juvenile stage. brooding with release of a planktonic larva, and freespawn- ing: Rouse and Fit/hugh. 1994) are represented in the spe- cies for which there is evidence of opposed ciliary bands. I conclude that opposed ciliary bands are probably wide- spread in the sabellins, a clade that includes over 400 species. In contrast, opposed ciliary bands do not appear in the direct-developing embryos of fabriciin sabellids (Rouse and Fitzhugh. 1994). Though metatrochal cilia have previously been identified by position in larvae of several species of sabellins (e.g., Rouse and Fitzhugh. 1994), their presence has more usually gone unnoticed. That metatrochal cilia may act with pro- totrochal and food-groove cilia as opposed-band systems in these larvae has not previously been described. It is possible that opposed ciliary bands are more widely distributed in nonfeeding annelid larvae. Indeed, the nonfeeding larvae of some serpulids also have opposed ciliary bands, at least by the criterion of position (Kupriyanova et 110 ;u.m: Rouse and Fitzhugh, 1994) and energy-rich (Fernet and Jaeckle, unpubl. data). The second step in this sequence is loss of the ability to feed. All known sabellid larvae are obligately nonfeeding. However, they retain some ancestral feeding structures (in particular, cilia of the food OPPOSED BANDS IN SABELLID LARVAE 305 groove and metatroch). These larvae can thus he interpreted as intermediates in the evolutionary sequence discussed above. Such intermediates have previously been considered rare (Hart. 1996: Wray. 1996). but my observations suggest that they may be common in this clade of annelids. These data may be useful in answering a question that has previously been poorly understood — that is, what specific events lead to the loss of larval feeding ability after an evolutionary increase in egg provisioning? Wray (1996) noted that loss of the ability to feed might be the result of any of many changes, such as loss of a particular digestive enzyme, loss of some aspect of ciliary coordination, or failure to complete morphogenesis of the larval mouth. He also noted that the rarity of nonfeeding larvae with only slightly derived morphology makes it difficult to identify the specific changes in morphology or behavior that render larvae unable to feed. Most putative intermediates (e.g., the sea urchin Phvlliictinllius hnpcrialis: Olson ft nl., 1993) have undergone changes in multiple feeding-related traits (c.t>.. number and shape of arms, structure of the larval gut) since divergence from a feeding ancestor, and it is thus difficult to identify any one key change that resulted in a loss of feeding ability. As putative intermediates, larvae of sabellins may pro- vide insight into how larval feeding ability is lost after evolutionary increases in egg size. Larvae of Schizo- bninchia insignis. and probably many other sabellins, pos- sess the ciliary bands needed to capture food particles from suspension. These ciliary bands remain capable of capturing particles and moving them to the mouth. The mouth, how- ever, does not connect to the midgut, which in any case has no (or, later in development, a very small) lumen because the cells that make up its wall are swollen with energy reserves. Loss of feeding in larvae of S. insignis may thus be related primarily to a change in the digestive system. This observation, along with a consideration of annelid development, suggests a specific hypothesis on the link between increased maternal provisioning of the egg and the loss of larval feeding ability. Embryos of annelids (and those of members of several other phyla, together known as spiralians) undergo spiral cleavage, a process in which the fates of some blastomeres are specified early in develop- ment. In spiralian embryos, the descendants of four cells — 3A. 3B, 3C. and 4D — form the endoderm. the embryonic tissue that becomes the larval midgut. As a result of unequal cleavages from the third through the fifth cleavage cycles, these cells, known as macromeres. are usually far larger than the remaining embryonic cells, the micromeres. At gastrulation, the macromeres and their descendants are in- ternalized, where they form the larval midgut (Kume and Dun. 1968: Anderson, 1973). Annelids that have feeding larval stages typically have small eggs (Schroeder and Hermans. 1975). In these spe- cies, the descendants of 3A-C and 4D form the wall of a larval midnut that has a substantial lumen. In annelids with nonfeeding larvae, however, eggs are larger. Increased egg volume is presumably due mainly to the addition of material used to fuel development of the larvae or juveniles. During early development most of this additional material is shunted to the macromeres for storage and gradual mobili- zation. In annelid embryos that develop from large eggs, then, macromeres are proportionally larger (relative to the micromeres) than those of embryos that develop from small eggs (Schneider ft ai, 1992). Construction of a functional midgut may be difficult when a large volume of endodermal cells must fit in the relatively small volume delimited by the remaining cells of the embryo. In this case, it may be impossible to assemble the endoderm into a midgut that has a substantial lumen, or any lumen at all. Indeed, nonfeeding larvae of annelids typically lack midgut lumens (Wilson. 1936; Anderson. 1973: Heimler. 1988). In addition to sim- ple size constraints, in annelid species with large eggs, division of endodermal cells (and subsequent gut morpho- genesis) may be delayed relative to species with small eggs (e.g.. Schneider el ul.. 1992). Thus, in annelids, a quantitative increase in egg volume, typically thought to be the initial step in the loss of larval feeding, may lead directly to a loss of larval digestive ability because of spatial constraints or delays in gut morphogen- esis. These effects may persist through larval development, resulting in nonfeeding larval development; alternatively, they may last only through part of larval development, delaying the onset of larval feeding until cells of the midgut wall shrink as energy stores are consumed. This hypothesis is attractive because of its simplicity and its vulnerability to test. One approach is the experimental reduction of endoderm volume by removal of one or more presumptive endodermal cells (f.g.. Boring, 1989; Clement. 1962; Martindale. 1986). Carrying out this manipulation in annelid embryos that normally develop into nonfeeding larvae with occluded midguts might yield larvae with open midguts. It is not clear how morphogenesis of the rest of the gut (e.g., mouth and stomodaeum) might be affected by macromere ablation, but it is possible that a simple reduc- tion of endodermal cell volume might permit the develop- ment of a complete larval gut. In species that retain ancestral particle capture systems, like the sabellids described here, this manipulation might result in conversion of a nonfeeding larva to a feeding larva. Another approach is to use intraspecific variation in egg size and larval nutritional mode to examine the effects of egg size on midgut development. For example, some indi- viduals of the annelid Streblospio benedicti (family Spio- nidae) produce small eggs (56-70 /u,m diameter) that de- velop into feeding larvae, while others produce large eggs ( 1 15-152 yum) that develop into nonfeeding larvae (Levin, 1984; Schulze el ai, 2000). If the hypothesis proposed above is correct, nonfeeding development in 5. benedicti should be a result of reduction in size of the midgut lumen or delayed development of the midgut in larvae that develop 306 B. FERNET from large eggs. This prediction can be tested with com- parative developmental data. This idea might also apply to other spiralian taxa such as molluscs u.id entoprocts. As their development is similar. they should be subject to similar effects of endodermal volume on midgut morphogenesis. Both approaches to test- ing the hypothesis — deletions of endodermal blastomeres and comparative studies within species with variable egg size and developmental mode (e.g., the ascoglossan Alderia modesta: Krug, 1998) — may be fruitful. This hypothesis links changes in development (in this case, egg size and allocation to endodermal lineages) to changes in the form and function of later stages (loss of larval feeding ability via delayed midgut morphogenesis). Such connections have long been sought by developmental biologists (e.g., Lillie, 1899; Freeman and Lundelius, 1992). In addition, it may be useful in explaining a peculiar obser- vation— that correlated intraspecitic variation in egg size and larval nutritional mode (a form of "poecilogony") ap- pears to be limited in distribution to spiralians. in particular annelids and molluscs (Chia et ai. 1996). I propose that annelid and mollusc species with great intraspecific varia- tion in egg size may show correlated variation in larval nutritional mode because of spatial constraints or hetero- chronic effects on midgut morphogenesis imposed by their conserved pattern of development. Acknowledgments I thank A.H. Whiteley for reminding me of Lee's studies of Schizobranchia insignis eggs, and B. Bingham and C. Mills for helping locate populations of Myxicola aesthetica. R.R. 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Development (suppl. i 97-106. Wray, G. A., and R. A. Raff. 1991. The evolution of developmental strategy in marine invertebrates. Trends Ecol. Evol. 6: 45-50. Reference: Bio/. Bull. 205: 30X-318. (December 2003) © 2003 Marine Biological Laboratory Cloning, Characterization, and Developmental Expression of a Putative Farnesoic Acid 0-Methyl Transferase in the Female Edible Crab Cancer pagurus CAROLYN J. RUDDELL1. GEOFFREY WAINWRIGHT1. AUDREY GEFFEN1, MICHAEL R. H. WHITE1, SIMON G. WEBSTER2. AND HUW H. REES1 * 1 School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom: and 2 School of Biological Sciences, University of Wales, Bangor, LL57 2UW. United Kingdom Abstract. Farnesoic acid methyl transferase (FAMTase) catalyzes methylation of farnesoic acid to yield the crusta- cean juvenoid, methyl farnesoate (MF). A full-length cDNA encoding a 275 amino acid putative FAMTase has been isolated from the mandibular organ of the female edible crab (Cancer pagurus) by reverse transcriptase-polymerase chain reaction in conjunction with cDNA library screening. A high degree of sequence identity was found between this and other putative crustacean FAMTases. Conceptual trans- lation and protein sequence analysis suggested that phos- phorylation could occur at multiple sites in the FAMTase. This finding is consistent with the recent observation that endogenous FAMTase activity in mandibular organ extracts can be regulated by phosphorylation in vitro. We demon- strated that the recombinant FAMTase could be expressed as a LacZ-fusion protein in Esclierichiu colt and have un- dertaken its partial purification from inclusion bodies. In an established assay system, the recombinant FAMTase lacked activity. Northern blotting demonstrated widespread expression of an approximately 1250-nucleotide FAMTase transcript in female C. pagurus tissues. Levels of FAMTase transcripts in mandibular organs of female C. pagurus were found to fluctuate during vitellogenesis and embryonic development. Throughout the spring of 2002. an HPLC-based method was used to measure hemolymph MF liters in more than 70 Received ' April 2002; accepted 5 September 2003. * To v - correspondence should be addressed. E-mail: reeshhfe'liv .... Ahbre\-iatitii,\. \ \MTase, S-adenosyl-L-methionine farnesoic acid O- methyl transferase: .". I . methyl farnesoate; MO-IH, mandibular organ- inhibiting hormone. female specimens of C. pagurus, which segregated into "high MF" and "low MF" groups. The high MF tilers, which occurred before or during early vitellogenesis, coin- cided with, or were preceded by, elevated levels of putalive FAMTase mRNA in the mandibular organs. Introduction Methyl farnesoate (MF), a sesquiterpenoid structurally similar to insect juvenile hormone III. is produced and secreted by the mandibular organs of crustaceans (Laufer et ai. 1987a, b;Borst et al.. 1987; Wainwright etui, 1996a, b, 1998). Just as the juvenile hormones maintain larval char- acteristics between successive molts in insects (for a review, see Riddiford, 1994), a recent report confirms that MF regulates larval metamorphosis in barnacles (Smith et al., 2000). In adult insects, juvenile hormone has been impli- cated in the regulation of ovarian development (Davey, 1996; Belles, 1998), and a growing body of evidence sug- gests an analogous role for MF in crustaceans. In the spider crab Libinia e/narginata and the edible crab Cancer pagu- rus, increased levels of MF synthesis in the mandibular organs (Laufer et al., 1987a) and elevated levels of MF in the hemolymph (Wainwright et al., 1996a) have been found to occur during vitellogenesis. In experiments where a va- riety of crustaceans were exposed to artificially enhanced levels of MF, either by the effects of eyestalk ablation (Jo et ul.. 1999), by direct injection of MF (Reddy and Ramamurthi. 1998, Rodriguez et al., 2002). or by adminis- tration of MF through the diet (Laufer et al., 1998), oocyte growth and ovarian development were stimulated. Simi- larly, a significant increase in mean oocyte diameter was reported when ovary explants from shrimp, Penaeus van- 308 METHYL TRANSFERASE CHARACTERIZATION 309 'i (Tsukimura and Kamemoto. 1991) and red swamp crayfish. Procambarus clarkii (Rodriguez el al.. 2002) were incubated in vitro with physiologically relevant concentra- tions of MF. This combined evidence strongly supports a role for MF in the reproductive development of female crustaceans. In crustaceans, ovarian development is broadly separated into two distinct phases, a pre-vitellogenic phase and a vitellogenic phase. During the pre-vitellogenic phase, pri- mary oocytes begin to accumulate rough endoplasmic retic- ulum. and endogenous glycoprotein content increases. At the end of this phase, oocyte development arrests at prophase-I of meiosis, which synchronizes the oocytes at the same stage of development (for a review, see Charniaux- Cotton and Payen, 1988). Synchronization is critical be- cause crustaceans fertilize and spawn all of their oocytes simultaneously. In locusts and shrimp, the meiotic block is released by physiological doses of ecdy steroids (Cledon. 1985; Lanot and Cledon. 1989). After meiosis resumes, germinal vesicle breakdown occurs, and vitellogenesis fol- lows. The vitellogenic phase is characterized by a signifi- cant increase in the size of the ovary and an accumulation of yolk protein within the oocytes of the developing ovary tissue. In C. pagurus. oocytes are fertilized as they are laid. by sperm that has been stored in the spermathecae since copulation, when the female was soft-bodied after molting. Eggs are brooded under the abdomen attached to ovigerous pleopod hairs until embryogenesis is complete, and the hatched larvae are released as free-swimming zoeae. Egg- laying is believed to occur during the winter, and the larvae hatch 6 months later (Warner, 1977). The precise timing of vitellogenic events in C. pagitnis has not been fully deter- mined, although vitellogenesis appears to begin during the spring, presumably in response to environmental cues, such as photoperiod and temperature. The exact role of MF in crustacean ovarian development is still unclear, but in C. pagurus the hemolymph MF concentration is reported to peak during the earliest stage of vitellogenesis, suggesting its involvement in the control of this process (Wainwright el nl.. 1996a). Previously, we investigated the regulation of MF produc- tion in the mandibular organs of C. pagurus. isolating and characterizing two 78-residue peptides — mandibular organ- inhibiting hormones (MO-IHs) — that down-regulate the production of MF by the mandibular organs (Wainwright et ill.. 1996b). Furthermore, we demonstrated that the action of MO-IH on mandibular organs ultimately regulates an S- adenosyl-L-methionine farnesoic acid O-methyl transferase (FAMTase). an enzyme that catalyses the final step of MF biosynthesis (Wainwright el ai, 1998). To date, putative FAMTase sequences from three decapod crustaceans have been published in on-line databases. Of these sequences, there is no evidence that the cloned gene products from the spiny lobster Paniilirus interruptus (GenBank accession number AF249871) or from the clawed lobster Hoimirus iimericanus (U25846) have FAMTase activity. However, the recombinant FAMTase from the shrimp Metapenaeus ensis (AF333042) has recently been reported to catalyze, in vitro, the conversion of farnesoic acid to methyl farnesoate (Silva Gunawardene et ai, 2002). No brachyuran decapod has been studied to date, but these findings with shrimp suggest that a homologous putative FAMTase from the crab Cancer pagurus might also have enzyme activity. In this report, we describe the isolation and characteriza- tion of a full-length cDNA encoding a 275-residue putative FAMTase from mandibular organs of female specimens of C. pagurus (GenBank accession number AY337487). The recombinant FAMTase was heterologously expressed, and enzyme activity was investigated in an established assay system (Wainwright et al., 1998). The distribution of puta- tive FAMTase expression is presented for a range of C. pagurus tissues, throughout the course of ovarian develop- ment and during embryogenesis. We also present further details regarding the previously reported peak of hemo- lymph MF (Wainwright et al., 1996a), which occurs prior to or during the earliest stage of vitellogenesis. Materials and Methods Animals Adult females of Cancer pagurus (Linnaeus), the edible crab, were obtained weekly from local fishermen and main- tained in a recirculating seawater system at ambient light and temperature. These wild-caught animals constituted a non-synchronized population; therefore, crabs at different stages of ovarian development could be encountered at any given time. Crabs were dissected after cold-anesthesia, and the stage of ovarian development was determined according to established criteria (Wainwright, 1995; Wainwright et al., 1996a). In brief, crabs were assigned a stage of ovarian development from 0 to 4. Stage 1 to 4 ovaries are vitello- genic, with steadily increasing organ size, oocyte diameter, and quantity of accumulated yolk protein (orange color). Stage 0 crabs, with cream-colored undeveloped ovarian tissue, are classed as either "pre-vitellogenic." if the hemo- lymph color is gray, or "vitellogenic," if the hemolymph color is orange; the orange color indicates the presence of circulating yolk protein. Two egg-carrying females were caught during the winter and maintained in individual tanks at the Marine Biological Laboratories at Port Erin, Isle of Man. Embryonic offspring were collected from the egg-bearing pleopod hairs in the abdominal brooding pouch using flat-ended forceps. Hatched larvae were collected by filtration of the tank water. Embryos, larvae, and dissected adult tissues were stored in Trizol reagent (Life Technologies, Inc.) at -80 °C prior to analysis. 310 C. J. RUDDELL ET AL. Isolation of u i • -1 tiii^ineni of FAMTase using a nested PCR appii The mai'.iiiluilar organs were dissected from three female C. />i/'s'i/n<.\ specimens (ovary stage 0, hemolymph orange), and total RNA was isolated using Trizol reagent. Nested degenerate PCR primers were designed based on conserved regions within the crustacean FAMTase sequences pub- lished online. First-strand cDNA synthesis was performed using I /j.g total RNA. as described in the SMART cDNA Library Construction Kit User Manual (version #PR92334) (Clontech Laboratories. Inc.). Most of this material was used to synthesize a mandibular organ cDNA library (see below). One-tenth of the first-strand synthesis reaction was used as cDNA template for PCR. with DMTSI sense (5'- GCNCAYGAYGCNCAYGTNGC) and DMTAS1 anti- sense (5'-GGYTCNGGRTCNGTCCAYTC) primers (Fig. 1 ). and with the following temperature profile: 94 °C for 2 min. followed by 25 cycles of 94 °C for 1 min. 53 °C for 2 min. 72 °C for 1 min. and a final extension step of 7 min. PCR was earned out in a 20-jnl reaction volume containing 0.5 units Taq DNA polymerase (Roche) in the manufactur- er's reaction buffer (with 1.5 mM MgCK) in the presence of 0.5 p.M PCR primers (MWG Biotech) and 0.25 mM each dNTP (Promega). Nested PCR was performed with DMTS2 sense (5'-CNCCNGAYATHYTNWSNGARGAR) and DMTAS2 antisense (5'-YTTNCKYTCYTCYTCRCARC) primers (Fig. 1 ). using 5 /u.1 of the first PCR reaction as a template. The following thermal cycle was employed: 94 °C for 1 min. 54 ' C for I min, 72 °C for 1 min for 25 cycles. Other PCR conditions and reaction mixture constituents were as described above. Agarose gel electrophoresis of the 241 561 641 721 881 961 1041 1121 taagttgttggtgcttctcctgtctgtacacacccccacacaccacacaccgagatcatggctgatgagattcccgccct MADEIPAL tggcacggacgagaacaaggagtaccgcttcagggagcttgatggcaagacccttcgattccaggtcaaaacagcgcacg GTDENKEYRFRELDGKTLRFQVKTAH DMTS1 attgtcatgtggcattcacgtcagccggtgaagagacagaccccatagtggaggtgttcattgggggatgggagggcgct DCHVAFTSAGEETDPIVEVFIGGWEGA DMTS2 gcctcggccatcaggttcaagaaagctgatgatctagtgaaggtggatapgccagacattcttagcgagggagagtaccg ASAIRFKKADDLVKVD(T)PDIL(S)EGE(Y)R GSP2 ncoV GSP3 GSP tgaattctggattgc tgtggaccacgacgaaataagagtaggcaagggcggagagtgggagccgctcatgcaggcgccca EFWIAVDHDEIRVGKGGEWEPLMQAP tcccagagcccttccctatcacccactacggctactccacaggctggggtgctgttggctggtggaagttcatgaacgac IPEPFPITHYGYSTGWGAVGWWKFMND agggtcctaaacactgaagactgcctcacctacaacttcgagcctgcctacggtgacaccttctccttcagcgtcgcctg RVLNTEDCLTYNFEPA(Y)GDTFSFSVAC cagtaacgatgctcatttggctcttacctctggcgccgaagagaccacgccaatgtacgagatcttcattggtgggtggg SNDAHLALTSGAEETTPMYEIF1GGW agaaccaacactccgccatccgcctcaataagggtgacgacatggccaaagtagagactccggacgcactgtgttgtgag ENQHSAIRLNKGDDNAKVETPDALCCE DMTAS2 ^ gaggagaggaagttcttcgtgtccttcaggaacggccatatcaaggtgggctAcaaggacac tgatcccttcctgcagtg E E R K F F v f s) F R N G H i K v G (V) K D T D P F L Q w DMTAS1 gactgaccctgagccctggaaggtaacccatgtgggatactgtacgggatggggcgctaccggcaagtggaagc ttgata TDPEPWKVTHVGYCTGWGATGKWKLD tctaagtgaacaaaaggtggaggtggtgatgtgatgtttgtcagtcatgcatcacactcaccaccactcgtcacactatc I * accacccctgctgccatgctatccactaccattggtacgtaataacgcttctatccttatctttgtcttcagtttataat aaagc ttccaaagcctgagaagccctatgagggtggagcgttgcgcacacacc tgttgctgcattaacctttaaatgtcc tcttacatggaattaaaagtgggagttatttttcgtactctttgtagcttcacgtoaataaaUcctgaaaac tag (a) 30 Figure 1. Sequence ol'cDNA from the mandibular organ encoding ihe putative FAMTase. The full-length cDNA was isolated by PCR and nucleotide sequence determined (see Methods I. The cDNA sequence shows an 825-bp open reading frame, which encodes a 275 amino acid protein. The stop codon is indicated by an asterisk. and a poly.idein lation signal (AATAAA) is enclosed in a box. Circles indicate potential phosphorylation sites in the mature protein Arrows indicate positions of primers, which are identified (see Methods). A single Eco RI site at 322 bp is underlined. 320 480 560 640 720 960 1040 1120 1225 METHYL TRANSFERASE CHARACTERIZATION 311 nested PCR products revealed a band of about 450 hp. which was cloned into the pGEM T-easy TA cloning vector according to the manufacturer's instructions (Promega). Se- quencing of the 450-bp cloned insert showed it to be very similar to the existing crustacean FAMTase sequences in databases. This cDNA fragment was used as a probe to isolate a full-length cDNA encoding C. pagunis FAMTase. Isolation of full-length FAMTase cDNAfroin <; mandihiilar organ cDNA lihrar\ From the remainder of the first-strand synthesis reaction (see above), a unidirectional, mandibular organ cDNA li- brary was constructed in bacteriophage ATriplEx2. The liter of the primary, unamplified library was 0.97 X 10h pfu/ml with >95% positive recombinanls. To isolate clones containing full-length cDNAs encoding C. pagiirus FAMTase, plaque hybridization screening was carried out using the 450-bp FAMTase fragment end-la- beled with [a-3-PrdCTP using the Ready-To-Go DNA Labeling Kit (Amersham Pharmacia) as a probe. Plaque lifts were carried out with BioTrace NT nitrocellulose blotting membrane (Gelman Sciences), and hybridizations and washes were carried out according to manufacturer's guide- lines. A feature of the ATriplEx2 vector is that it contains an embedded bacterial plasmid DNA, pTriplEx2. Excision and circularization of the plasmid DNA from the linear bacte- riophage DNA is readily achieved by a process involving /;; vivo excision using Cre recombinase-mediated site-specific recombination at two loxP sites flanking the embedded plasmid. This is carried out by incubating ATriplEx2 at 31 °C in the presence of E. coli BM25.8 (Clontech Labo- ratories, Inc.). The resullanl plasmid can be used lo express LacZ fusion protein variants of the encoded proteins under the regulation of a LacZ promoter. Positive clones from the library screening were converted into plasmid clones and analyzed by restriction enzyme digestion with EcoRl and Sail. 5' -Rapid amplification of cDNA ends (5' -RACE) To obtain the 5'-end of the FAMTase cDNA clone, 5 '-RACE was carried oul using total RNA from mandibular organs. A 5 '-RACE system version 2.0 (Life Technologies) was used to amplify the 5' terminus of the message for sequencing. Briefly, a gene-specific primer (GSP1, 5'- ACTCTCCGCCCTTGCC) was hybridized to ihe mRNA, and cDNA was synthesized using Superscript II reverse transcriptase. The RNA was Ihen degraded with RNase mix (RNase H and RNase Tl ). and the cDNA was purified using a GlassMax spin cartridge supplied with the kit. A poly(dC) tail was added to Ihe 3 '-terminus of the purified cDNA using dCTP and terminal deoxynucleotidyl transferase. and the cDNA region corresponding to the 5'-end of the mRNA was amplified by two successive rounds of PCR using additional gene-specific primers (GSP2, 5'-TACTCT- TATTTCGTCGTGGTCC; GSP3, 5'-ACAGCAATCCA- GAATTCACGG), together with anchor primers supplied by the manufacturer. The second-round PCR product of about 380 bp was cloned into pGEM-T Easy Vector, and the nucleotide sequences of several clones were determined. Expression of FAMTase protein Expression of protein encoded by cDNA within pTripl- Ex2 plasmid was carried out, as follows, in E. coli TOP 10 F'. Bacteria containing the pTriplEX2 plasmid were grown to a density of OD6m 0.5 to 0.6. at 37 °C, in LB medium supplemented with 50 ju.g/ml of ampicillin. Expression of LacZ fusion protein was achieved by addition of isopropyl j3-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM (Brent. 1994). SDS PAGE analysis of proteins Extracts of proteins from E. coli were prepared to yield both soluble protein fractions and insoluble inclusion-body fractions, according to a published method (Brent, 1994). In both cases, E. coli cells were harvested by centrifugation (3000 X g. 10 min, 4 °C). washed in PBS buffer, and centrifuged (3000 X g. 10 min, 4 °C). The pellet was resuspended in HEMGN buffer (100 mM KC1, 25 mM HEPES [pH 7.6], 0.1 mM EDTA [pH 8.0], 10% [v/v] glycerol, 0.1% [v/v] Triton X-100) containing protease in- hibitors (1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium metabisulfite and protease inhibi- tor cocktail [Sigma. #P8340] added at 10 jul per 100 ml of original culture volume) and lysozyme (0.5 mg/ml). and lysed by sonication. After centrifugation (27,000 x g. 15 min, 4 °C) to separate soluble (supernatant) and insoluble fractions (pellet), the pellet was extracted into HEMGN buffer containing 8 M guanidinium-HCl, and centrifuged (87,000 X g, 30 min, 4 °C). The supernatant was dialyzed once against HEMGN buffer containing 1 M guanidinium- HCl and protease inhibitors (see above), Ihen Iwice against HEMGN buffer containing protease inhibitors. The dialy- sate was centrifuged (12,000 X g. 5 min, 4 °C) to yield 8 M guanidinium-HCl soluble (supernatant) and insoluble (pel- let) protein extracts (see Fig. 3). Portions of all extracts were analyzed by eleclrophoresis on a 10% polyacrylamide gel. Protein bands were visualized by staining wilh colloidal Coomassie blue G250. FAMTase assays Broken-cell extracts of E. coli were assayed for FAMTase activity using assay conditions previously pub- lished (Wainwright et al., 1998). Briefly, extracts (200 /u.1) were dialyzed against a hypotonic HEPES buffer (0.037 M HEPES, 0.3 M sucrose, 0.01 M KF, pH 7.4) before the 312 C. J. RUDDELL ET AL addition of 2.4 ^M [12-3H] farnesoic acid and 250 S-adenosyl-L-in. tl ionine, in a final reaction volume of 50 ju,l. Incubation- were carried out at 37 °C for 1 h and were terminated rr. t! •- addition of 150 ju.1 of acetonitrile. The reaction products were analyzed by reversed-phase HPLC with on-line radioactivity monitoring, as described previ- ously (Wainwright et til., 1998). Expression of FAMTase in C. pagurus tissues Total RNA from a variety of tissues (see Results) was isolated using Trizol reagent (Life Technologies). For Northern blotting, about 10 /ig of total RNA from individ- ual tissues was electrophoresed on a formaldehyde/ 1% aga- rose gel for 3 h at 75 V. The RNA was blotted onto Electran nylon membrane (BDH) with 20 x SSC (SSC is 0.15 M NaCl/0.15 M sodium citrate) and RNA cross-linked to the membrane by UV radiation. The FAMTase probe was pre- pared, as described above, and hybridization was carried out in QuickHyb solution (Stratagene) for 1.5 h at 68 °C. After hybridization, the blot was washed three times, at room temperature, for 10 min each, in 2 X SSC containing 0.1% SDS, and twice, at 45 °C, for 10 min in 0.1 X SSC containing 0.1% SDS. Autoradiographs were exposed at -70 °C. To compare relative levels of expression of FAMTase in samples, a mouse 18S rRNA probe (Takeuchi et al.. 2000) was co-hybridized under the same conditions. This proce- dure provided an internal calibration for each sample and allowed for differences among lanes in the loading of RNA. In preliminary control experiments, when Northern blotting was carried out with individual probes alone, using identical hybridization and wash conditions, the hybridization pat- terns showed single bands of appropriate sizes for each probe (18S rRNA. 2000 nf, FAMTase mRNA, 1250 nt). Under the conditions used, therefore, the 18S rRNA and FAMTase mRNA probes did not cross-hybridize with their respective target transcripts. So we co-hybridized the blots in subsequent experiments. Densitometric analyses were earned out using Quantity One software (Bio-Rad) and amounts of FAMTase mRNA normalized against those of 18S rRNA. Preparation of hemolymph methyl farnesoate extracts for HPLC Hemolymph samples were taken from adult female spec- imens of C. pagurus with a hypodermic syringe; the arth- rodial membrane at the base of a walking leg was punctured, and 2 ml of hemolymph was extracted. Methyl farnesoate isomers were extracted into hexane using a triphasic proce- dure (Borst and Tsukimura, 1991) incorporating modifica- tions according to Wainwright et al. (1996a). Briefly, he- molymph samples (2 ml) were added to tubes containing 2 ml NaCl 4% (w/v) in H,O, 5 ml acetonitrile (Merck, far UV HiPerSolv grade), and 100 ng cis, trans-MF isomer as an internal standard. The mixture was partitioned against 2 ml n-hexane (Merck, HiPerSolv grade), achieving phase sepa- ration by centrifugation at 500 X g for 10 min at 20 °C. The hexane layer (top) was removed and 300 ;u,l subjected directly to HPLC analysis. HPLC quantification of methyl farnesoate Levels of all-trans-MF contained in hemolymph hexane extracts were determined by adsorption HPLC on a Varian Pro Star chromatography workstation, with a modification of the method previously described by Borst et til. (2002). Separation of MF isomers contained in hexane extracts (300 /LI!) from hemolymph was achieved on a Rainin MicroSorb MV silica adsorption column (5 /nm, 250 X 4.6 mm internal diameter) using isocratic elution at 2 ml/min for 45 min in 0.47r diethyl ether in /i-hexane (Merck, HiPerSolv grade) that had been dried overnight after the addition of 50 g of molecular sieve 4 A (bead), 8-12 mesh (Sigma, #M1760), per 2.5 1 of solvent. Eluted compounds were detected by UV absorbance at 229 nm. Peak areas were calculated using Star workstation software (Varian). All-trans-MF content of he- molymph samples was calculated by comparison of the all-tnins-MF peak area to the cis, trans-MF peak area (internal standard 100 ng). Results Isolation, characterization, and expression of FAMTase from C. pagurus inantlibular organ Isolation and characterization of FAMTase. To obtain full- length cDNA encoding FAMTase, nested PCR was fol- lowed by isolation of full-length cDNA from a mandibular organ cDNA library. Previously published putative FAMTase sequences provided information for the design of degenerate oligonucleotide primers for the isolation of a ca. 450-bp fragment of cDNA that encoded a putative FAMTase. Subsequent sequence analysis suggested that this was, in fact, a putative FAMTase cDNA fragment of 442 bp. This partial cDNA was used to screen a mandibular organ cDNA library. Initially, approximately 8 X 104 re- combinant bacteriophage were grown in two 15-cm petri dishes and screened. This initial screen identified four pos- itive clones that were subsequently isolated and purified. All four phage clones were converted to their corresponding plasmid clones as described, and the inserts were analyzed by restriction enzyme digestion with Sail and EcoRl: the products were then separated electrophoretically on a 1% agarose gel. Of the clones analyzed, two distinct types were apparent: one type, on digestion, produced two products (900 and 300 bp) originating from the cloned insert cDNA, while the other type produced only a 900-bp insert. Se- quencing and BLAST searching revealed the former type. METHYL TRANSFERASE CHARACTERIZATION 313 Group 1. to be putative FAMTase sequence clones. The complete sequence of the putative FAMTase clone was obtained (see Fig. 1 ). The clone was 1216 bp in length, and conceptual translation indicated an open reading frame of 825 bp encoding a 275 amino acid protein with a predicted molecular weight of 31114. The 5'-UTR was 48 bp long. The 3'-UTR was 336 bp long and contained a polyadenyl- ation signal. A ATA A A, 13 bp upstream of the poly(A) tail. 5 '-RACE demonstrated that the isolated and sequenced clone was 9 bp shorter than the full length of the 5'-UTR. A single EcoKl restriction enzyme cutting site occurs at 322 bp. ClustalW alignment of the isolated putative FAMTase from C. pagitrus, with sequences identified in other crusta- cean species, demonstrated a high degree of sequence iden- tity (Fig. 2). Analysis of the protein sequence with Signal? ver. 1.1 software (http://www.cbs.dtu.dk/services/SignalP/) suggested that the protein does not contain a signal peptide cleavage site. Further analysis of the protein sequence, with NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/) and ScanProsite (http://ca.expasy.org/tools/scanprosite/). for po- tential posttranslational modifications shows multiple high- scoring (score > 0.8) sites for possible phosphorylation at serine, threonine, and tyrosine side chains within the mol- ecule (Fig. 2). Expression and activity of FAMTase protein To determine whether the protein encoded by the isolated FAMTase cDNA clone was indeed an active FAMTase. expression of the protein in E. coli was carried out as described (Methods). Just prior to the addition of 1PTG to C. pagurus H. americanus M. ensis P. interruptus MA-DEIPALGTDENKEYRFRELDGKTLRFQVKTAHDCHVAFTSAGEETDPIVEVFIGGWE MGDDNWASYGTDENKEYRFRDISGKTLHFQVKTAHDAHVALTSGAEETDPMVEIFIGGWE MA-DNWPAYGTDENKEYRFRIIKGKTLRFQVKAAHDAHIALTSGEEETDPMLEIFIGGWE MGDDNWPSYGTDENKEYRFRDIGGKCLRFKVKTAHDAHVALTSGAEETDPIVEVFIGAWE *********** *.**.*** * * * * * C . pagurus H. americanus M. ensis P. interruptus C. pagurus H. americanus M. ensis P. interruptus GAASAIRFKKADDLVKVE(T^DII@EGX^IREFWIAVDHDEIRVGKGGEWEPLMQAPIPEPF GAASAVRFKKGEDLVKVE@PDII@EEB^REFWIAFDHDEIRVGKGGEGEPFMQCPIPEPF GAASAIRFKKADDLTKVT(T^DILNAE^REFWIAFDHDNVRVGKGGEWEPFMSATVPEPF GAASAIRFKKADDLAKVE(TpDILNEE^REFWITFDNDEVRVGKAGDWEPFMMSPSQSHS *****.**** •** ******** ******* *.*..**** *. **.* PITHYGYSTGWGAVGWWKFMNDRVLNTEDCLTYNFEP?(Y)GDTFSFSVACSNDAHLALTSG GITHYGYSTGWGAVGWWQFHAEKSYNTEDCLTYNFIPV(Y)3DTLEFSVSCSNriAHVALTSA EITHYGYSTGWGATGWWQFHSEMHFQTEDCLTYNFVP\(Y)3DTFSFSVACSNDAHLALTSG KSPTMAIPLAGVLSAGGSFIMR-DFHTEDSQAYKFEPV^DSLTFSVSCGHDAHLALTSG ******** C. pagurus H. americanus M. ensis P. interruptus AEETTPMYEIFIGGWENQHSAIRLNK GDDMAKVETPDALCCEEERKFF\(S)FRNGH AEETTPMYELLLGGWENQHSAIRLNK GDDMIKVDTPDILCCEEERKFW\(S)FKNGH PEETTPMYEVFIGGWENQHSAIRLSKEGRSSGEDMIKVDTPDIVCCEEERKFT$|)FKDGH PEETTPMYEVFIGGWENQHSAIRLNK GDDMIKVDSPDIVCSEEERKFWI(£)FKNGR ******** ************ * . ** ** * ****** C. pagurus H. americanus M. ensis P. interruptus IKVQ@KDTDPFLQWTDPEPWKVTHVGYCTGWGATGKWKLDI IRVd^KDTDPFMEWTDPEPWKITHIGYCTGWGATGKWKFEY IKVG@QDSDPFMEWTDPEPWKITHVGYCTGWGASGKWKFEF IRVQYkDSDPFMEWTDPEPWKVTHVGYTTAWGAAGKWMLEI ** * . ** * Figure 2. Amino acid sequence alignment of putative FAMTases from four crustaceans. ClustalW align- ment of FAMTases from Cancer pagurus (this report, GenBank accession number AY337487), Hamarus americanus (U25846). Metapenaeus ensis (Silva Gunawardene el al., 2001. AF333042). and Panulirus inler- ni/>m\ (AF249871 ). Identical amino acids at a particular position, in all sequences, are denoted by an asterisk. Colons denote alignment of amino acids with strong similarities; periods indicate aligned residues with weaker similarities, according to their physicochemical properties. Hyphens denote gaps introduced to maximize the sequence alignment. Circles indicate potential phosphorylation sites in the mature protein. EMBL ClustalW default alignment settings were used (www.ebi.ac.uk/clustalw/). 314 C. J. RUDDELL ET AL the cultures, a 1-ml sample (lime = 0 h) was taken and protein extract i v pared as described (see Methods). Sam- ples (I n. nal culture were taken at 1, 2 and 5 h post-IPTG ..ition. Both soluble and insoluble extracts of the bar . • ia were prepared and analyzed by SDS-PAGE. Analysis of the samples taken 5 h after the addition of IPTG clearly shows that recombinant protein expression is in- duced by IPTG. and that the recombinant protein is pro- duced predominantly in the insoluble inclusion body frac- tion (Fig. 3). Molecular weight analysis demonstrated the induction of a protein of approximately 40 kDa. This size is entirely consistent with the expected size of the FAMTase- LacZ fusion protein. To determine whether the recombinant fusion protein exhibited any FAMTase activity, the protein extracts were assayed for their ability to convert farnesoic acid into methyl farnesoate using conditions previously described (see above; Wainwright et al., 1998). The broken cell ex- tracts exhibited no detectable FAMTase activity (results not shown). Expression of FAMTase during ovarian development and embryogenesis Expression of FAMTase in tissues and developmental stages. To determine the tissue distribution and develop- mental expression of the putative FAMTase, Northern blot- ting was carried out using the 450-bp clone as a probe. The probe detected a single band of approximately 1250 nt in a number of tissues, including muscle, eyes, mandibular or- gans, epidermis, gills, heart, ovary, hepatopancreas and gut (Fig. 4). The extremely low signals detected by Northern blotting in Y-organs and sub-epidermal adipose tissue pre- KDa markers Soluble fraction Insoluble fraction - == Plasmid clone IPTG 40 kDa 75 50 37 25 15 Figure 3. Expression of recomhinant putative FAMTase. pTriplEx2 plasmids containing cDNA inserts encoding full-length FAMTase were gri in £. CD// TOPIO F'. Protein expression was induced by addition of IPTG (see Methods). Extracts of E. coli prepared 5 h after induction with IPTG w .•: e analyzed for expression of recombinant protein by SDS-PAGE analy-: • Methods). Soluble and insoluble post-dialysis fractions are shown. absence (-) or presence ( + ) of either IPTG (to induce recomhm., 'em expression when plasmid is present) or plasmid clone in the original . '// culture conditions is indicated in the table above the gel image. eluded estimation of relative FAMTase expression levels in these tissues; and in hemolymph RNA, the FAMTase tran- script was undetectable by Northern blotting. To determine the developmental profile of expression of FAMTase in mandibular organs, RNA was extracted from the mandibular organs of female crabs at different stages of ovarian development, and Northern blotting was carried out as described. A representative blot (Fig. 5a) demonstrates a distinct variation in the level of expression of FAMTase in the mandibular organ during ovarian development. Follow- ing densitometric analysis of the Northern blot autoradio- grams, the ratio of FAMTase to 18S rRNA was determined, and the results were displayed graphically (Fig. 5b). Expres- sion of FAMTase from the mandibular organ is significantly higher before the onset of vitellogenesis than after vitello- genesis has begun (unpaired t test, P = 0.03). During the mid and late stages of ovarian development, the levels of mandibular organ FAMTase expression appeared to fluctu- ate, but definitive trends could not be identified at these stages. Female methyl farnesoate hemolymph liters. As part of an investigation aimed at further characterizing events that occur during the transition from the pre-vitellogenic to vitellogenic phases of ovarian development, we measured the hemolymph methyl farnesoate (MF) tilers of over 100 female specimens of C. pagitnis throughout the spring of 2002. In stage 0 crabs (n == 70). hemolymph MF tilers segregaled inlo two groups: "low" (93%) and "high" (7%) MF, wilh a cul-off liter of about 150 ng/ml between the groups (Fig. 6). It was noted that although one high MF crab was at an early stage of vilellogenesis (stage 0, orange hemolymph), four of the five high MF crabs fell into the stage 0 pre-vitellogenic category (slage 0, gray hemo- lymph). FAMTase expression during embryonic and lan'al develop- ment. As an extension of our investigations, samples of developing embryos, up to hatching, were collected and analyzed for expression of FAMTase Iranscripts. The resulls (Fig. 7) show lhal, in bolh groups of embryos sampled from each of the individual brooding females, levels of FAMTase transcript increased noliceably during developmenl, and fell to near the basal level just before halching. Discussion Here we report the isolation and characterization of a putalive farnesoic acid methyl Iransferase cDNA from man- dibular organs of the edible crab Cancer pagurus. Using a combination of a nested PCR-based approach and screening of a mandibular organ cDNA library, a 12 16-bp cDNA was isolated that encodes an approximately 31 -kDa protein mol- ecule (Fig. 1). The putative FAMTase of C. pagurus ex- hibits a high degree of sequence similarity wilh those MF.THYI. TKANSFFRASF. CHARACTERIZATION 315 V // / 18SrRNA FAMTase (~1250nt) Figure 4. Northern blot analysis. Approximately 10 /Mg of total RNA from a variety of Cancer \itiv.uni\ tissues and two organ equivalents of Y-organ RNA was electrophoresed. blotted onto a nylon membrane, co-hybndi/ed at 68 ' C with a '2P-labeled-FAMTase probe and a inouse IXS rRNA probe, and washed at 45 °C (see Methods). The Northern blot shows the tissue distribution and si/e of the C. /xtxiirux FAMTase transcript. a) 18SrRNA FAMTase (~1250nt) b) 1.2 Stage of ovarian development 0* 0* 0 0 0 0 I <•* 1 i HF ° 8 ^ 'E C/5 3 1 2 °6 < -2- 0.4 LL 0.2 p = 0.03 Pre- Early Vitellogenic Vitellogenic ^e (0) (0'-1) Stage of ovarian development Figure 5. Profile of expression of putative FAMTase mRNA in mandihular organ throughout ovarian development in Cancer paxiiru.\. (a) A typical Northern Not analysis of RNA isolated from mandibular organs from female crabs at different stages of ovarian development (0* indicates stage 0 vitellogenic animals). Stage 4 (late viiellogenic stage) not shown, (b) Autoradiograms developed from Northern blotting experiments were .imilyzed by computerized densitometry. Images were acquired with a GS710 scanning densitometer (Bio-Radi and analyzed with Quantity One software (Bio-Rad). The relative expression of FAMTase was normalized to the 18S rRNA signal to take account of unequal loading between samples in different lanes. Results are grouped as follows: pre-vitellogenic (stage <>i. early vitellogenic (stage 0* vitellogenic and stage 1). mid-stage vitellogenic (stages 2 and 3), and late vitellogenic (stage 4) animals. All values are mean ± standard error of the mean for n = 3-6 samples. 316 C. J. RUDDELL ET AL re O in 20 Low MF High MF 400 15 • — i O \ c 10 J c 350 . o- 5. 0> D o U- -i r~H~n 300 . ooooo o o o o o o CM ^ to co o r\j *» to eo o o OJ-WtOOOOOOO « oo o c^ ^ v ,1 and tagged 261 colonies of P. elisa- hcilnu' ;il e transects totaling 70 m2 of substratum located at depths of 12-15 m on San Salvador, Bahamas. The colonies were photographed in place at roughly 6-month intervals between December 1997 and December 1999; and the growth of individual branches was deter- mined by measuring changes in branch lengths through sequences of images, as in Figure 2. Branch generation and type (i.e.. mother vs. daughter) were assessed from the images, and the number of branches that originated in each time interval was recorded as well as the branches they developed from. Colony height was determined from either the images or from direct measurements in the field. analvsis and branch measurements Pseudopterogorgia elisabethae forms colonies with most side branches oriented in a single plane. Therefore, readily measured images could be obtained by positioning colonies between a grid 10 cm X 10 cm and a clear acrylic plastic cover, which held the branches against the grid (Fig. 2). If a colony was small (<20 cm height), the entire structure was photographed; if it was large, an arbitrarily selected branch containing 15-25 branches was followed. In Decem- ber 1997, photographs were taken with a Nikonos V under- water camera and Kodachrome 200 film. Those images were later digitized and converted to TIFF. In subsequent observations, photographs were taken at a resolution of 640 X 480 bits with a Sony Mavica digital camera (either MVC-7 or MVC-83) in an underwater housing. Distortion created when photographs were shot at a slight angle from the perpendicular was corrected in Photoshop (Version 4.0, Adobe). A 250 X 250 pixel grid was overlaid on the image, and the shape of the original image was adjusted with the free transform function of the program until the 10-cm grid in the photograph matched the 250-pixel grid. The length of each branch in the photograph was mea- sured with the program SCION (Scion Corporation, Fred- erick, MD). Although the program measures distance with high accuracy, variation is introduced into the measure- ments by several steps in the measurement process. First, although the tip of a branch is easily discerned in the images, it was also necessary to define its point of origin. We chose, as the point, the intersection of the branch with the line running along the middle of its mother branch, and that point had to be identified in each image. Second, the branches are curvilinear structures and were measured as segmenteJ lines. Small differences in measurements are created b\ iation in the number and placement of those line segnu o assess the magnitude of measurement variation, three Afferent observers measured each of 130 branches. The bctween-measurement standard deviation was 0.3 cm. When an entire colony was included in the image, height was measured as the length of the longest branch. If the entire colony was not included in the image, height (length of the longest branch) was measured in the field with a flexible tape measure, to the nearest 0. 1 cm. For purposes of analysis, colony heights were categorized into six size classes: 0.1-10.0 cm, 10.1-20.0 cm, 20.1-30.0 cm. 30.1- 40.0 cm. 40.1-50.0 cm, >50.1 cm. Growth rates Over the 2 years, 261 colonies with 5870 branches were monitored, and 23,478 individual length measurements were made. Growth rate — the difference between succes- sive measurements — was then determined, and those values were extrapolated to annual rates based on the number of months between the measurements. Measurement error for growth rates was 1.2 cm y~', which combines the effects of extrapolation of the 6-month intervals to 1 year and the additive effect of variance in each of the two measurements of colony length. The individual growth measurements were categorized by the time interval in which the measurement was made and by branch age, based on when the branch originated. Branch ages were categorized into one of five classes; <6, 6-12, 13-18, 19-24 months, or present at the start of monitoring. In some analyses, we also designated branches that originated during the study as "new" branches. This latter category distinguished branches that were less than 2 years old from those branches present at the start of the study. Each of the growth rates was also clas- sified according to the branch's generation order and branch type (mother or daughter). Negative growth rates To reduce the effects of grazing on the analyses, cases in which growth was <0.0 were dropped from the data set. By rejecting these cases of negative growth, the most severe effects of grazing were eliminated. Grazing also may have reduced the observed growth of some branches with posi- tive growth rates, but since scars from grazing heal rapidly, such branches could not be identified. Rejecting the nega- tive values may have inflated the calculated growth rates of branches that were otherwise not growing. Since the mea- surement error was 1.2 cm y '. some branches that had not grown would, through measurement error alone, have small negative growth rates, and some would have small positive growth rates. Exclusion of branches with growth less than 0.0 cm y ~ ' would have eliminated the underestimates of the zero growth branches but not the overestimates. Statistical analyses Growth rates were compared by analysis of variance (ANOVA, functions UNIANOVA and MANOVA. SPSS OCTOCORAL COLONY GROWTH 323 version 10. 1). In those analyses, branches were classified using the five independent variables: branch order, branch type (mother or daughter), branch age. time interval in which the measurement was made, and colony height. Branch length was also included as a covariate in some of the analyses. Due to the size and complexity of the data set. it was impossible to examine all of the effects in a single analysis. Our strategy was to conduct multiple tests, each including the greatest number of variables possible, and to use Bonferroni corrections to significance testing when mul- tiple tests were conducted on the same data. Between-colony variation and tinu1 interval effects. The same branches were measured multiple times, so a repeated- measures ANOVA was the most appropriate design. How- ever, because of the large number of branches nested within colonies, an ANOVA that included all five categorical vari- ables could not be computed within a single repeated- measures design. We therefore conducted a repeated-mea- sures ANOVA that tested for the random effects of branches within colonies and time interval (the 6-month interval in which the measurement was made). The effects of inter- colony variation were again examined in an ANOVA of the growth of branches that were less than 6 months old (i.e.. growth during the time interval in which the branch had originated). Growth rates in this analysis were compared with respect to colony and time interval (Table I A). A simple two-way ANOVA was used for this analysis, as data from no single branch was included in more than one observation in this analysis. Between-colony variation and branch a^e. Next, a series of analyses that simultaneously considered colony and age of the branch were conducted (Table IB through IE). The analysis was repeated for each of the four time intervals, \\hich again led to a single branch being used only once in each of the analyses. Multi-way ANOVA. Finally, a multi-way ANOVA that included all of the positive growth rates was conducted using branch type, branch order, branch age, and colony height as the independent variables (Table 2). All of the ANOVA results are reported for untransformed data. Growth rates were heteroscedastic even after a variety of transforms (Levene's, Bartlett's or FmM tests of homo- geneity of variances, depending on the structure of the data set). ANOVA results were almost always concordant with a parallel ANOVA using the rank transformed data and with nonparametric analyses, which could only consider a single factor at a time. Results General observations grew at dramatically slower rates. This is evident in Figure 2A. where the rapid extension of daughter branches (e.g., branches 22. 23. and 24) near the tip of the primary branch is in marked contrast to that of branches closer to the base, many of which exhibited no apparent growth (11, 13, 14). A simplified characterization of the results is that mother branches grew faster than daughters, new branches grew faster than those that were more than 6 months old (Fig. 3), and branches on small colonies had higher growth rates than those on large colonies (Fig. 4A). The measured rates of branch growth were highly vari- able, ranging from negative values to 17.8 cm y~'. Large negative values were often associated with almost total branch loss, as in the case of branch 10 in Figure 2A; this colony, for instance, lost two branches between December 1998 and July 1999. The presence of some uncharacteristi- cally short daughter branches that did not grow during the study suggested that severely damaged branches did not grow further. When cases of negative growth rates were eliminated, the data set was reduced to 8341 individual growth rate measurements. Statistical anal\ses of branch growth Between-colony variation and time-interval effects. The repeated-measures analysis of variance was restricted to the 608 branches for which measurements were available from D }c 1 Mother Branches p ^ ' Daughter Branches E 4 11 ~ 1 (D 735 1 2 ^ 7 2041 ^ o n 1034 ^ 305 60 4149 Growth of Pseudopterogorgia elisabethae colonies was not indeterminate. Branches exhibited a distinct develop- mental cycle in which they arose, grew rapidly, and then 012345 Age Class Figure 3. Branch growth rates for Pseudopterogorgia elisabethae colonies from San Salvador. Bahamas (mean ± standard error) plotted as a function of branch age class. Branches in age classes 1 through 4 originated during the course of the monitoring: class 1. 6 months; 2. 6-12 months; 3. 12-18 months; 4, 18-24 months. Branches in class 5 were present at the start of the 2-year monitoring program. Since mother and daughter branches cannot be distinguished when they originate, there are no age-class 1 (<6 months old) mother branches. The data set also did not include any age-class 4 (18-24 months old) mother branches. Only non- negative growth rates were used in the analysis, and the number of measurements for each class is indicated above or below the error bar. 324 H. R. LASKER ET AL. 5.0 2.5 § O Colony Height (cm) 7.5 5.0 2.5 B _L 2 4 6 8 10 Branch Length (cm) 12 Figure 4. Growth rates of Pseudopterogorgia elisubethae branches from San Salvador. Bahamas, as a function of colony height (A) and branch length (B). Values in (A) are means ± standard error. (B) O denotes daughter branches, and • denotes mother branches. The figure presents a random subset of the over 10.000 points. all four time intervals. (Branches were excluded from this analysis if they did not originate until later in the experi- ment, were lost during the experiment, or had a single interval in which the measurement could not be made due to poor photo quality.) The analysis identified significant ef- fects of both time interval and colony, as well as an inter- action of time interval by colony (all effects. P < 0.001 ). The same result was obtained when the analysis was re- stricted to the middle two time intervals, thereby allowing the inclusion of a total of 2496 branches. When the growth rates of newly originated branches were analyzed separately, growth rates differed among colonies, and there was also an interaction between colony and time interval, but no significant effect of time interval alone (Table 1A). These analyses indicate that the growth rates differed between colonies, and that the magnitude of differ- ences between colonies varied between time intervals, but there was no simple additive difference in variance based on time inu al ;\lone. For instance, one colony may have had its highest growth rates in one time interval, while a differ- ent colon) had iiv lowest growth in the same time interval. Between-colon , va: union and branch ai>e. Despite the effects of colony and time interval on growth, significant fixed effects were detected when the data were partitioned into subsets based on branch type. Separate analyses of the growth of daughter branches during each of the time inter- vals (Table 1. B-E) identified a significant effect of branch age in three of the four time periods (C-E). There were significant interactions between colony and branch age in all four intervals, and significant colony effects in two of the time intervals (Table 1. D and E). Multi-way ANOVA. The effects of branch type, genera- tion, age, and colony height were compared in this large analysis (Table 2). Branch growth rates were significantly affected by branch type (growth rates of mother branches > daughter branches. Fig. 3), branch age (younger > older. Fig. 3). and colony height (short > tall. Fig. 4A). In addi- tion, there were significant interactions between branch type and age. branch type and colony height, and in the three- way interaction between branch type, order, and colony height. The significant interactions reflect the nonlinearity of the patterns seen in Figures 3 and 4; that is. the effects of the different factors were not additive. Variation between colonies was differentiated in these analyses due to the computational limits of incorporating colonies as a fourth independent variable with 260 degrees of freedom (i.e., 261 colonies). Branches from the different colonies were represented in almost all of the combinations of branch type and age, which should have reduced the confounding effects of not incorporating colony identity as an independent variable. Time interval was not tested in these analyses because it is confounded with the age of the branches (i.e., the later intervals for a given branch also record the growth of an older branch). Generation, which was marginally not significant, was confounded with branch type because first-generation branches are by definition mother branches. When daughter and mother branches were analyzed separately in an analysis that was otherwise iden- tical to that in Table 2, generation was not a significant factor (P = 0.52 and 0.40. respectively). The effect of branch age on growth rates of the new branches is underestimated in Figure 3 because it assumes that new branches grew over a 6-month period. If we assume that branches originated continuously over the 6-month interval, then the average age of a new branch would be 3 months, and the mean growth rate would be twice that reported in Figure 3. Furthermore, as noted in Materials and Methods, excluding negative values from the analysis has the effect of slightly inflating growth estimates when the true value is near zero. When cases of negative growth were included, older (>12 mo) daughter branches had growth rates close to zero. With branch length as a covariate, growth rates of daugh- ter branches were analyzed separately for the effects of branch age. Both increasing age and increasing branch length (Fig. 4B) had significant negative effects on branch OCTOCORAL COLONY GROWTH Table 1 Analysis of variance tables testing colony ami temporal variation in gnm-th rates of Pseudopterogorgia elisabethae at Sun Salvador, Bahamas 325 Source of variation df Mean square F />' A. Growth of newly originated branches during their first 6 months over tour successive time intervals COLONY 166.0 5.27 1.52 0.002 Error 207.4 3.46 TIME INTERVAL 3.0 6.25 2.11 0.099 Error 343.3 2.96 COLONY x TIME INTERVAL 1 59.0 3.87 1.93 0.000 Error 1702.0 2.00 B. Growth of daughter branches as a function of branch age Dec 1997-June 1998 COLONY 62.0 7.76 1.59 0.075 Error 33.0 4.88 AGE- 1.0 7.55 2.29 0.135 Error 68.9 3.30 COLONY x AGE 39.0 4.34 2.27 < 0.001 Error 790.0 1.91 C. Growth of daughter branches as a function of branch age June 1998-Dec 1998 COLONY 125.0 5.49 1.48 0.015 Error 120.3 3.71 AGE 2.0 34.82 14.16 < 0.001 Error 193.0 2.46 COLONY x AGE 121.0 3.68 3.36 < 0.001 Error 2131.0 1.10 D. Growth of daughter branches as a function of branch age Dec 1998-July 1999 COLONY 153.0 5.48 2.12 < 0.001 Error 231.7 2.58 AGE 3.0 85.86 40.23 < 0.001 Error 435.8 2.13 COLONY x AGE 199.0 2.75 1.87 < 0.001 Error 2248.0 1.47 E. Growth of daughter branches as a function of branch age July 1999-Dec 2000 COLONY 122.0 6.08 2.13 < 0.001 Error 258.6 2.86 AGE 4.0 26.77 10.73 < 0.001 Error 373.9 2.49 COLONY x AGE 195.0 3.28 2.10 < 0.001 Error 1303.0 1.56 1 Significant P values listed in bold. Since some of the same branches are tested in each of the time intervals (B-E), Bonferroni correction of significance levels would suggest that P = 0.0125 be used as the highest significant P value. F = Mean square/Error mean square. : Age classes were 0-6. 7-12. 13-18. 19-24. and >24 months. The number of classes present increased as the experiment progressed, those branches aged, and new branches originated. growth rates. However, the two variables are confounded, and cases of long, young branches do not exist, so deter- mining whether age or branch size is the functional factor is difficult. Patterns of branch and colony growth Mother-daughter comparisons. Mother branches were not identified until they had produced a side branch, but their growth rates suggest that their growth behavior changes before their daughter branches are produced. A retrospective analysis of branches that eventually became mother branches indicates that they had higher growth rates than daughter branches 1 year prior to the start of branching (Fig. 5). During the 6 months in which branching was first observed, these branches had the greatest growth rates of any of the groups of branches that we distinguished. In contrast to the mother branches, daughter branches exhib- ited decreasing growth rates as they aged and elongated (Fig. 5). After a year of growth, rates were near 1 cm y~', and when negative growth is incorporated into the analysis, the rates were not significantly different from zero. The 326 H. R. LASKER ET AL. Table 2 Analysis <>/ r. >! branch growth rates as a function of branch ami ..in.cteristics uice of variation df Mean square F P Branch age 4 11.95 5.06 0.000 Generation 3 6.05 2.56 0.053 Branch type 1 68.44 29.00 0.000 Colony height 5 13.14 5.57 0.000 Branch age x Generation 7 2.26 0.% 0.459 Branch age x Branch type 4 16.14 6.84 0.000 Order X Branch type 2 7.01 2.97 0.051 Branch age X Generation • Branch type 3 2.59 1.10 0.349 Branch age x Colony height IS 1.42 0.60 0.902 Generation x Colony height 15 5.96 2.52 0.001 Branch age v Generation x Colony height 2] 3.25 1.38 0.117 Branch type x Colon) height 5 6.16 2.61 0.023 Branch age x Branch type x Colons height 5 2.43 1.03 0.399 Generation x Branch type x Colony height 5 7.45 3.16 0.008 Error 8236 2.36 Total 8335 Significant P values listed in bold. F Square. = Mean square/Error Mean different growth trajectories for the two branch types, per- haps from their origination, also suggests that the estimated growth rates of daughter branches may have been inflated by the behavior of mother branches that had not started branching and were thus misidentified. The existence of a determinate developmental sequence in the growth of daughter branches is again suggested by the distribution of branch sizes observed at San Salvador. If branch growth were indeterminate, then continuing growth of branches on a colony would have led to a steady accu- mulation of ever-larger branches, as well as to the presence of many branches with intermediate branch length. Most branches ceased growing when they reached a length of 5-8 cm (Figs. 4B and 6B). With time, colonies added height and new branches at their distal end (Fig. 2B), but colonies did not widen continuously at their base. The development of a plumelike morphology requires that the side elements of the plume show determinate growth. The few branches that became larger (Fig. 6B) were mother branches. Colony size. Branch formation and extension control both the form and size of the colony, and colony size was also subject to determinate growth. Branching did not occur indefinitely among P. elisaheilnic branches. Colonies al- most never had more than fourth-order branches (we have observed only one colony with fifth-order branches), and the number of branches produced by mother branches de- creased with branch order. Branch origination rates were 4.7 (standard error = 0.2) new branches for each 6-month monitoring period on first-order branches, and 3.2 (0.2) and 2.4 (0.5) on second- and third-order branches, respectively. Origination rates were significantly different between branch generations (two-way analysis of variance of natu- ral-log-transformed data. F289 = 15.9, P --•- 0.001). Neither the time interval nor interaction effects were signif- icant (P = 0. 1 and 0.29, respectively). Branch growth also declined as a function of colony height independent of branch generation (Fig. 4A). The net effect of these pro- cesses was that, on San Salvador, colonies seldom exceeded 50 cm in height (Fig. 6A), and all of the monitored colonies were less than 70 cm in height. Discussion The results lead to conclusions in three interrelated areas, all of which suggest that Pseudopterogorgia elisahethae. and by extension other gorgonians, have body plans that are under greater developmental control than generally consid- ered in discussions of modularity. First, although colonies are built through the iterative generation of polyps, both the branch and the whole colony behave as integrated units. Second, the tempo and mode of branch origination and growth generate branches and colonies of finite size, as well as predictable form. Third, branches on P. elisabethae col- onies appear to fall into two distinct developmental catego- 4.0 3.0 o O 2.0 1.0 0.0 a Old New -1y -0.5y New Old Daughter Branches Mother Branches Figure 5. Growth of Pseudopterogorgia elisabethae branches from San Salvador. Bahamas. Daughter branches were divided into those present at the start of observations (old daughter) and those that were <0.5 y (new daughter). Branches that transformed from daughter to mother branches are included as mother branches, and their growth rates at 1 y and 0.5 y before they started branching, are denoted as - 1 and -0.5 y. respectively. "New" mother branches started branching during the 6 months in which the growth rate was determined. Branches that were already mother branches at the start of the observations are labeled "Old." All values are means ± standard error. OCTOCORAL COLONY GROWTH 327 05 50 • A c - 1 40 - O - 30 I 20 E n nn 20 40 60 Colony Height (cm) Jg 1600 "O TO 1200 i_ DO (D .0 E D 800 400 80 B 2 4 6 8 10 12 14 16 >16 Branch Length (cm) Figure 6. Size-frequency distribution of colonies (A) and branches (B) from 261 Pseudopterogorgia elisabethae found within 70 m2 of belt transects at 12-16 m depth at San Salvador, Bahamas. Heights were measured as the length of the tallest branch, and the measurement was made either from photos or in the field with a tape measure. Height classes are centered on the upper limit of the size class; 0-5 cm and >50 cm classes were lumped with adjacent size classes in the statistical analyses. Branch lengths were determined from digital images either of entire colonies or, in the case of large colonies, from digital images of an arbitrarily selected subset of branches. Branch measurements are from December 1999. Branches include both daughter and mother branches. ries, mothers and daughters, which exhibit different growth characteristics from the time they first originate. In addition to those findings, the results also have implications for colony regeneration following disturbance, and in a final section, we discuss the implications of the study for the harvest of P. elisabethae, which is already being conducted in the Bahamas. Branches as modules Growth of P. elisahethae colonies is best described in terms of branches, and each branch behaves as an integrated unit, or module. Daughter branches follow a predictable developmental sequence in which they first grow rapidly. then slow as they age, and eventually stop growing. Mother branches follow a sequence in which they grow and gener- ate both daughter and mother branches, but their growth and the rate at which they generate new branches also slow as the colony grows. Concepts of integration and modularity have been used in describing the development and evolution of suites of mor- phologic features among solitary organisms (Pagliucci, 2002). This approach emphasizes the developmental rela- tionships of traits. While not all groups of integrated traits need be "modules" as used in the invertebrate and plant literature, the modules that make up invertebrate colonies should exhibit the statistical correlations indicative of inte- grated development and evolution (i.e., Magwene. 2001). Our analysis of branch growth rates suggests that both branches and colonies develop as integrated units. Simi- larly, correlations among five traits in 21 gorgonian species also differentiated polyp-level traits from those at the branch or colony level (Sanchez and Lasker, 2003). In the context of this broader definition of modularity, three levels of modularity or integration can be recognized in P. elisa- bethae: polyps, branches, and the colony. The polyp has always been recognized as a distinct unit with a well- defined ontogeny and determinate growth. Our data on branch growth suggest that the branches of P. elisabethae also exhibit a well-defined ontogeny. The colony must also be considered as a level of organization, because growth of the branches is also dependent on colony-level traits, that is, on colony height and generational order. Determinate growth among modular organisms Daughter branches on P. elisabethae stop growing as they age, and the growth rates decrease as the colony increases in height. Furthermore, origination rates of branches decrease with branch generation. In concert, these changes in developmental rates conserve a colony's size and form, a pattern that is functionally equivalent to determinate growth. The pattern of determinate growth observed in P. elisabethae colonies could be generated by set developmen- tal programs or through predictable responses to microen- vironmental variation around the colony. Both are, at some level, growth in response to cues and are not mutually exclusive. We argue that a developmental program is the principal factor controlling the determinate growth of branches, while the size of whole colonies probably reflects a mix of both developmental effects and environmental and historical factors (Rinkevich, 2000). Branches have a clear developmental cycle which, among the daughter branches, leads them to stop growing long before they reach 10 cm in length. Kim and Lasker (1997) report that interior branches of the gorgonian Plexaura homomalla have lower growth rates than those on the pe- rimeter of the colony, a pattern they attributed to nutrient 328 H. R. LASKER ET AL. supply and self-interference. A number of realistic models of form in rr^Jular organisms have been developed, in which grov is controlled by the local responses of the individu J modules to their local environment (Braverman, 1974; Graus and Maclntyre, 1976; Colasanti and Hunt. 1997; Kaandorp and Kiibler, 2001; Oborny et al. 2001). However, the decrease in branch growth among P. elisa- bethae colonies is best described as an age-dependent de- crease. Thus, although the smallest branches had the great- est growth rates, many small branches also exhibited low growth (Fig. 5). Furthermore, daughter branches stop grow- ing while adjacent mother branches continue to grow, which indicates that position alone does not control branch growth. Colony size in P. elisabethae also appears to be determi- nate, and observations of determinate growth have been reported among a wide range of colonial taxa. As noted, octocoral descriptions often include maximum colony sizes (i.e.. Bayer, 1961), and decreasing growth with increasing colony size has been reported for a number of gorgonians (Grigg, 1974; Velimirov, 1975; Mitchell etui.. 1993: Coma et al., 1998; Cordes et ai, 2001). Among scleractinian corals, determinate growth of independently growing branches generates colonies with determinate form and size (Rinkevich, 2002). Among botryllid tunicates, groups of zooids, referred to as systems, undergo synchronous senes- cence (Sabbadin, 1969), and whole colonies, including iso- lated explants from a common source, undergo simulta- neous senescence (Milkman. 1967; Rinkevich et al., 1992). In addition, graptolite colonies are believed to have had determinate growth leading to distinct species-specific forms and sizes (Mitchell, 1988). Maximum size alone does not demonstrate determinate growth. In a manner functionally equivalent to determinate growth, modular organisms may also stop growing, not according to a genetically determined developmental plan, but due to size-dependent interactions between the colony and environment, such as the balance between nutrient uptake and metabolic rates. Size-dependent change in col- ony growth that is mediated by metabolic rate and resource capture has been modeled by Sebens (1982. 1987) and by Kim and Lasker (1998). Taxa that exhibit growth patterns consistent with these simple models have been reported in octocorals (McFadden. 1986; Kim and Lasker, 1997) and tunicates (Holyoak, 1997). Ecological processes such as size-dependent mortality also could generate a maximum colony size and the appear- ance of determinate growth. Colonies are susceptible to being knocked over because drag forces increase with col- ony size and bioerosion weakens the substratum around the holdfast (Birkeland. 1974). During most of this study, how- ever, mortality decreased with colony height (mortality per 6 months: 0-10 cm, 0.146: 11-20 cm, 0.108; 21-30 cm, 0.071; 31-40 cm, 0.048; >40 cm, (1.000). That pattern of mortality would have led to the accumulation of large colonies within the population. Mortality of colonies taller than 40 cm increased from 0.0% to 40.2% when Hurricane Floyd, a Category 4 hurricane, struck San Salvador on 13 September 1999. Although mortality events generated by such storms reduce the number of large colonies, the dis- tribution of colony sizes observed in the San Salvador population would require that the mortality of large colonies be high almost every year, not only in those occasional years with severe hurricanes. Decreased growth rates among the larger colonies appears to be a more parsimonious explanation of the size-frequency distribution. Because ag- ing and size are often correlated and the ages of most of the colonies were not known, the causative variable is difficult to distinguish. Mothers and daughters — two developmental classes of branches Age. generation, and colony height all affect rates of branch growth and origination, but the most striking differ- ences in the growth of branches are those between mother and daughter branches. The data indicate that the two branch types are fundamentally different. First, mother branches continue growing while adjacent branches stop growing; for instance, compare branch 2 to branches 4 or 17 in Figure 2A. The self-shading effects hypothesized for Plexaura homomalla (Kim and Lasker. 1997) do not ac- count for the continued growth of mother branches, which were otherwise indistinguishable from daughter branches. Second, mother branches exhibit high growth rates as soon as they originate, well before they have produced their first daughter. However, the two branch types are not immutable. When colonies are damaged, branches that were previously daughter branches begin to extend and generate new branches (Castanaro and Lasker, 2003). Understanding whether and how branches "become" mother branches will be essential to our understanding of developmental pro- cesses among these colonial organisms. Applications Modular growth is an especially advantageous growth strategy, when transplants are used to remediate populations (Rinkevich, 2000), when explants are used as stock in mariculture, and for the sustained harvest of wild popula- tions where colonies are cropped at regular time intervals (Castanaro and Lasker. 2003). Understanding the pattern of growth of P. elisabethae colonies is particularly important because this species is harvested and extracted for a class of natural products called pseudopterosins (Mayer et al,, 1998). Material for extraction is collected by cropping branches from colonies. If growth is inversely related to the size of the colony, then reductions in colony size will enhance growth and productivity. Among harvested popu- lations, this suggests that colonies can be maintained at an OCTOCORAL COLONY GROWTH 329 optimal size, and that naturally occurring populations might recover from disturbance at rates greater than the growth rates observed before the disturbance. Alternatively, if there is also an age-based component to growth regulation (i.e.. Hughes and Connell. 1987), then recovery from either an- thropogenic or natural disturbance may not be as great as suggested by colony size alone. Detailed understanding of colony growth patterns is essential to determining whether a species is suitable for sustained harvesting and whether remediation following anthropogenic disturbance is likely to succeed. Conclusions Knowlton and Jackson (1994) have argued that — far more often than generally acknowledged — the apparent plasticity of coral reef cnidarians reflects genetic differen- tiation. Indeed, the literature provides numerous hints of genetic controls on size and form: genetically based, spe- cies-level differences in colony form within the Montas- traea annnlahs complex (Weil and Knowlton. 1994); dif- ferences in regeneration among clones of the reef coral Stylophora pistillata (Rinkevich, 2000); age effects on the sur\ ival of stony corals (Hughes and Connell, 1987): grap- tolites with highly determinate patterns of colony size (Mitchell, 1986); and botryllid tunicates that exhibit zooid and colony senescence (Rinkevich et ? . Knowlloi :id J. B. C. Jackson. 1994. New taxonomy and niche paft!tio,,iut on coral reeds — jack of all trades or master of some? Trcr..i:, Ecol. Evol. 9: 7-9. Lasker, H. R., and J. A. Sanchez. 2002. Allometry and astogeny of modular organisms. Pp. 207-253 in Reproductive Biology of Inverte- hi'tites. Vol. XL Progress in Asexual Reproduction, R. N. Hughes, ed. John Wiley & Sons, New York. Magwene, P. M. 2001. New tools for studying integration and modu- larity. Evolution 55: 1734-1745. Mayer, A. M. S., P. B. Jacohson, W. Fenical, R. S. Jacobs, and K. B. Glaser. 1998. Pharmacological characterization of the pseudo- pterosins: Novel anti-inflammatory natural products isolated from the Caribbean soft coral, Pseudopterogorgia elisabethae. Life Sci. 62: 401-407. McFadden, C. S. 1986. Colony fission increases particle capture rates of a soft coral — advantages of being a small colony. J. Exp. Mar. Biol. Ecol. 103: 1-20. Milkman. R. 1967. Genetic and developmental studies on Botryllus schlosseri. Biol. Bull. 132: 229-243. Mitchell, C. E. 1986. Morphometnc studies of Climacograprus (Hall) and the phylogenetic significance of astogeny. Pp. 1 19-129 in Palaeo- ecology and Biostratigraphy of Graptolites, C. P. Hughes and R. B. Rickards, eds. Geological Society Special Publication 20, Geological Society of America, Boulder. CO. Mitchell, C. E. 1988. The morphology and infrastructure of Brevigraptus quadrithecatus n. gen., n. sp. (Diplograptacea), and its convergence upon Dicaulograpttis hystrix (Bulman). J. Paleont. 62: 448-463. Mitchell, N. D., M. R. Dardeau, and W. W. Schroeder. 1993. Colony morphology, age structure, and relative growth of two gorgonian cor- als. Leptogorgia hebes (Verrill) and Leptogorgia virgulata (Lamarck), from the northern Gulf of Mexico. Coral Reefs 12: 65-70. Oborny, B., T. Czaran, and A. Kun. 2001. Exploration and exploitation of resource patches by clonal growth: a spatial model on the effect of transport between modules. Ecol. Model. 141: 151-169. Pagliucci, M. 2002. Touchy and bushy: phenotypic plasticity and inte- gration in response to wind stimulation in Arahidopsis thaliana. Int. J. Plant Sci. 163: 399-408. Rinkevich, B. 2000. Steps towards the evaluation of coral reef restora- tion by using small branch fragments. Mar. Biol. 136: 807-812. Rinkevich, B. 2002. The branching coral Stylophoru pistillahi: contri- bution of genetics in shaping colony landscape. Isr. J. Zool. 48: 71-82. Rinkevich, B., R. J. Lauzon, B. VV. M. Brown, and I. I,. Weissman. 1992. Evidence for a programmed life-span in a colonial protochor- date. Proc. Null. Acad. Sci. USA 89: 3546-3550. Sabhadin, A. 1969. The compound ascidian Botrvlliis schlosseri in the field and laboratory. Publ. Stn. Zool. Napoli 37(suppl.): 67-72. Sanchez, J. A. 2002. Dynamics and evolution of colony form among branching modular organism, Ph.D. dissertation. University at Buffalo. State University of New York. Buffalo. NY. 137 pp. Sanchez, J. A., and H. R. Lasker. 2003. Patterns of morphologic integration in marine modular organisms: supra-module organization in branching octocoral colonies. Proc. R. Sot: Loud. DOI: 10.1098/ rspb.2003.2471. Sanchez, J. A., W. Zeng, V. R. Coluci, C. Simpson, and H. R. Lasker. 2003. How similar are branching networks in nature? A view from the ocean: Caribbean gorgonian corals. J. Theor. Biol. 222: 135-138. Sebens, K. P. 1982. The limits to indeterminate growth: an optimal size model applied to passive suspension feeders. Ecology 63: 209-222. Sebens, K. P. 1987. The ecology of indeterminate growth in animals. Anmi. Kc\: Ecol. S.v.v/. 18: 371-407. Velimirov, B. 1975. Growth and age determination in the sea fan Emii- cella clavolinii. Oecologia 19: 259-272. Weil, E., and N. Kmmlton. 1994. A multi-character analysis of the Caribbean coral Monlaslraea anntdaris (Ellis & Solander. 1786) and its two sibling species, M. faveolata (Ellis & Solander. 1786) and M. fnmksi (Gregory, 1895). Bull. Mar. Sci. 55: 151-175. Reference: Biol. Bull. 205: 331-338. (December 2003) '. limatula were maintained in 0.45-ju.m filtered, 359ft artificial seawater (ASW; Instant Ocean, Aquarium Sys- tems, Mentor, OH) supplemented with 50 ju,M ammonia. Geukensia demissa was maintained in filtered, 30%r ASW supplemented with 50 i^M ammonia. The ASW solution in the chambers was kept mixed with magnetic stir bars (60 to 100 rpm). ASW flow rates ranged from 0.18 to 0.19 ml Maintaining the clams in a flow-through respirometry system allowed for constant monitoring of experimental conditions. The oxygen concentrations in the chamber out- flows were determined with a polarographic O2 sensor (POS), whereas the concentrations of sulfide and, in some experiments, ammonia were measured by flow injection analysis (FIA). Throughout this paper, sulfide refers to SHiS (primarily the sum of HS~ and H:S), and ammonia refers to SNH, (the sum of NH3 and NH4+ ). Outflows were pumped directly through a flow cell containing a sulfide- insensitive gold microcathode POS (Orbisphere 2120, Ge- neva) followed by a series of FIA injection sample loops SULFUR AMINO ACIDS IN SOLEMYA VELUM 333 F pump Figure 1. Schematic of flow-through respirometry system. (A) CO,, N2, and O2 gas mixture. (Bl Seawater-gas equilibration column with pH regulation. (C) Syringe pump for metering sodium sulfide stocks into seawater entering the chambers. The syringe pump is also used to meter inhibitor into chamber 2. (D) Water-jacketed chambers containing clams, w ith at least one empty chamber to function as a control. Outflows from the chambers are pumped to a pneumatically actuated six-way stream-selector valve (E). The position of this valve determines whether the outflow goes to waste or to the analysis system. The analysis system consists of a flow injection analyzer for sulfide and ammonia determination and an O2 sensor mounted in a flow cell (F). The analyzer and stream-selector valve are linked to a data acquisition and automated control system (G). (Rheodyne Type 50 valves with Rheodyne 5701 pneumatic actuators, Rohnert Park, CA). The FIA determination of sulfide involved derivitizing the sample stream with 1.5 mM 2,2'-dithiodipyridine, which forms a stable product with an absorbance maximum at 343 nm (Svenson, 1980). The derivitized sample was loaded into an injection loop and sent to a variable wavelength detector (Hyperquan VUV-20, Colorado Springs, CO). This method produces 343-nm absorbance peaks proportional to sulfide concentration (Svenson. 1980). Ammonia was de- termined by a modification of the FIA protocol of Willason et al. ( 1986). The sample was loaded into an injection loop and then treated with NaOH to raise the pH. converting ammonium to ammonia gas. Ammonia passes through a Teflon membrane into a carrier stream containing phenol red, raising the pH of the phenol red solution. The resulting color change (absorbance at 560 nm) is monitored with a detector fabricated from a green LED and a phototransistor. POS and FIA detector outputs were continuously recorded with a data acquisition system (Sable Systems, Datacan V, Henderson, NV), which was also used to control FIA injec- tion valve actuation. Three respirometer chambers containing clams, one con- trol chamber, and two standards were connected to a six- way stream-selector valve (Fig. 1). Every 0.13 h the valve was automatically switched, allowing sampling from all six channels every 0.8 h. Peak height (for FIA) or average output (for POS) was calculated to determine concentration differences between chambers containing clams and the control chamber. These data were continuously monitored to ensure, in the case of oxygen measurements, that the clams were not exposed to hypoxic conditions. Direct mea- surements of sulfide in samples taken from the respirometer outflow were used to standardize sulfide data obtained by FIA. Fluxes were calculated as follows: /imol sulfide • g~' wet weight • h ' = (concentrationexi,enmentau.hamher - con- centrationcl,nm,Uh;miht,r) X (flow rate in 1 • h ') X (g 'wet weight). Experimental treatments In all experiments, the clams were acclimated for 24 h in ASW prior to sulfide or inhibitor exposure. In experiments in which clams were exposed to sulfide, a syringe pump (Harvard Apparatus 944, South Natick, MA) was used to meter a 20 mM Na2S solution (Fig. 1C) into the ASW solution before its entry into the respirometer chambers. Final sulfide concentration in the chambers was 0.45 ± 0.05 mM. Clams assayed for amino acid levels were exposed to sulfide for 24 h. In some experiments, a metabolic inhibitor was added to the ASW solution after the 24-h acclimation period. The final concentrations of these inhibitors were 0.9 mM chloramphenicol (Sigma, St. Louis, MO). 0.02 mM cycloheximide (Sigma), and 0.25 mM methionine sulfoxi- mine (MSX; Sigma). In experiments in which sulfide con- sumption was examined, the clams were acclimated for 24 h in ASW, then exposed to sulfide for up to 100 h. During the final 24 h of the sulfide exposure, metabolic inhibitors were added to the ASW solution. The clams were weighed before and after treatment. Amino acid analyses After experimental treatment, the bivalves were opened by severing their adductor muscles. The gills and foot of each clam were dissected free of other tissues, blotted briefly on a paper towel, then individually frozen in liquid nitrogen and stored at -80 °C. The gills and feet were individually homogenized in distilled water (1:25. tissue/ dH2O) on ice. To precipitate proteins, the homogenates were treated with 5% sulfosalicylic acid (Sigma) in a 1:10 ratio of sulfosalicylic acid/homogenate (Lee and Slocum. 1988). The solutions were centrifuged for 5 min at 16,000 X g, and the supernatants were stored at —80 °C. Total free amino acids were quantified in a Beckman 334 J. L. JOYNER ET AL. 6300 amino acu; ai alyzer following a protocol modified from Lee and , (1988). The samples were diluted (1:30, by vol A ith Li-S buffer (96.8% H2O. 1% LiCl, 1% thu . 0.7% HC1, 0.5% benzoic acid; pH 2.2: Becki i. her. Inc.. Fullerton. CA). Of the 240-250 jul of sa : or standard loaded onto the sample loops. 50 ju.1 of eacii solution was analyzed. The amino acids were sep- arated in Li-A buffer (98% H2O. 1% Li citrate. 0.5% LiCl. 0.5% HC1; pH 2.8; Beckman Coulter) on a 10-cm ion exchange column and reacted in-line with ninhydrin solu- tion. Absorbances were monitored at 570 nm and 440 nm. After preliminary experiments, only taurine, hypotaurine, and thiotaurine were quantified. The standards were 200 /xA/ taurine (Sigma) and 20 /u,A/ hypotaurine (Sigma) in Li-S buffer. The thiotaurine standard was prepared by dissolving 0.0011 g hypotaurine and 0.05 g Na,S • 9H2O (Fisher Scientific. Fair Lawn, NJ) in deionized water, heating to 100 °C. acidifying the solution with 1 M HC1, and evaporating the solution (Cavallini et «/., 1963). This standard was verified by mass spectrometry. The temperature of the column affected the separation of thiotaurine from taurine and the reactivity of hypotaurine with ninhydrin. Thiotaurine could be detected only at 70 °C. The values of hypotaurine reported here are only from analyses run at 45 °C, because hypotaurine was not as reactive with ninhydrin at 70 °C. The temperature did not affect taurine levels. Reported values for taurine are aver- ages between levels detected at 45 °C and 70 °C. Results are presented as the mean ± the standard error and as average rates of synthesis per gram wet weight over a 24-h period [(amino acid level in g~ ' wet weight in clams exposed to sulfide) — (amino acid level in clams not ex- posed to sulfide)/24 h] assuming the synthesis rate was linear over the 24-h period. Differences among means were detected using one-way ANOVA for each amino acid in S. velum samples (Statistica. Statsoft, Inc., Tulsa. OK). The appropriate comparisons were analyzed with Fisher's LSD procedure (Statistica). The G. demissa and Y. limatitla amino acid data were analyzed with two-sample t tests (Statistica). Results Sulfide exposure increased taurine and thiotaurine levels in S. velum hut not in two nonsymbiotic bivalves Specimens of Solemya velum exposed to sulfide had significantly more taurine (P = 0.0034) and thiotaurine (P < 0.000 1 ) in their gill tissue than clams not so exposed (Table 1 ). These values equate to average rates of change in taurine and thiotaurine levels of 0.89 fimol • g~' wet weight • h"1 and 0.22 jumol • g ' • h"1 over the 24-h incubation period. Hypotaurine levels were not significantly affected (P = 0.064). Cysteine and methionine, two other sulfur-containing amino acids, were below the limits of detection in all samples. Levels of the most abundant non- sulfur-containing free amino acids — alanine. glutamate, and aspartate — were unaffected by sulfide exposure (data not shown). In preliminary experiments with sulfide-exposed clams, free amino acid profiles of S. velum foot (symbiont- free) and gill tissues were similar (data not shown), as observed previously (Conway and McDowell Capuzzo, 1992). In subsequent experiments with metabolic inhibitors, only taurine, hypotaurine, and thiotaurine levels in the sym- biont-containing gill tissues of S. velum were quantified. Gill tissue from the nonsymbiotic bivalve species Geu- kensia demissa and Yoldia limatitla contained less taurine (P < 0.0001, non-sulfide-exposed) than S. velum (Table 1), but comparable levels of hypotaurine (P = 0.068) and thiotaurine (P -- 0.648). The concentrations of these amino acids were the same whether or not the bivalves were exposed to sulfide (all comparisons, P > 0.05). Metabolic u/liihiturs decreased taurine and thiotaurine levels in S. velum gills To investigate the role of the chemoautotrophic symbi- onts, clams were exposed to chloramphenicol, a specific Table 1 Effects of sulfide exposure and inhibitors on tauriin; liypukiurine. and thiotaurine levels in gills of three bivalve species Species Taurine Hypotaurine Thiotaurine Treatment ( n ) -Sulfide + Sulfide -Sulfide + Sultide -Sulfide Data are mean ± SEM in /imol amino acid/g wet weight of gill tissue. (/(), Number of replicates; MSX. methionine sulfoximine. •' Significant differences (/' - 0.05) between clams exposed to sulfide ( + Sulfide) and not exposed (-Sulfide). h Significant differences (P • 0.05) between clams treated with inhibitor and not treated. + Sulfide Solemya velum No inhibitor (16) 100.3 ± 5.5a 120 ± 5.4Jh 13.7 ± 1.4 11.4 ± 1.3 0.35 ± 0.2J 5.4 ± 0.6iLb Chloramphenicol (9) 96.5 ± 6.5 102.2 ± 4.5h 12 ±0.8 12.9 ± 1.4 0.42 ± 0.2a 3.6 ± 0.8a'b Cycloheximide (5i 100.1 ± 6.6 109.5 ± 3.9 20.3 ± 2.6 15.1 ± 2.2 1.4 ± 0.2" 5.5 ± 0.7a MSX (7) 91.7 ± 10 96.1 ± 5.7h 18.5 ± 3 12.7 ± 4 0.79 ± 0.3" 6.5 ± 0.6a Geukensia dt'n:. No inhibitor (7) 37.9 ± 1.9 39.9 ± 1.2 4.7 ± 0.7 4.5 ± 0.6 0.65 ± 0.2 0.95 ± 0.4 Yoliliti limuniUi No inhibitor (3) 62 ± 5.5 56 ± 6.7 10.9 ± 1.5 9.9 + 1.3 0.16 + 0.16 0.24 ± 0.03 SULFUR AMINO ACIDS IN SOLEMYA VELUM 335 inhibitor of bacterial protein synthesis (Burnap and Trench. 1989). at a concentration previously determined to disrupt symbiont metabolism but to be nontoxic to the host (R. W. Lee, unpubl.). To examine the role of the host, clams were exposed to cycloheximide. a specific inhibitor of eukaryotic protein synthesis (Burnap and Trench. 1989). at a concen- tration nontoxic to the host for the duration of the treatment. In additional experiments, the effects of the ammonia as- similation inhibitor, methionine sulfoximine (MSX; Rees. 1987) were examined: MSX inhibits glutamine synthetase. which has been detected in 5. velum tissues (Lee et al.. 1999). To ensure complete inhibition of ammonia assimila- tion, the MSX level was 10-fold higher than that utilized by Rees (1987). Taurine. hypotaurine. and thiotaurine levels in clams not exposed to sulfide were not affected by exposure to any of the inhibitors (Table 1). Additionally, the wet weights of whole clams were not altered by treatment with sulfide or metabolic inhibitors. When clams were treated with the three metabolic inhib- itors, the usual sulfide-induced increase in taurine levels was not observed (P values for comparisons between —sulfide and + sulfide. in the presence of inhibitors: chlorampheni- col, P = 0.552; cycloheximide. P = 0.451: MSX. P = 0.707). Hypotaurine levels were not altered by sulfide exposure or treatment with inhibitors (P = 0.064). Thio- taurine levels increased after sulfide exposure, even in the presence of metabolic inhibitors (P values for comparisons between —sulfide and + sulfide in the presence of inhibitors: chloramphenicol. P < 0.001; cycloheximide. P < 0.001: MSX. P < 0.0001 ). This sulfide-stimulated increase, how- ever, was reduced by treatment with chloramphenicol (P = 0.016. sulfide-exposed control clams versus chloramphen- icol-treated sulfide-exposed clams). Chloramphenicol and MSX decreased S. velum sulfide and ammonia consumption The average rates of sulfide and oxygen consumption in the absence of inhibitors were 2.57 ± 0.06 (5) /nmol • g~' wet weight • h~' [mean ± SE (n of experiments)] and 3.98 ± 0.01 (5) ju,mol • g~' • h~'. respectively. Chloram- phenicol exposure completely halted sulfide consumption after 10 h (Fig. 2A. hours 120-130). Cycloheximide treat- ment did not affect sulfide consumption rates. Treatment with MSX inhibited ammonia uptake (Fig. 2B, hours 10- 30). Discussion This study demonstrates that taurine and thiotaurine lev- els in Solemva velum gills increase after sulfide exposure. These two amino acids may function as nontoxic sulfide storage compounds. Inhibition of symbiont and host protein synthesis and host ammonia assimilation blocked sulfide- stimulated taurine synthesis; in contrast, only the inhibition I ~ 5 5 §1 u > o> v 3 "o co I 1 - 0 - -1 - -2 -3 -4 - -5 - inhibitor added sulfide added chloramphenicol control cycloheximide 50 100 hours 150 MSX stopped B Figure 2. Effect of inhibitors on Solemya velum ammonia and sulfide fluxes. (A). Chloramphenicol (0.9 mA/). an inhibitor of prokaryotic protein synthesis, blocked sulfide consumption. Cycloheximide (0.02 mA/). an inhibitor of eukaryotic protein synthesis, had no effect. (B). Methionine sulfoximine (MSX. 0.25 mA/). an inhibitor of ammonia assimilation, blocked ammonia uptake and resulted in ammonia excretion. Negative fluxes denote uptake from the medium. of symbiont metabolism decreased the sulfide-stimulated thiotaurine synthesis. The maintenance of free amino acid pools depended upon the presence of functioning symbi- onts, sulfide consumption, and host ammonia assimilation. Thus, sulfur-containing free amino acids also may be a link between cycling of nitrogen and sulfur in chemoautotrophic symbioses. The magnitude of changes in taurine and thiotaurine pools observed in the present study is sufficient for these amino acids to be physiologically significant sulfide storage compounds. In experiments in which clams were exposed to sulfide for 24 h, the increases in taurine and thiotaurine levels corresponded to synthesis rates of 0.89 ju.mol • g~' wet weight • h"' and 0.22 jLtmol • g~' • h~', respectively. Since taurine contains one S atom and thiotaurine contains two S atoms, this corresponds to a potential sulfide incor- poration rate of 1.33 ;u,mol • g~' • h~'. The average rate of whole animal sulfide consumption measured under the con- trol (no inhibitor) conditions was 2.57 jumol • g • h '. which is similar to the sulfide consumption rate of Solemya reidi under similar experimental conditions (Anderson et al., 1987). Therefore, the contribution of taurine and thio- 336 J. L. JOYNER ET AL taurine to sulfide detoxification could account for up to 50% of the total sulfido r. i\ Treatment Jiloramphenicol. cycloheximide. and MSX PR : the sulfide-induced increases in taurine leveK i by the control clams. The lack of detectable taurine x\iuhesis in chloramphenicol-treated clams likely can be attributed to the chloramphenicol-induced cessation of sulfide consumption. Additionally, chloramphenicol may act to prevent synthesis of mitochondria! proteins that are not nuclear encoded. Therefore, effects of chloramphenicol treatment might also be ascribed to impairment of mito- chondrial metabolism. However, in preliminary experi- ments, S. velum tolerated treatment with 0.9 mM chloram- phenicol for at least 9 days, suggesting that the effects due to chloramphenicol treatment are most likely the result of disrupted symbiont metabolism rather than toxicity to the host. Treatment with cycloheximide, which inhibits eukary- otic protein synthesis (Burnap and Trench. 1989) and is functionally analogous to chloramphenicol. decreased tau- rine synthesis in the presence of sulfide, but did not affect sulfide consumption. These results suggest that taurine is synthesized by the host and that the cycloheximide treat- ment did not affect any sulfide consumption which may occur in host tissues (Powell and Somero, 1986). Exposure to MSX blocked ammonia assimilation, probably contrib- uting to the decreased taurine levels in MSX-treated clams. These results mirror those reported by Lee and coworkers (Lee et al., 1997), who found a direct relationship between external ammonia availability and taurine levels in S. reidi. Hypotaurine levels were not altered by exposure to sul- fide or the metabolic inhibitors. These results suggest that hypotaurine is not directly involved in sulfide detoxifica- tion, nor is it an intermediate in the taurine synthesis path- way in 5. velum (Cavallini cr al.. 1976). Alternatively, hypotaurine could have protective functions, such as by serving as a compatible osmolyte (Yin et al., 2000) or by scavenging free radicals (Huxtable, 1992). Thiotaurine levels were greater in the gills of sulfide-ex- posed clams than in non-sulfide-exposed clams, regardless of exposure to metabolic inhibitors. Treatment with chloram- phenicol reduced, but did not prevent, sulfide-induced thiotau- rine synthesis. This reduction likely resulted from the chlor- amphenicol-induced cessation of sulfide consumption. Inhibition of host metabolic activity with cycloheximide did not affect thiotaurine levels in sul fide-exposed clams. Thiotau- rine may be produced abiotically in host tissues, in which case host enzymati. pathways may not be necessary. Despite the MSX-inducecJ inl ''.ion of ammonia assimilation, thiotaurine levels in MSX-u clams increased following sulfide expo- sure. Again, these re nil suggest that thiotaurine may be produced abiotically from precursors already present in the gill tissue and depend less upon ammonia availability. Taurine and thiotaurine synthesis in S. velum It is not known whether symbiotic bivalves maintain free amino acid pools by absorbing amino acids from the envi- ronment or by synthesizing them. We do know that S. reidi can take up free amino acids from sediment interstitial water (Lee et al., 1992). However, sulfur-containing amino acids were not detected in the pore water samples from 5. reidi burrows (Lee et al., 1992) and were not present in the incubation medium in the experiments presented here, sug- gesting that solemyid clams synthesize taurine and thiotau- rine. The biosynthesis pathways of taurine and thiotaurine in solemyid clams are unknown, but the results from this study suggest that these pathways require ammonia assim- ilation, sulfide consumption, and active symbiont metabo- lism. Ammonia is present at elevated levels in the burrow environment of solemyid clams (Lee et al., 1992; Krueger, 1996). and the clams assimilate it into amino acids, which may then serve as precursors for sulfur-containing amino acids (Lee and Childress. 1994). The present study indicates that glutamine synthetase is the primary enzyme in the assimilation pathway, since MSX treatment blocked ammo- nia uptake and caused ammonia excretion, similar to what was demonstrated in an algal-cnidarian symbiosis (Rees, 1987). The product of ammonia assimilation, glutamate, can then be used as a precursor in the production of taurine, which is a major product of ammonia assimilation in S. reidi tissues (Lee et al., 1997; thiotaurine production was not tested for). Therefore, in S. velum, taurine and thiotaurine production in response to sulfide, as demonstrated in this study, may be facilitated by the ability of the bacterium- mollusc association to synthesize glutamate from inorganic nitrogen. Just as glutamate likely contributes organic nitrogen to the synthesis of taurine and thiotaurine, the probable source of organic sulfur is cysteine. All of the demonstrated taurine synthesis pathways in mammalian and invertebrate tissues incorporate cysteine (Jacobsen and Smith. Jr.. 1968; Bender, 1975; Bishop et al., 1983; Huxtable. 1992), which apparently cannot be synthesized by molluscs (Bishop et al.. 1983). Although some intertidal molluscs utilize external cysteine sources to maintain taurine pools (Allen and Awapara. 1960; Jacobsen and Smith. Jr.. 1968; Allen and Garrett, 1972; Bender. 1975), it is unlikely that solemyid clams take up cysteine from their environment (Lee et al.. 1992). Therefore, the most likely source of cysteine or other taurine precursors is the symbiotic bacteria. Gram-negative bacteria cannot synthesize hypotaurine, taurine, or thiotau- rine (Jacobsen and Smith. Jr., 1968; Huxtable, 1992). but can make cysteine (Kredich, 1996). Cysteine synthesis in E. coli requires sulfur in the form of sulfide or thiosulfate (Stauffer, 1996) and assimilated ammonia in the form of glutamate (Reitzer, 1996). Translocation of the essential SULFUR AMINO ACIDS IN SOLEMYA VELUM 337 Environment NH3 HS Host -*• Glutamate Cysteine Taurine Thiotanrine Figure 3. Proposed model of taurine and thiotaurine biosynthesis in Solfin\;i vfliiin. The clams extract ammonia and sulfides from the burrow einitonmcm. Ammonia is assimilated into glutamate. which is probably utilized by the xymbionts in cysteine synthesis. Cysteine is translocated to the host and utili/cd in the synthesis of taurine and thiotaurine. amino acid cysteine from gram-negative symbionts to host may occur in 5. velum gill tissue as modeled in Figure 3. Such translocations have been demonstrated in bacteria- aphid and algal-cnidarian associations (Wang and Douglas. 1999: Douglas etui, 2001). The results of this study suggest that S. velum relies upon its symbionts as a source of taurine precursors, as modeled in Figure 3. The inhibition of symbiont metabolism with chloramphenicol, therefore, may equate to a loss of cysteine metabolism, thus decreasing sulfide consumption and tau- rine and thiotaurine synthesis by the host. Ammonia limi- tation, either by MSX treatment or reduced exogenous am- monia resources, may limit glutamate availability in S. velum tissues, thereby limiting cysteine production in the symbionts (Fig. 3). This could result in the lower taurine levels seen in the solemyid clams in this study and in previous work (Lee et al., 1997). Thus, taurine and thiotau- rine may be a link between nitrogen and sulfur cycling in chemoautotrophic symbioses and serve as nontoxic sulfide storage and transport compounds. The absence of similar patterns in nonsymbiotic sulfide-tolerant molluscs (Geuken- sia demissa and YolJin limatula) suggests these functions of sulfur-containing free amino acids may be limited to sym- biotic molluscs. 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These associations occur in the photic zones of both temperate and tropical benthic habitats, and they thrive in nutrient-poor tropical marine environments because they conserve and recycle nutrients. In its associations with dinorlagellates, the host cnidarian most commonly houses symbionts within gastrodermal cells, in a vacuole of phagosomal origin. The initial infec- tion, during which the dinoflagellate symbionts are internal- ized, typically occurs when dinorlagellates that enter the host's mouth are taken into phagocytic gastrodermal cells that line the gastric cavity (Colley and Trench, 1983; Fitt and Trench, 1983: Schwarz et ai. 1999, 2002). The phago- some, through unknown mechanisms, fails to fuse with lysosomes (Fitt and Trench. 1983). and the dinoflagellates remain undigested within the vacuole. Ultimately, the dinoflagellates reside within a compartment delineated by multiple membranes (Taylor. 1968. 1987; Wakefield et ai. 2000). Historically, the origin of the multiple membranes that surround the symbiont has been uncertain. Recently, immunolocalization studies using host-specific and dinoflagellate-specific monoclonal antibodies suggest that only the outermost membrane originates from the host, and all of the inner membranes originate from the dinoflagel- lates, perhaps accumulating from repeated cycles of ecdysis within the host vacuole (Wakefield and Kempt'. 2001 ). We have been interested in identifying host genes that play roles in symbiotic interactions in cnidarian- dinoflagel- late symbioses (Weis and Levine, 1996; Weis and Reyn- olds. 1999; Reynolds et ai, 2000). We have used as a model system the temperate symbiotic sea anemone Anthopleura elegantissima (Brandt. 1835), which is abundant in the intertidal region of the eastern Pacific, from Mexico through Alaska. This species is able to form associations with two species of dinoflagellates, Symbiodiniiim californium and S. inuscatinei. The presence of one or more of the symbionts within a particular host depends upon microhabitat differ- ences that are created along temperature and light gradients that occur along latitudinal and intertidal ranges. Along the Oregon coast, most hosts contain only a single symbiont, S. inuscatinei (LaJeunesse and Trench, 2000). Using biochemical and molecular approaches, we have identified a number of host genes that are likely to function in mediating host-symbiont interactions (Reynolds et al., 2000; Weis and Levine, 1996; Weis and Reynolds, 1999). One of these, s\m32. encodes a protein that belongs to a class of cell adhesn>n proteins called fasciclin domain pro- teins (Reynolds et ai. 2000). Fasciclin domain proteins share low overall sequence identity, but all possess between one and four repeats of three highly conserved "fasciclin" domains that are believed to function as adhesive domains in cell-cell and cell-extracellular matrix interactions (Hu et ai, 1998). Members of this family have been identified in organisms as diverse as mycobacteria (Harboe and Nagai, 1984; Harboe et ai, 1995; Terasaka et ai, 1989), Volvox (Huber and Sumper. 1994), insects (Zinn et ai, 1988), sea urchins (Brennan and Robinson. 1994), and humans (Skonier et ai, 1992; Takeshita et ai, 1993). To further investigate the role played by s\in32 in sym- biotic interactions between cnidarian hosts and dinoflagel- late symbionts, we used immunocytochemical techniques to examine the distribution of the sym32 protein within the A. elegantissima-S. inuscatinei association. In this paper we demonstrate that sym32 protein is differentially distributed in symbiotic and aposymbiotic A. elegantissima both at the cellular and subcellular level. Of particular interest, sym32 antiserum labels the multiple layers of membrane that sur- round the symbiont within the host cell. We also show that anti-sym32 antiserum specifically labels the accumulation body of dinoflagellates residing within host gastroderm. Western blots of proteins from two Symbiodiniitin species revealed a protein doublet of 45 and 48 kDa (45/48 kDa) that cross reacts with the sym32 antiserum; but in immu- nolocalization studies, the antiserum was insufficiently spe- cific to detect the target protein in cultured specimens of S. bermudense. Possibly, both the host and symbiont produce fasciclin domain proteins that interact via the fasciclin ad- hesive domain. Materials and Methods Animal maintenance Symbiotic and aposymbiotic specimens of A. elegan- tissima were collected at low tide from the intertidal zone at Seal Rock, Oregon. Aposymbiotic anemones were taken from under rock overhangs or crevices where there was little or no light to support the growth of symbiotic dinoflagellates. Symbiotic anemones were taken from the open rock benches that are exposed to light. Anemones were transported to the laboratory, where they were maintained in an 11 °C recirculating seawater aquarium on a 12:12 h light:dark cycle. Anemones were fed previously frozen adult brine shrimp about once a week. Light-level immunocytochemistry Anemones. Tentacles from both symbiotic and aposym- biotic specimens of A. elegantissima were clipped and im- mediately transferred to tissue freezing medium (Triangle Biomedical Sciences) and frozen at -80 °C. Tentacles were cryosectioned at —20 °C on a Reichert-Jung cryostat (20-jum sections) and placed on polylysine slides, then immersed in 4% paraformaldehyde fixative in phosphate SYM32 LOCALIZATION IN ANTHOPLEURA 341 buffered saline (PBS: 10 mM phosphate buffer, pH 1.2. + 150 mM NaCl) for 1.5 h. Sections on slides were rinsed three times, 5 min each time, in PBS with 0.5% BSA; dehydrated in a methanol series (25%, 50%, 75%, 100%, 75%. 50%. 25%): and rinsed again in PBS/BSA. Sections were incubated in a blocking solution of 1:200 dilution of goat serum:PBS/BSA for 30 min at room temperature and then rinsed three times, 5 min each time, in PBS/BSA. Slides were incubated for 1 h in either a 1:2000 dilution of sym32 antiserum from rabbit (antibody development de- scribed in Reynolds <•/ at.. 2000) in PBS/BSA or in a 1 :2000 dilution of preimmune serum from the same rabbit. Sections were rinsed three times, 5 min each time, in PBS/BSA and then incubated for 1 h in a 1 :200 dilution of goat anti-rabbit IgG-5-nm colloidal gold conjugate (Ted Pella). Slides were rinsed as above. Gold particle labeling was silver-enhanced using a silver enhancement kit (Ted Pella). To stop color development, slides were washed in ePure water. Coverslips were affixed with a glycerol mount and sealed with finger- nail polish. Cultured Symbiodinium bermudense cells. Immunofluo- rescence was used to investigate whether the symbiont- produced 45/48 kDa cross-reactive protein (identified from Western blots, described below) could be localized to sym- bionts free from host cells. Cells of 5. bermudense (CCMP830) were grown in sterile filtered seawater enriched with f/2-Si media at about 100 p.E light on a cycle of 12 h light to 12 h dark at 25 °C. Cells were collected by centrif- ugation from liquid media and resuspended into 3% para- formaldehyde in PBS. After fixation for 30 min, cells were given two 10-min washes in PBS and transferred to PBS. Cells were incubated 30 min in blocking solution (PBS + 3% BSA): 30 min in PBS/BSA/0.2% Triton X-100: 1 h in preimmune serum or anti-sym32 antiserum diluted with PBS/BSA/Triton X to 1:50. 1:200. 1:2000: 5 min in PBS, repeated five times: I h in 1:200 Alexa Fluor 488 goat anti-rabbit IgG in PBS/BSA; and 5 min in PBS. repeated five times. After incubation, the cells were viewed under an Olympus BX-60 fluorescence microscope. EM-level immunocytochemistry Anemones. Immunocytochemistry was performed on three occasions using symbiotic anemones collected at dif- ferent times and on one aposymbiotic anemone. Tentacles from aposymbiotic and symbiotic anemones were clipped and immersed in 1% paraformaldehyde, 1% glutaraldehyde fixative in PBS for 1.5 h. Tentacles were rinsed three times, 10 min each time, in PBS and then dehydrated for 15 min in each concentration of a methanol (MeOH) series (15%, 30%, 50%, 85%. 95%. 100%, 100%). Tentacles were infil- trated with LR White resin on a rotating table in a series of MeOH dilutions (1:3 LR White:MeOH overnight, 1:1 over- night, 100% LR White for 3 h), and then placed in gelatin capsules in fresh LR White. LR White was allowed to polymerize at 52 °C for 2 days. Ultra-thin, gold-silver sections were cut with a diamond knife and placed onto Formvar-coated nickel grids. These grids were processed for immunocytochemistry as follows: they were immersed in blocking solution (PBS + 5% BSA) for 15 min, incubated in a 1:1000 dilution of sym32 anti- serum or a 1 : 1000 dilution of preimmune serum in PBS for 1.5 h, rinsed 3 times. 10 min each time, in PBS/BSA + 0.1% Tween 20, incubated in a 1:75 dilution of EM grade goat anti-rabbit lgG-15-nm colloidal gold (Ted Pella) in PBS for 1 h, rinsed as above, rinsed in ePure water for 5 min, and then allowed to dry. Grids were stained in 2% uranyl acetate for 5 min. rinsed by dipping in water 10 times each in three changes of water, then immediately stained in 0.4% lead acetate for 3 min, with water rinses as above, and then air dried. Between 5 and 10 grids of each type of anemone were viewed under 60 kV using a CM- 12 Phillips transmission electron microscope. Cultured Symbiodinium bermudense cells. Cells were collected as described in the light microscopy section, fixed as described for anemone tentacles, and processed essen- tially as described for anemone tentacles. Several dilutions of anti-sym32 antiserum and preimmune serum were tested (1:10, 1:50, 1:200. 1:500, 1:1000, 1:2000), as well as vari- ous dilutions of either Tween-20 or Triton X (0%, 0.1%. 1%) in the following solutions: blocking solution, primary antibody solution, wash solution. Preparation of anemone and dinoflagellate proteins: one- and nvo-dimensional SDS-PAGE and Western analysis Proteins were isolated from the host as follows: a host anemone was removed from an 1 1 °C recirculating aquar- ium and flash-frozen in liquid nitrogen. The anemone was minced with a razor blade, and placed into a glass grinder with a Teflon pestle driven by a hand drill in four volumes ice-cold grinding buffer (100 mM Tris. 100 mM NaCl. 10 mM EDTA) with protease inhibitors (Sigma: 5 /id per 10 ml buffer). The homogenate was placed into a centrifuge tube and the grinder was rinsed with two volumes of buffer (v:w anemone tissue), which was added to the tube and mixed well. The remaining homogenate was centrifuged at 16,000 X g for 10 min at 4 °C to remove algal cells and host cell debris and membranes from the homogenized anemone tissue. The supernatant was removed to a new tube and centrifuged again. Protein concentration was determined on this cleared homogenate using the Bradford assay (Pierce Coomassie reagent). Host proteins prepared according to this protocol are free from contamination by symbiont pro- teins (Weis and Levine. 1996). Proteins were isolated from symbionts that had been either continuously maintained in culture with no host con- tact for many generations or freshly isolated from an 342 J. A. SCHWARZ AND V. M. WEIS A. elegantissim,: !X>M. Many species of Symbiodinium can be isolated l^ir hosts and brought into culture in nutrient-si]."': ^ U seawater. These symbionts are there- fore t . •-!'. any host cell contact. We obtained frozen pellete.i \ :nbionts from cultures of S. benmtdense, which was oi igmally isolated from the tropical sea anemone Aipta- siti/>iillida. A chunk of the pelleted symbionts (about 100 /xl in volume) was briefly ground in a glass tissue grinder with a Teflon pestle in an equal volume of grinding buffer with protease inhibitor cocktail. Examination under a light mi- croscope revealed that nearly all symbionts were still intact after this step. An equal volume of acid-rinsed glass beads (Sigma: 425-600 /j,m) was added and the mixture alter- nately vortexed for 15-30 s and placed on ice for about 30 s, for a total of 20 times. With repeated vortexing. the homog- enate became intensely orange, likely indicating the release of the major water-soluble accessory pigment, peridinin- chlorophyll protein. By this means, at least 75% of symbi- onts were broken open, as determined by light microscopy. With a syringe and a 24-gauge needle, the homogenate was removed from the glass beads and centrifuged at 16,000 X g for 10 min at 4 °C to remove cellular debris. The superna- tant was placed into a new tube and again centrifuged. This cleared supernatant fraction was then assayed for protein concentration, as described above, and then prepared for one-dimensional or two-dimensional SDS-PAGE, as de- scribed below. Proteins from freshly isolated symbionts were prepared by isolating symbionts from a host and then extracting proteins using glass beads to fracture the symbiont cell wall and release the contents of the cytoplasm. A host anemone weighing about 2 g was removed from an 1 1 °C recirculat- ing aquarium and flash-frozen in liquid nitrogen. The anem- one was minced with a razor blade and placed into a glass grinder with 3 ml of grinding buffer plus protease inhibitor. All subsequent steps were performed on ice. The anemone was ground with a Teflon pestle instead of a ground glass pestle to homogenize the animal tissues without shearing the cell walls of the symbionts. This homogenate was cen- trifuged at 2000 x g for 10 min to pellet the symbionts. The pellet, about 300 /xl in volume, was partially cleaned of anemone debris by regrinding the pellet in grinding buffer using a glass tissue grinder with a Teflon pestle (this re- grinding step was sufficient to break up the pellet, but not break open the symbionts) and reconcentrating the symbi- onts by centrifugation. The partially cleaned pellet was ground a final time before adding 500 jul of buffer with protease inhibitor cocktail and an equal volume of prerinsed glass beads. This resuspended pellet contained a significant amount of host tissue, as revealed by the presence of nu- merous nematocysts. We then followed the same vortexing and centrifugation protocol as described above. The cleared supernatant fraction (intensely orange in color) was used for one-dimensional SDS-F AGE, as described below. One-dimensional SDS-PAGE with 10% Nu-PAGE Bis- Tris gels (Invitrogen) was performed on proteins from host tissue and from freshly isolated, and cultured symbiont proteins. Samples were denatured and prepared for electro- phoresis using LDS buffer + DTT (Invitrogen) according to manufacturer's instructions; 10 /u,g of protein was loaded for each sample. Electrophoresis was performed in MOPS buffer according to the manufacturer's instructions. Gels were transferred to 12.5 mM Tris, 100 mM glycine, 10% MeOH for 20 min, and proteins were electrophoretically transferred onto nitrocellulose membrane for 1.25 h at 100 V in a BioRad chamber. Two-dimensional SDS-PAGE was performed using pro- teins extracted from freshly isolated symbionts. Proteins were extracted as described above, except that no NaCl was used in the buffer, as salt interferes with isoelectric focus- ing. The SDS-PAGE was carried out on a Multiphor II system (Amersham Pharmacia), according to the manufac- turer's instructions and as described in Reynolds et al. (2000). Thirty microliters of symbiont homogenate contain- ing 60 /jig of protein was used for isoelectric focusing on an 180-mm IPG strip. pH 3-10. After isoelectric focusing, the IPG strip was equilibrated and placed on a 12% ExcelGel; electrophoresis was performed according to manufacturer's instructions. After electrophoresis, the gel was placed into transfer buffer (50 mM Tris. 40 mM glycine, 0.04% SDS, 20% methanol) for 20 min. The gel was removed from the plastic backing, and proteins were transferred to a nitrocel- lulose membrane under a discontinuous buffer system (Mul- tiphor II system from Amersham). For Western analysis, the membranes from one- and two-dimensional SDS-PAGE were incubated at 4 °C over- night in blocking buffer (TBS: 20 mM Tris. 500 mM NaCl, Ph 7.5. + 5% powdered milk + 0.1% Tween-20). The following morning, the membrane was washed for 1 5 min in TBS + 0.5% Tween-20 (TBST); incubated for 45 min in a 1:1500 dilution of anti-sym32 antiserum:block buffer; rinsed 10 min each in TBS. TBST, TBS; incubated 45 min in a 1:5000 dilution of HRP-antirabbit IgG (Amersham Pharmacia); and washed as before. Sym32 protein was detected by chemiluminescence using ECL detection re- agents (Amersham Pharmacia) and exposing membranes to film for 1 min. Results Microscopy of anemone tentacles Cryosectioned tentacles. Cryosectioned tentacles of apo- symbiotic and symbiotic anemones were incubated with ( 1 ) preimmune serum as a negative control for endogenous staining or (2) sym32 antiserum. Staining for sym32 in aposymbiotic tentacles was distinct from that in symbiotic tentacles, relative to preimmune controls (Fig. 1). In both symbiotic and aposymbiotic tentacles, preimmune controls ep m ga SYM32 LOCALIZATION IN ANTHOPLEURA , 343 B ep m ga Figure 1. Light micrographs showing immunolocalization of sym32 protein within cryosections of tentacles from aposymbiotic (A and B) and symbiotic (C and D) Antlwpleura elegantissima. The spherical symbionts can be clearly seen within the gastroderm of symbiotic tentacles. Sections were incubated in either preimmune serum (A andC)orsym32 anti-serum (B and D). and sym32 was visualized using silver enhancement of colloidal gold labeling. Sym32 levels are high in the gastrodermis of symbiotic anemones, low in the gastrodermis of aposymbiotic anemones, faint in the epidermis of both types of anemones, and absent in the mesoglea of both types of anemones. Host tissue layers are marked as ep = epidermis, ga = gastrodermis. m = mesoglea. Scale bar = 0.3 mm for all panels. showed light brown staining in epidermal and gastrodernial tissues, and no staining in the mesoglea. In aposymbiotic tentacles incubated in sym32 antiserum. staining in the epidermal and gastrodermal layers was slightly darker than in the preimmune controls. In symbiotic tentacles, staining in the epidermis was also slightly darker than in the preim- mune controls, and in the gastrodermis, where dinoflagel- lates are housed, it was significantly darker. The mesogleal layer of both aposymbiotic and symbiotic tentacles re- mained unstained. Electron micniscopv. To further examine the location of sym32 within the host-symbiont association, we performed EM-level colloidal gold immunocytochemistry using sym32 antiserum to label the sym32 protein within thin sections of resin-embedded tentacles from one aposymbiotic and three symbiotic anemones (Figs. 2-4). We examined between 15 and 25 sections from each anemone. We also performed negative controls with preimmune serum to check for non- specific labeling of the tissues. In all cases, the preimmune controls were almost completely free of gold-sphere label- ing (data not shown). In aposymbiotic tentacles. sym32 gold-sphere labeling was associated exclusively with medium-density vesicles located within both epidermal and gastrodermal cells (Fig. 2). There was no evidence of any sym32 label within the mesogleal layer, and the pattern of distribution of the sym32-containing vesicles in the epidermal cells was distinct from that in the gastrodermis. The vesicles were relatively uncommon in the epidermis (Fig. 2 A, B) but more abundant in the gastrodermis. where they were concentrated along the apical end of the gastroderm, near the interface between the gastroderm and the gastric cavity (Fig. 2C. D). In symbiotic tentacles, the pattern of distribution in the epidermis was the same as in aposymbiotic tentacles; sym32 gold-sphere labeling was contained within vesi- cles that were relatively sparsely distributed, most com- monly occurring near nematocysts. In contrast, the dis- tribution of sym32 within the gastrodermis was dramatically different. The sym32-containing vesicles that were so abundant in aposymbiotic gastroderm were not present in symbiotic gastroderm. Instead, the sym32 344 J. A. SCHWARZ AND V. M. WEIS Figure 2. Transmission electron micrographs of iramunogold-labeled sections from tentacles of aposym- biotic anemones. Gold spheres, \isible as black dots, indicate the presence of s\m?2. lA) Epidermal cells with nematocysts (n). (B) Enlargement of the boxed section of A. with gold spheres labeling \esicles located near nematocysts. (C) Gastrodermal cells adjacent to the gastric cavity (go. (D) Enlargement of the boxed section of C. showing gold spheres within vesicles in the gastrodermal cells. Scale bars = 2 p.m (A. C) and 1 ^m (B. D). label was associated with the multiple membranes that enclose the dinoflagellate sxmbiont within the host cell (Fig. 3A, B). Gold spheres were diffusely arranged within these membranes, not clearly associated with any single membrane layer. To confirm that this labeling was specific tn :he membranous lasers, we quantified the staining rela \ .• to areas outside the membranes. The membranous l.i contained an average ot 12.2 gold spheres ± 4.65 uiiti.\siitui. I A) Illustrates the presence of dinoflagellates within host gastrodermal cells. (Bi Enlargement of boxed area in A. showing gold spheres associated with the multiple layers of membrane that surround the dinoflagellates. The arrows delineate the margins of the multiple membranes surrounding the dinofiagellate. Preimmune controls showed virtually no gold-sphere labeling (not shown), c == symbiont chloroplast. cw = s\rnbiont cell wall, g = gastrodermal cell of the host, s = dinoflagellate symbiont. Scale bar = 2 .m. Western Nuts The presence of gold-sphere label within the accumu- lation bodies of the symbionts suggested that the symbi- onts were producing a sym32 homolog. We performed Western analysis using anti-sym32 antiserum to look for cross-reactive proteins in homogenates from symbionts freshly removed from a host anemone and from cultured symbionts that were not in contact with host cells. Anti- sym32 Western blots of one-dimensional gels from ho- mogenates of freshly isolated Symbiodinium nniscatinei revealed three bands (Fig. 5A. lane 2). The 32-kDa band, identical in size to a band in host-only homogenate (lane 1). is probably due to contamination from host sym32. This was expected, as protein preparations of the symbi- onts are invariably contaminated by host proteins (Weis et ill., 1998). A 48-kDa band and a faint 45-kDa band below it suggest the presence of cross-reactive proteins that are produced by the symbionts. Homogenates of cultured algae (Symbiodinium bermudense) that were never in contact with host tissues (lane 3) also contained the same 45/48-kDa doublet but lacked the 32-kDa host band. Western blots of two-dimensional gels of freshly isolated 5. muscat inei homogenates revealed two spots with distinctly different molecular weights and isoelec- tric points (pi) (Fig. 5B). A cross-reactive spot at 32 kDa. 8.2 pi. corresponds exactly with host sym32 (Reynolds el al.. 2000), and again is probably due to host contamina- tion. In addition there was a 48-kDa spot with a pi range of 4.3 to 4.5. The 45/48-kDa protein doublet that is present in cultured S. bermuilense and in S. muscatinei freshly harvested from a host, and is also faintly visible in the host lane, therefore represents a protein produced by S\mhi<>dininm both when it is in symbiosis with a host, and when it is free-living. Microscopy of cultured Symbiodinium bermudense cells We were interested in determining the location of the symbiont-produced 45/48-kDa protein doublet. We there- fore used the anti-sym32 antiserum (which was devel- oped against recombinant host sym32 protein), to local- ize the target protein in cultured specimens of S. bermudense. We used two immunolocalization methods. For intact cells, we used immunofluorescence, in which a fluorescent secondary antibody is detected by fluores- cence microscopy. For sectioned cells, immunoelectron microscopy allowed us to detect any patterns in staining 346 J. A. SCHWARZ AND V. M. WEIS CW -. Figure 4. Transmission electron micrographs of immunogold -labeled sections ot Jinoflagellalc symhionts contained within host gastrodermal cells. (A) Dinoflagellate contained within a host gastrodermal cell. (B) Enlargement of the accumulation body, shown boxed in A, illustrating sparse gold labeling of the dinoflagellate accumulation body. (C) Region around the accumulation body of another symbiont (section not counterstained) showing intense labeling specific to the accumulation body. The cell wall (cw) and membrane layers (m) are visible as concentric gray rings around the symbiont. Sections incubated with preimmune serum showed virtually no gold-sphere labeling (data not shown), m = membranes surrounding the dinoflagellate, cw = dinoflagellate cell wall, c = dinoflagellate chloroplast, ab = accumulation body of the dinoflagellate. Scale bar = 2.5 /urn (A). 1.0 /iin (B, C). at the subcellular level. Despite testing many concentra- tions of antiserum and buffer solutions, we were unable to detect a pattern of staining of the target protein in cultured cells of 5. hcrmudcnse. With the immunofluo- rescence method, preimmune controls looked identical to antiseruiii incubated specimens at all dilutions of serum that we i sted. With immunoelectron microscopy, we could deu > specific staining to any particular com- partment wi!1;.' the cell, even though the background staining varied Imost no gold-sphere labeling at the 1:2000 dilutions h heavy labeling at the 1:10 dilutions. In the preimmune controls, labeling was restricted to a few, widely scattered spheres. Discussion The sym32 protein is distributed among different subcel- lular compartments in symbiotic and aposymbiotic anemo- nes. Most notably, the sym32-containing vesicles that are so abundant in the gastroderm of aposymbiotic hosts are absent from symbiotic hosts; instead, sym32 localizes to the sym- biosome membranes. This suggests that the internalization of symbionts is accompanied by a transfer of sym32 from gastrodermal vesicles to the symbiosome membranes. This would most likely occur during phagocytic uptake of the symbiont. by fusion of the sym32 vesicles with the phago- some. The presence of sym32 within the dinoflagellates them- SYM32 LOCALIZATION IN ANTHOPLEURA 347 _43 kDa _30 kDa 1 2 3 B 4.5 8.3 pi - 67 kDa -43 -30 -20 Figure 5. Ami-sym32 Western blots of protein homogenates from Symbiodinium. (A) Western blot of a one-dimensional gel. All lanes contained ]() /Mg of soluble protein. Lane I. host-only proteins, shows a 32-kDa band and a faint 48-kDa band. Lane 2. Svmbiot/iniiim nniscatinei freshly harvested from a host anemone (and therefore contaminated with host tissues), shows three bands: a strongly staining 32-kDa band, a strongly staining 48-kDa band, and a faint 45-kDa band. Lane 3. cultured Symhiiuliniiim hcrimuli'iisi: contains two equal intensity bands at 45-kDa and 48-kDa. (B) Western blot of a two-dimensional gel of protein homogenate (60/ng) from S. nniscatinei freshly harvested from an anemone host. Two spots are present: a 32-kDa. pi 8.2, spot, identifiable as sym32, presumably from contaminating host tissue, and a 48-kDa spot, pi range 4.3-4.5. selves complicates the picture and adds a new dimension to our studies of sym32. The accumulation body in dinoflagel- lates is postulated to function as a lysosome. although this organelle has not been studied in many species, and its function has not been examined in Symbiodinium. The free- living dinoflagellate Prorocentrum has multiple accumula- tion bodies with features characteristic of eukaryotic lyso- somes. These bodies contain electron-dense material, fibrous material, and membranous material, and they pos- sess acid phosphatase activity, react positively with the periodic acid/Schiff reagent, and stain with acridine orange (Zhou and Fritz. 1 994). Symbiodinium has a single accumu- lation body that varies in size and is postulated to function as a molecular "trash dump" (Taylor. 1987; Wakefield el ai, 2000). We observed that the accumulation body is invari- ably located adjacent to the nucleus, often appearing to displace the edge of the nucleus. If the accumulation body is a lysosome or a trash dump, host sym32 may be trans- ported from the vacuolar membranes, across the dinoflagel- late cell wall, and into a degradative pathway within the dinoflagellate. This ability to transport molecules from the host cell, across the vacuolar membrane, into the cytosol or organelles of an intracellular inhabitant is common in par- asitic protozoans, and there are many mechanisms by which this occurs (Schwab ct o, K. Tezuka, and E. Amann. 1993. OSF-2: cloning and chara i .ii'ini of a novel protein expressed in bone with sequence ' h insect protein fasciclin I. Biochem J. 294: 271-27? Tamur . vtiaclii, J. Osuga, K. Ohashi, N. Yahagi, M. Sekiya, H. Ok •!/:!•.. S. lomita, V. lizuka, H. Shimano, R. Nagai, S. Kimura, M. 'isujimoto, and S. Ishibashi. 2003. FEEL-1 and FEEL-2 are endocvtic receptors tor advanced glycation end products. J. Biol. Chem. 278(15): 12613-12617. Taylor, D. L. 1968. In situ studies of the cytochemistry and ultrastruc- ture of a symbiotic marine dinoflagellate. J. Mar. Biol. Assoc. UK 48: 349-366. Taylor, F. J. R., ed. 1987. The Biology of Dinoflagellates. Botanical Monographs. Vol. 21. Blackwell Scientific Publications. Oxford. Terasaka, K., R. Yamaguchi, K. Matsuo, A. Yamazaki, S. Nagai, and T. Yamada. 1989. Complete nucleotide sequence of immunogenic protein MPB70 from Mycubacterium bovis BCG. FEMS Microhiol. Lett. 49: 273-276. \\akefield, T. S., and S. C. Kempf. 2001. Development of host- and symbiont-specific monoclonal antibodies and confirmation of the origin of the symbiosome membrane in a cnidarian-dinorlagellate symbiosis. Biol. Bull. 200: 127-143. Wakefield, T. S., M. A. Farmer, and S. C. Kempf. 2000. Revised description of the fine structure of in situ "Zooxanthellae" genus Symbiodinium. Biol. Bull. 199: 76-84. Wang, J. T., and A. E. Douglas. 1998. Nitrogen recycling or nitrogen conservation in an alga-invertebrate symbiosis'? J. E.\p. Biol. 201: 2443-2453. Wang, J. T., and A. E. Douglas. 1999. Essential amino acid synthesis and nitrogen recycling in an alga-invertebrate symbiosis. Mar. Biol. 135: 219-222. Weis, V. M. 1993. The effect of dissolved inorganic carbon on the photosynthesis of the symbiotic sea anemone Aiptasia pulchella: role of carbonic anhydrase. J. Exp. Mar. Biol. Ecol. 174: 209-225. Weis, V. M., and R. P. Levine. 1996. Differential protein profiles reflect the different lifestyles of symbiotic and aposymbiotic Antliopleura elegantissima, a sea anemone from temperate waters. J. Exp. Biol. 199: 883-892. Weis, V. M., and W. S. Reynolds. 1999. Carbonic anhydrase expression and synthesis in the sea anemone Anthopleura elegantissima are en- hanced by the presence of dinoflagellate symbionts. Physiol. Biochem. Zool. 72: 307-316. Weis, V. M., J. von Kampen, and R. P. Levine. 1998. Techniques for exploring symbiosis-specific gene expression in cnidarian-algal asso- ciations. Pp 435-448 in Some Molecular Approaches to the Study of the Ocean. K. E. Cooksey. ed. Chapman and Hall, London. Zhou, J., and L. Fritz. 1994. The PAS/accumulation bodies in Proro- cciitnini lima and Pi'oroct'iitntm niticiilosum (Dinophyceae) are dinoflagellate lysosomes. J. Phycol. 30: 39-44. Zinn, K., L. McAllister, and C. S. Goodman. 1988. Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila. Cell 53: 577-587. Reference: Biol. Bull. 205: 351-366. (December 20(Ml (0 2003 Marine Biological Laboratory Columellar Muscle of Neogastropods: Muscle Attachment and the Function of Columellar Folds REBECCA M. PRICE Department of Geophysical Sciences, University of Chicago, 5734 S. Ellis Ave., Chicago, Illinois 60637 Abstract. Malacologists often assume that ornamentation on snail shells is functional, and therefore adaptive. I con- ducted the first comprehensive test of the widely accepted hypothesis that columellar folds, a type of internal orna- mentation, enhance the performance of the columellar mus- cle, which attaches the snail to its shell. Careful dissections of live, non-relaxed specimens reveal that the physical at- tachment between the columellar muscle and the columella is not restricted to a small, circular patch located deep within the shell. Instead, the attachment is long and narrow, extending approximately a full whorl along the length of the columella. I developed a novel technique for preparing three-dimensional reconstructions from photographs docu- menting the dissections. These reconstructions were then used to measure four parameters that describe the muscle: ( 1 ) the surface area of the physical attachment between the muscle and columella. (2) the total contact area between the muscle and the columella, (3 1 the depth of attachment, and (4) the length of attachment. None of these parameters differed significantly between species with and without folds. In light of the biomechanics of muscular hydrostats, values of the first parameter indicate that columellar folds probably do not guide the columellar muscle as the animal moves in and out of its shell. Values of the other parameters indicate that columellar folds neither increase an animal's ability to maneuver its shell nor facilitate deeper with- drawal. These results, and the fact that folds have evolved convergently several times, might indicate that folds are an easily evolvable solution to many functional problems, none of which are currently understood. Received 22 October 2002; accepted 29 August 2003. E-mail: rmprice@uchicago.edu Introduction Malacologists often assume that gastropod shell orna- mentation is adaptive, but experiments that demonstrate the function of these presumed adaptations are rare (Morton. 1967; for notable tests of ornamentation function, see Palmer, 1977. 1979; Bertness and Cunningham, 1981; Appleton and Palmer, 1988; Marko and Palmer. 1991; West et a I., 1991; Carefoot and Donovan, 1995; Donovan el al.. 1999). Columellar folds, plications on the columella. or central column of a gastropod shell (see Fig. 1 ). are partic- ularly intriguing ornaments because they evolved repeatedly in a number of clades, such as the Caenogatropoda. Opis- thobranchia. and Pulmonata (Price, 2001). In the subclade Neogastropoda of the caenogastropods, the columellar folds may be an adaptation that is intimately related to the colu- mellar muscle (Fig. 1). In fact, the muscle does attach the animal to the columella (Signer and Kat, 1984; Fretter and Graham, 1994) and has grooves that fit between each fold. In this paper, I test three hypotheses that purport to explain a functional relationship between the columellar folds and the columellar muscle. Columellar folds are readily visible on the inner lip of many shells, and systematists have used the impressive diversity of fold shapes to distinguish species and higher taxa. I consider a fold to be any ridge on the inner lip of the aperture that extends along the columella for a number of whorls, usually extending all the way to the apex. Some folds occur at the bottom edge of the aperture, as in Nas- sarius vibex, whereas others, such as those in Terebru dislocata, are located in the center of the inner lip. Folds can be wide (Triplofiisus giganteus) or narrow (Nassarius vibex), subtle (Busycon contrarium) or prominent (Vasum muricatum). The columellar muscle conforms exactly to the shape of the folds where it lies over them, and this conformation has inspired the hypotheses that are most commonly cited to 351 352 R. M. PRICE explain the fimction of folds. These hypotheses must be considered in li;:h, of the fact that the columellar muscle functions a .1 muscular hydrostat (Thompson ct al, 1998), controlling protraction from, and retraction into, the shell. Like hydrostatic skeletons in general, the volume of a mus- cular hydrostat remains constant, so a contraction in one direction induces elongation in an opposing direction (Kier and Smith, 1985). The columellar muscle is composed of muscle fibers that are oriented longitudinally, transversely, and obliquely with respect to the long axis of the columellar muscle (Thompson el al., 1998). Thus, the columellar mus- cle controls its own twisting, shortening, and elongating in addition to protraction and retraction. Dall (1894; restated by Fretter and Graham, 1994) pub- lished the only nonfunctional explanation of folds. His explanation does not pertain to the columellar muscle, re- lying instead on the nature of the mantle, the tissue that secretes the shell. Dall surmised that folds, and all other internal ornamentation including the parietal ridge, lirae, and teeth on the outer lip, were deposited in the wrinkles that would form when an overly large mantle retracted into the shell. This idea predicts that ornamentation would be more pronounced at the aperture (Dall, 1894), but ornamen- tation around the aperture where the mantle is largest is exaggerated only in some species with determinate growth (Paul, 1991; Vermeij and Signer, 1992; Vermeij. 2001a); furthermore, contrary to Dall's prediction, not all of these animals have columellar folds. Indeed, the internal mor- phology of gastropods is highly stereotyped, not random, and it is unlikely that the mantle would always wrinkle uniformly (Signer and Kat. 1984). There are also no obvi- ous differences in mantle size between species with and without internal ornamentation in general, or columellar folds in particular (pers. obs.). Three interrelated hypotheses have been presented to explain how the folds affect the function of the columellar muscle, thereby explaining both why folds have evolved and why so many neogastropod lineages maintain them: 1 . Guidance. Columellar folds guide the columellar mus- cle as the animal moves in and out of its shell (Signor and Kat, 1984; Ponder, 1972). The analogy here is that the folds act like a railroad track guiding a train; that is, they prevent the animal from slipping in its shell. One manner in which the folds may guide the muscle is by protruding far into the columellar muscle, re- stricting the muscle movement along the folds. In this event, the area of contact should be greater in species with folds than in those without. I test this prediction. Signor. and Kat (1984) limited their guidance hy- pothesis to high-spired, narrow (turritelliform) spe- cies, implying that the columellar muscle would slip within the shell unless folds guided its movement. Any attempt to extend this hypothesis to low-spired shell shapes must account for the absence of folds in some large neogastropods, such as S\rinx ama- niix. whose shell can reach almost a meter in length (Harasewych and Petit, 1989). 2. Maneuverability,'. Columellar folds enhance a snail's ability to maneuver its shell (Fretter and Graham, 1962; Vermeij, 1978). A number of authors have assumed that the shape of the muscle's physical at- tachment to the columella is small and circular, like the adductor muscle attachment in bivalves (e.g.. Lin- sley. 1978; Signor and Kat. 1984; Morita, 1993; Thompson et al.. 1998). Vermeij (1978) assumed that the attachment occurs immediately over the folds and predicted that animals with folds would consequently have a larger surface area of muscle attachment. An animal with greater attachment area might be better at maneuvering its shell, for example when it swings the shell back and forth to fend off a predator (Thompson et al., 1998), or when it pries open the shells of prey (Taylor et al., 1980). This hypothesis has remained untested because dissections usually begin by remov- ing and discarding the shell, and because the muscle easily detaches from a cracked shell. 3. Predator avoidance. Snails with columellar folds can withdraw more deeply into their shells (Dall, 1894), increasing their ability to escape from predators. Dall (1894) commented that snails with columellar folds retract more deeply, but presented no data in support of this idea. Since then, others have documented that snails that retract more deeply are better at escaping predators: they are harder to reach, and they take so long to handle that the predator eventually abandons the task (Vermeij, 1978. 1987). Although it is not immediately obvious why these behaviors might be associated with columellar folds, the data required to confirm Dall's claim are easy to collect. I have developed procedures for dissecting gastropods while keeping the columellar muscle intact; employed a novel mathematical algorithm that converts a photograph of a snail into a three-dimensional surface from which I can measure areas; and examined neogastropod species that represent a range of columellar fold morphologies, as well as species with smooth columellae. Contrary to what has been assumed, the muscle attachment to the columella is complex and leaves no scar. None of the three hypotheses outlined above adequately explain the functional relation- ship between the columellar folds and the columellar mus- cle. Materials and Methods Sample Quantitative data on the columellar muscle were col- lected from seven species with folds (from five genera and FUNCTION OF COLUMELLAR FOLDS 353 tour families) and five species lacking them (from five genera and three families), affording phylogenetic breadth (Table 1). One of the species without folds. Strombus ala- tus, is not a neogastropod; it is a caenogastropod (a clade that contains the neogastropods) with shell shape similar to other species considered here. At least two and up to five specimens were studied for each species, for a total of 31 specimens for most measurements (36 specimens for attach- ment depth). These data were supplemented with qualitative observations from an additional nine species. All specimens were stored in 70% ethanol and deposited at the Field Museum of Natural History (FMNH). Chicago, Illinois. Except for Oliva xtiyana. all species have a functional operculum, eliminating any possible bias introduced by a relationship between folds and an operculum. The sample includes species with a variety of columellar fold shapes and numbers (Table 2), but all have fairly modest folds. Prominent folds, such as those in the Mitridae. Volutidae, and Cancellariidae could not be included, be- cause they were too difficult to collect or purchase alive. Oliva sayana has plications on the inner lip of the aperture that some authors describe as columellar folds (e.g.. Abbott, 1974), but these do not continue inside the shell, and they therefore do not contact the columellar muscle, even when the animal is fully protracted. As such, these apertural plications cannot affect the function of the columellar mus- cle, so I consider Oliva sayana to be without folds. Terminology describing orientation A description of fold morphology requires terms that orient the reader to the snail shell (Fig. 1 ). In this paper, all terms refer to a snail shell in a standard orientation, with apex up and aperture visible (and on the right for dextral species). Top and bottom refer to positions along the coiling axis. The bottom of a whorl is farthest from the apex, whereas the top of the whorl is closest to the apex. The width of a feature is measured along a line parallel to the top-bottom axis, following the convention that the short axis of the columellar muscle represents width, whereas the long axis of the columellar muscle represents length (Fig. 1A, E, Fig. 2E). Apical and apertural refer to directions along the Table 1 Species examined Species Collection site* Family Folds Museum ID Data type Busycon contrarium (Conrad. 1840) FS Melongenidae Yes Busycon spiratum (Lamarck. 1816) FS Melongenidae Yes Fasciolaria hunteria (Perry. 1811) FS. WFS, SM Fasciolariidae Yes Fasciolaria tulipa (Linnaeus, 1758) HT Fasciolariidae Yes Nassarius vibex (Say. 1822) FS, WFS Nassariidae Yes Triplofusus gigante us ( Kiener. 1840) HT Fasciolariidae Yes Camharus cancellarius (Conrad. 1846)t DIR Muricidae Yes Chiciiri'us florifer distans (Adams, 1855) WFS Muricidae No Melongena corona (Gmelin, 1791) FS, WFS Melongenidae No Sirainniiita haemaxtoma (Linnaeus, 1767) WFS Muricidae No Strombus alalus (Gmelin, 1791) P Strombidae No Urosalpinx perrugala (Conrad. 1846) WFS. SM. ST Muricidae No Columbella ritsticoides (Heilprin, 1886) DIR Columbellidae Yes Leucozonia nassa (Gmelin, 1791) S Fasciolariidae Yes Opealostoma pseudcdon (Burrow, 1815) IV Fasciolariidae Yes Pleurnpltica salmo (Wood. 1928) B Fasciolariidae Yes Terebra dislocata (Say, 1822) HT Terebridae Yes Vusum muricatum (von Born. 1778) G Vasidae Yes Cypruea < en-Hi (Linnaeus. 1771 ) G Cypraeidae No Oliva sayana (Ravenel. 1834) SM. HT Olividae No Pnlysrira nobilis (Hinds. 1843) GC Turridae No 299449, 299459 Quantitative 299444, 299463, 299468, 299472 Quantitative 299450, 299492. 299493 Quantitative 299448. 299456 Quantitative 299475. 299477 Quantitative 299441, 299442 Quantitative 299486, 299487 Quantitative 299451, 299458 Quantitative 299453, 299457 Quantitative 299452, 299454. 301941 Quantitative 301942, 301943 Quantitative 299481. 299482. 299483, Quantitative 299484, 299485 299473. 299474 Qualitative 299506, 299520, 299521, Qualitative 299522, 299526. 299527, 299530, 299531 299496 Qualitative 299494 Qualitative 299488, 299489. 299490. 299491 Qualitative 299500 Qualitative 299495 Qualitative 299460, 299461, 299466. 299469 Qualitative! 299497, 299499 Qualitative * Collection sites: B, Bique. Panama (Pacific Ocean); DIR. Dog Island Reef. Florida (Gulf of Mexico); FS. Florida State University Marine Lab, Turkey Bayou. Florida (Gulf of Mexico); G, Galeta. Panama (Caribbean Sea); GC, Golfo de Chiriqui, Panama (Pacific Ocean); HT. Hammock Trail, near St. Joseph Bay. Gulf County, Florida (Gulf of Mexico); IV. Isla Venado near Playa Veracruz. Panama (Pacific Ocean); P. Purchased from Gulf Specimen Aquarium and Marine Biological Supply, Panacea. Florida; S. Sebastian. Florida (Indian River County); SM. St. Mark's Wildlife Refuge, Florida (Gulf of Mexico); ST. Beach at St. Teresa. Florida (Gulf of Mexico); WFS. West side of Florida State University Marine Lab on oyster bar. t Tentatively placed in Soteneistra by Vermeij (20()lb). f Only the attachment depth was measured. 354 R. M. PRICE Table 2 fold inorphi i/i . , \iimined Sn Fold morphology inirium Busycon spiratum Ctintharus cancellarius Columbella rusticoides Fasciolaria himteria Fasciolaria tu/ipa Leucozonia nassa Nassarius vibex Opeatostoma pseudodon P/europloca salmo Terebra dislocala Triplofusus giganteus Vasum nniricatiim I subtle fold at the bottom of the whorl, distinguished by a groove and angled obliquely; difficult to observe at aperture, but more prominent in older whorls As in B. contrariuiii. but slightly more defined 1 fold at the bottom of the whorl, angled obliquely; broader than in other species 1 fold immediately at the bottom of the whorl 2 folds at the bottom of the whorl, angled obliquely; the lower edge of the bottom fold coincides with the edge of the columella As in F. himteria, but with 3 folds 3 folds, angled obliquely and closely spaced 1 well-defined fold at the bottom of the whorl, angled obliquely; square, rather than rounded, profile As in L. nasssa As in F. tulipa 2 large, broad folds spread throughout whorl; the bottom fold is more prominent than the top one As in F. tulipa. but top fold is more subtle than the others 5 folds in "1 prominent — 1 weak — 1 prominent — 1 weak — 1 prominent" pattern; folds angled more perpendicular to coiling axis than in any of the other species and spread throughout the bottom two-thirds of the whorl spiral of the shell towards the tip or opening respectively: the columellur muscle narrows apically, but is wide aper- turally. A junction occurs where the bottom of one whorl meets the top of another. The depth of a feature indicates the number of revolutions between it and the aperture, where one revolution is 360°; for example, the columellar muscle attachment might begin at a depth of 300°. Dissections Dissections were performed on live, untreated animals. Surprisingly, any treatment of animals, such as freezing, storing in ethanol, or relaxing in magnesium sulfate (Epsom salts), caused the muscle to detach from the shell, even when tugged only slightly. Fresh material is therefore es- sential to study columellar muscle attachment. To expose the soft tissues while keeping them and the columella intact, I used a Dremel rotary tool with a 1.6-mm-diame'ter carborundum abrasive wheel to cut away the exterior. The size of the blade limited the specimens that could be dissected with precision to those with whorls larger than half a centimeter. Thus, the attachment morphology could be quantified in the largest specimens of Nassarius vibex, but the available speci- mens of Columbella rusticoides were too small. The largest whorl of Terebra dislocata, a high-spired species, is only a few millimeters tall, so it could not be quantified with these methods. Although the most apertural point of the attachment was documented in Oliva sayana. the morphology of attachment throughout the rest of its length was inaccessible, because the delicate columella cannot withstand even slight pressure. To distinguish between muscle that was physically at- tached and muscle that was simply pressed against the columella, I used a blunt but flexible 34-gauge copper wire to probe between the tissue and columella. In all cases, only the top of the muscle (that closest to the junction between two whorls; see Figs. 1,2) was physically attached. Since the bottom of the muscle was free, the attachment was not disturbed when probed. Measurements The measurements required to test the hypotheses (Table 3) were the standardized (see below) total area of muscle physically attached to the columella, the standardized total area of muscle in contact with the columella throughout the attachment, the depth of attachment, and the length of the attachment. It was especially difficult to measure the total area of muscle physically attached to the columella (ATT in Figs. 1, 2). In these species, the columellar muscle attachment does not scar the shell, so the attachment area had to be observed directly. I tried removing strips of columellar muscle that were not attached to determine how much muscle was left on the columella, but this procedure inevitably destroyed the attachment. However, the attachment was so narrow relative to the total width of the columellar muscle that, when measured from digital photographs, it could be rea- sonably estimated as a thin but long box, only one pixel wide but many degrees long. The small bias introduced by this approximation should be the same in species with and without folds. Another challenge was to measure the surface area of contact between the columellar muscle and the columella (CM in Figs. 1, 2G). The apertural end of the muscle grades into the foot (Voltzow. 1990: Thompson et al., 1998), so the only point that could be consistently identified at the apertural end of the columellar muscle was the point at which the muscle attachment began (ATT in Fig. 2E). I measured the total area of muscle in contact with the colu- mella: beginning with the attachment, extending down the width of the muscle to the bottom of the whorl, and extend- ing across the columella to include the most apical part of the muscle (CM in Figs. 1, 2G). Because the muscle always spanned the columella between the attachment and the FUNCTION OF COLUMELLAR FOLDS 355 Apert A. B. CA 180 CF D Figure 1. Definition of terms describing columellar morphology illustrated with Triplofusus gigunteus. (A) Top refers to the part of a whorl relatively closer to the apex along a vertical axis dulupicul in some literature, e.g.. Vemieij. 1978); bottom refers to the lower part of that same axis (abapicul). The open arrows wrap around the axis from the aperture to the apex. The apertural point of the muscle attachment is that closest to the aperture (also see D). and the a/>iV. its shell. Furthermore, the muscle probably does n<-. need to be guided, because it is a muscular hydro- stat. The muscle probably mirrors the morphology of the folds simply because it is physically adjacent to them. I reject the hypotheses that animals with folds maneuver better because their attachment area is larger, and that these animals avoid predators by retracting deeper. While I reject these hypotheses as general explanations for columellar fold function in neogastropods as a whole, future work may demonstrate that some of them adequately explain function within groups of species with similarly shaped folds. Methodology I developed two novel methods in this research: one is a practical dissection technique, and the other is an analytical approach for measuring distances and areas from photo- graphs of specimens. With the dissection technique, I de- scribed the columellar muscle attachment in 20 neogastro- pods and one caenogastropod (Table 1). The soft tissues of other gastropods can be dissected without damaging the columella, and future applications of this approach will provide a more general understanding of how soft tissues are situated within the shell. With the analytical approach, I determined the relative position of soft tissues and shell features. I quantitatively characterized the columellar mus- cle in 1 1 species of neogastropods and one caenogastropod (Tables 1, 3, 4). This algorithm will work for any organism arranged as a stack of circles — echinoderms, cnidarians, foraminifera, diatoms, and mushrooms, for example. The measurements and their analysis rest on three as- sumptions. First, to measure the area of contact between the columellar muscle and the columella, I assumed that the amount of muscle apertural to the attachment varies equally in species with and without folds. Second, I also assumed that the attachment area was only one pixel wide, which seems justified by the observation that the attachment was quite thin relative to the total width of the muscle. Third, areas were inflated by the algorithm that transformed the two-dimensional data, because the columella was assumed to be perfectly symmetrical (see Appendix). All of these assumptions were justified, especially in this first attempt to quantify differences in the columellar muscle. Furthermore, there is no a priori reason to believe that they would affect species with folds differently than species lacking them. Note that the measurements are coarse (Table 4), and that subtle differences in contact area, attachment area, depth of attachment, and length of attachment cannot be resolved with these methods. Columellar muscle attachment The complexity and extent of the columellar muscle attachment has gone unrecognized because the attachment itself is remarkably delicate. A slight tug in the wrong direction (away from the aperture, toward the apex) tore the muscle from the columella. and the strength of the attach- ment deteriorated rapidly in preserved or relaxed animals. Its tenuous nature probably reflects the interplay between the physical mechanism of adhesion and the shape of the adhesive surface. Although the muscle was easily detached from the columella, the animal never naturally experiences a force pulling on the attachment from the angle or location I used during dissection; if the shell is opened as I have opened it, the animal is already too exposed to save itself from predators. An adhesive joint that can withstand large shear stresses will frequently fail when peeled (Portelli, 1986). The muscle attachment is analogous to a long piece of tape on a tabletop. When peeled backward, perpendicular to the table, the tape detaches easily, but when pulled along its length, it has great strength. Surprisingly, none of the species studied here have mus- cle scars, even though scars are found in all major groups of shelled molluscs throughout their history (e.g., Abbott, 1974; Lindberg, 1985; Pojeta, 1985; Doguzhaeva and Mutvei, 1996; Isaji et al., 2002). In vertebrates, however, muscles frequently do not leave attachment scars (McGowan, 1982; Bryant and Seymour, 1990), especially when they insert directly into bone instead of attaching to a tendon which inserts into bone. In snails, the columellar muscle inserts into an epithelium that in turn inserts into the shell (Tompa and Watabe, 1976). Additional work is re- quired to determine whether neogastropods and vertebrates lack muscle scars for similar reasons. The fact that the muscle attachment is so much longer than previously thought (by authors such as Linsley, 1978; Morita, 1993; Thompson et al.. 1998) has interest- ing implications for how the columellar muscle func- tions. Thompson et al. (1998) calculated, on theoretical considerations, the force required to buckle the columel- lar muscle and the amount of torsion the muscle could exert. The muscle, which has a crescentic cross section, buckled more easily and could not exert as much tor- sional force as a cylindrical muscle with a circular cross section. However, these authors assumed that the muscle was attached at only one end rather than throughout its length. The true, side-long attachment should help the muscle resist compressive forces (J. T. Thompson, St. Joseph's University, pers. comm.), so that the mechanical disadvantage due to buckling may not be so severe. Similarly, the net torsion exerted must be reconsidered in light of the new attachment data. FUNCTION OF COLLIMELLAR FOLDS 361 Guidance I hypothesized that folds guide the columellar muscle during protraction and retraction by protruding far into the columellar muscle, compelling the muscle to move along the folds. I reasoned further that, if folds act as struts, then they should protrude far enough into the muscle to signifi- cantly increase the amount of contact between the muscle and columella. For the species analyzed here, however, there was no significant difference in the contact area or length of attachment between species with folds and those lacking them. Therefore, the folds-as-struts hypothesis, as explained here, was not supported. From another perspective, the methods were relatively coarse, and the data were poorly resolved. If these methods are applied to species with a subtle fold topology, then no significant increase in the contact area may be detectable. In species with folds, I compared the unadjusted contact area to the contact area after it had been adjusted with the fold modifier. The two metrics differed by more than 2<7r in only one specimen, and the average difference was only 0.2% (n = 17). This difference is less than the precision of the measurement of contact area, which is only to one decimal place. Therefore, although the topography of folds does not increase the contact area in the specimens considered here, the contact area may be greater in species with more prom- inent folds, such as those in the Volutidae, Mitridae. and Cancellariidae. Thus, additional work with these species and with methods providing higher resolution may lend support to the guidance hypothesis and its corollary that folds act as struts. A consideration of the association between the area of contact and the presence of folds suggests other functional relationships. For example, if the advantage offered by strut-like folds were offset by the benefit of a large contact area, species with folds should have a significantly smaller area of contact than those without. Similarly, species with folds could have the same area of contact as species without folds, provided that the increase in contact due to the protruding folds was offset by a shorter attachment length. However, no such significant differences were observed. The guidance hypothesis assumes that, without the resis- tance offered by folds, the muscle would slip along the columella during both retraction and protraction, presum- ably causing the animal to expend more energy. However, the arrangement of fibers within the muscle, including some fibers that are wound obliquely around the robust, apertural end of the muscle, suggests that the fibers, and not the columellar folds, control the path of the muscle when it contracts (Thompson et cil., 1998). Because the muscle is attached along its top edge (Fig. 2), it will shift across the columella when the oblique fibers contract. In light of this newer evidence about the columellar muscle in particular and the muscular hydrostats of molluscs in general (Kier and Smith. 1985, 1990; Hodgson and Trueman. 1987; Kier, 1988; Marshall ct ni. 1989), the muscle seems to function in the way that was previously thought inefficient (Signer and Kat, 1984). Signor and Kat (1984) formulated the guidance hypoth- esis in part because Signor (1982) correlated burrowing behavior in high-spired gastropods with columellar folds; 55 of 59 burrowing species had folds, but only 1 in 46 non-burrowing species did. He concluded that columellar folds guide the columellar muscle in burrowing animals (Signor and Kat, 1984). However, 40 of his 59 burrowers were in the Terebridae, and only 4 of his 1 1 families included non-burrowing species. Thus, his results may be explained by phylogenetic bias and should be reanalyzed with comparative methods based on phylogenetic contrasts (as in Harvey and Pagel. 1991) once the appropriate esti- mates of relationship are available. The advantage of folds to burrowing animals is not obvious, especially if the guid- ance hypothesis is not true. Furthermore, many species with folds do not burrow (G. J. Vermeij. University of California, Davis, pers. comm.). The species considered here cannot be used to explore the relationship between folds and burrow- ing behavior, because species were not sampled randomly across burrowing and non-burrowing habitats. In conclusion, since the inner surface of the columellar muscle is an exact impression of the columellar folds, it is only reasonable to assume that the muscle moves along the folds. This assumption does not require, however, that the folds dictate the muscle's motion. I suspect that the simi- larity in the shape of the muscle and folds is due simply to their proximity, and that the direction the muscle moves is governed instead by attachment morphology and muscle fiber orientation. Maneuverability As with contact area, the folds added insignificantly to the attachment area. The attachment was so long, and so much of it was distant from the folds, that the folds would need to protrude into the muscle six times more than they do (i.e., multiply the fold modifier by 6) to significantly increase attachment area in species with folds at the a < 0.05 level. If animals with folds are better able to maneuver their shells. it is not because they have a greater surface area of muscle attachment. Signor and Kat (1984) suggested that the columellar muscle is divided by the folds into functionally discrete units joined only by connective tissue. However, judging by the observations presented here, the divisions they describe are probably part of a gradation between the robust, most apertural, part of the muscle and the weak, more apical part (left and right sides of Fig. 2E-G). Since Signor and Kat ( 1984) did not mention the frequency or placement of their histological sections, nor illustrate their results, it is difficult 362 R. M. PRICE to reevaluate thei. conclusions in the light of the newly recognized attaci ich. presumably as a defense mech- anism. While it is ell understood that aeolid nudibranchs store cnidanan nematocysts from their prey, the dynamics of nematocyst selection are not explicit. There are more than 25 types of nematocysts in cnidarians (see Mariscal, 1974), each with different functions in prey acquisition and de- fense, and those present in a given cnidarian are a function of the species. Therefore, specific nematocysts are present in varying combinations and proportions among nudibranch prey species (Mariscal, 1974: Calder, 1988). Nudibranch nematocyst incorporation is a function of availability in the diet, so depending on the cnidarian prey they consume and on which parts of the prey, nudibranchs sequester different kinds of nematocysts within their cnidosacs. Additionally, they can preferentially select specific types from what is available (Grosvenor. 1903; Day and Harris. 1978). The nematocyst complement serves as a measure of feeding history, as nudibranchs incorporate a small number of all nematocyst types found in the prey they have been consum- ing. However, individuals of a given nudibranch species feeding on the same diet sequester varying proportions of those nematocyst types (pers. obs.), indicating that factors other than strict availability must be involved in selection. Predation pressure may influence nematocyst uptake, and therefore different nematocyst types may be selected in response to specific predators. Since nudibranch nemato- cysts purportedly function as predator deterrents, predator cues may affect nematocyst incorporation such that nudi- branchs maintain weapons capable of combating predators specific to the area in which they live. Edmunds (1966) suggested that certain nematocyst types may be more effec- tive against some predators — penetrants for use against fish and adherents against crustaceans, for example — so nema- tocyst incorporation may be based on nudibranch predators in the vicinity. Despite studies of individual species popu- lations, few studies have examined variation with respect to the predation pressure encountered by the organism in ques- tion. The objective of this study was to identify changes in nematocyst uptake by nudibranchs in response to chemical cues from potential predators. My work examines the in- corporation of nematocysts by nudibranchs in response to individual predator species and considers both predation pressure and the nudibranch' s previous experience. To fur- ther elucidate the relationship between nematocysts in nudi- branch cnidosacs and predation pressures, I examined nematocyst uptake in the nudibranch Flabellimi verrucosa with and without exposure to potential predators. I hypoth- esize that (a) the presence of specific predators differentially affects nematocyst uptake, (b) response depends on the nudibranch's previous exposure to the predator, and (c) response depends on the predator's ability to prey on the nudibranch. By testing these hypotheses I will determine whether population-level variation in nematocysts seques- tered by F. verrucosa provides a link between nematocyst incorporation and predation pressure. Materials and Methods Study organisms Flabellimi (formerly Coryphelld) verrucosa (= rufi- branchialis) (Sars, 1829) is a common aeolid nudibranch in the shallow marine subtidal throughout the Gulf of Maine. Its distribution is circumboreal: in the Atlantic its range includes northern Europe (British Isles, Norway, and Ice- land) and Greenland south to the Gulf of Maine; in the Pacific F. verrucosa can be found off British Columbia and the coast of Russia (Bleakney, 1996). A generalist predator. F. verrucosa consumes numerous athecate hydroid species, scyphistomae, and tunicates (Kuzirian, 1979). No predators are known to prey primarily on this nudibranch. In the following experiments, F. verrucosa was exposed to a variety of predators, and nematocyst uptake was com- pared with uptake in the absence of predator cues. The predators to which F. verrucosa was exposed included the wrasse Tautogolabrm adspersus (Walbaum, 1792). the sea star Crossaster papposus (Linnaeus, 1767), and the green crab Carcinux maenas (Linnaeus, 1758). Crossaster pappo- sus is present in cold deep waters in the southern Gulf of Maine collection area, though none were observed at the depths where nudibranchs were collected for this study. While not a nudibranch specialist, C. papposus feeds on nudibranchs, including F. verrucosa, when they are encoun- tered in the field (Mauzey et at., 1968) (pers. obs.). Among its other prey, the wrasse T. adspersus (cunner) is known to feed voraciously on some nudibranch species, but shows an aversion to consuming F. verrucosa (Harris, 1986; pers. obs. ). Cunner are seasonally abundant in the southern Gulf of Maine in the summer and fall when water temperatures are mild, but they are absent during the colder winter and spring seasons. The omnivorous green crab Carcinus mae- nas is common both intertidally and subtidally throughout the Gulf of Maine, but unlike some crab species reported to prey on certain nudibranchs (Harris, 1970; Ajeska, 1971), C. maenas is not a known nudibranch predator. The experience that specimens of F. verrucosa collected in the southern Gulf of Maine have had with the predators used in this study ranges from common exposure (C. mae- nas and T. adspersus) to no probable exposure (C. pappo- sus) for nudibranchs of the collected generation. Depending on collection location, there may be differences in exposure to cunner: nudibranchs collected from mooring chains at Shoals are less likely to have encountered T. adspersus than those from Nubble (description below). At Nubble, fish forage and live on the wall where nudibranchs were col- lected, but at Shoals, the mooring lines do not support substantial populations of the predatory fish. For all collec- NUDIBRANCH RESPONSE TO PREDATORS 369 tion sites, nudibranchs are unlikely to be found deep enough to have been exposed to C. papposus prior to collection. However, parental generations of F. verrucosa may have been exposed to the entire suite of experimental predators in other areas within the species' range, presenting the possi- bility that nematocyst uptake could be influenced by a revolutionary response based on exposure of previous generations to the predators used in this study. Specimen collection Nudibranchs were collected from two locations within the southern Gulf of Maine: Cape Neddick (Nubble) in York. Maine (43°9'54"N. 70°35'29"W). and Gosport Har- bor near Appledore and Lunging Islands of the Isles of Shoals island group located about 10 km off the coast of New Hampshire (42°59'21"N, 70°36'54"W). The shallow subtidal of Cape Neddick and the Isles of Shoals are both algal-dominated, gradual slopes containing vertical rock surfaces and undercuts dominated by animal communities. Nudibranch populations at these sites are of the same ge- netic stock due to site proximity and widespread dispersal of planktonic veliger larvae, but their post-settlement feeding histories may differ due to the availability of prey items at the two locations. Between 30 and 50 specimens of Flabellina verrucosa were collected from vertical rock wall surfaces at Nubble in 3-8 m of water, and also from blooms of the hydroid Tubularia crocea on mooring ball lines near the Isles of Shoals (Appledore and Lunging Islands) in October and November 2001 and January 2002. Animals were main- tained following collection and for the duration of all ex- periments at 10 °C in a constant-temperature room at the University of New Hampshire. Tanks were filled with nat- ural seawater obtained from the Coastal Marine Laboratory at the Portsmouth Coast Guard Station at the mouth of Portsmouth Harbor <43°4'20"N. 70°42'37"W). Animals for each site were kept together and used separately in each experiment. Experimental setup Experimental tanks containing a predator were estab- lished 1-2 days before experiment inception. Control tanks did not contain a predator. Following initial nematocyst counts (procedure described below) for each nudibranch population, nudibranchs were placed in individual flow- through containers with two hydroid food sources. The hydroids used were Obelia geniculata (Linnaeus. 1758) and Tiihiilnria spp. (crocea and/or indivisa), each present in excess so that food was not a limiting factor. Examination of the tissues of these cnidarian prey species revealed mutually exclusive nematocyst complements (Table 1 ), so they offer a variety of nematocyst types. Tubularia spp. were collected from the nudibranch collection sites at the Isles of Shoals, Table 1 Nematocvst types found upon examination of cnidarian prey tissues Hydroid species Nematocysts apparent in tissue* Obelia geniculaiti Ttthuiiiria indivisa, T. crocea MM. MA ST. DS. HeA. HME. BI * Nematocyst types are abbreviated as follows: MM. microbasic mas- tigophores; MA, microbasic amastigophores; ST. stenoteles; DS, des- monemes; HeA. heterotrichous anisorhizas; HME, heterotrichous micro- basic euryteles; BI, basitrichous isorhizas. and O. geniculata from pilings at the Portsmouth Coast Guard Station. The hydroids used as prey were not fed during the experiment and have no direct ecological rela- tionships with the predators used in the study. Experimental flow-through containers remained in the tanks for 2 weeks, with a fresh replacement of hydroid food and a partial water change in the tanks after 7 days. Control tanks had five containers for each nudibranch population randomly distributed among the tanks. After 2 weeks of exposure to the experimental conditions, the nematocyst content of each nudibranch was evaluated by examining ceras squashes for three cerata per animal via light micros- copy. Cerata were removed from the anterior region in the first or second ceratal cluster by using forceps combined with the animals' propensity to autotomize these projec- tions. For each ceras, 100 nematocysts (identification ac- cording to Mariscal, 1974; see reference for visual repre- sentations) were categorized on the basis of visual characteristics (if the field of view included more than 100 nematocysts, all in the field were counted). Counts included both fired and encapsulated (unfired) nematocysts. The setup was repeated with each predator and for each nudi- branch collection site, adjusted as described below. The numbers of replicates for each experiment are summarized in Table 2. For the C. papposus experiment, ten 2.5-gallon tanks containing a sea star measuring 2.7-6.5 cm and three 2.5- gallon tanks without a sea star were established. Each tank contained one container for each nudibranch collection site. The sea stars were starved for the duration of the experiment because they refused to eat regularly once brought into the laboratory. The experiment using the cunner T. adspersus was conducted similarly, but due to limited numbers of captive fish, it involved six 10-gallon tanks, each containing two flow-through containers for each experimental site. The cunner in the tanks measured between 7 and 17 cm and were fed locally collected Mytilus edulis meat every other day. When the green crab Carcinus maenas was used as the predator, tank setup and size was equivalent to that used for the C. papposus experiment, with crab carapace size rang- ing from 5.5 to 7.0 cm. Crabs were fed frozen cooked 370 K. FRICK Table 2 Summary of experimental design, including number of replicate flow-through containers for each population inui each treatment Number of Number of Number of Number of Predator treati experimental containers experimental tanks control containers control tanks ••us Ttlllt. 'S.', '/J/VIIN lll/.S/'t ' »:i! Carcinus maenas 10 12 10 10 6 10 5 6 5 3 4 shrimp two to three times weekly for the duration of the experiment. Total numbers of nematocysts for each condition were anahzed using chi-square tests of independence to identif\ differences in the distribution of nematocysts. Pairwise ANOVA anahses were performed on average nematocyst proportions for each nudibranch using the PC program Systat 9.0 to determine the nematocyst types that were exhibiting changes for each comparison of conditions. Results Southern Gulf of Maine populations of Flabellina verru- cosa responded to the presence of some experimental pred- ators by changing which nematocyst types they incorpo- rated. Field-collected nudibranchs showed a wide range of incorporated nematocysts and high variability between in- dividuals and between sites (Fig. 1). with stenoteles (ST). desmonemes (DS). and heterotrichous microbasic eunteles (HME) sharing dominant percentages. The results for pred- ator exposure are subdivided below, and nematocyst incor- poration response for all predators is summarized in Table 3. All results marked as being significant are based on P values less than 0.05. Southern Gulf of Maine predator responses Flabellina verntcosa responded to the presence of Cross- aster papposus with significantly increased incorporation of microbasic mastigophores (MM) and depressed uptake of heterotrichous anisorhizas (HeA) (Fig. 2 A). In response to Tautogolabms adspersus (cunner). incorporation of MM was also significantly increased (Fig. 2B>. while uptake of heterotrichous microbasic eunteles (HME I and stenoteles (ST) was depressed. The presence of the green crab Card- mis maenas evoked no significant differences in nematocyst incorporation compared to control nudibranchs (Fig. 2C>: all differences were attributable only to change due to the provided diet. Site-dependent responses Trends in nematocyst incorporation depended on the predator used, not the site where the nudibranchs \\ere collected. However, these trends were not always signifi- cant. When exposed to the sea star Crossaster papposus. nudi- branchs from Shoals significantly adjusted their nematocyst incorporation. (Fig. 3A) following the same trends as the combined data for the two sites (Fig. 2). Those from Nubble showed no significant differences attributable to the pres- ence of C. papposus, but showed a propensity for increased incorporation of MM (Fig. 3B). Exposure to the cunner T. adspersus also triggered sig- nificantly increased incorporation of MM in the Shoals population when compared to the control group (Fig. 4A). while uptake of three other nematocyst types was depressed. Nudibranchs from Nubble showed similar trends of increas- ing nematocyst incorporation, but the only significant dif- ference was lower uptake of microbasic amastigophores (MA) in the experimental nudibranchs (Fig. 4B). As in the combined test (see Fig. 2C). the presence of Carcinus maenas did not elicit differences in nematocyst incorporation for experimental nudibranchs in comparison with control animals for either the Shoals or Nubble popu- lations. Control comparison Individual southern Gulf of Maine populations incorpo- rated nematocysts similarly from the provided hydroid di- ets. \et exhibited some differences in nematocyst uptake in the absence of predator cues. However, results were not consistent between experiments when comparing control groups from the different collection sites (Fig. 5). In the experiment with C. papposus. nudibranchs collected from Shoals had significantly higher incorporation of ST (Fig. 5 A i. In the T. adspersus experiment. Nubble nudibranchs retained a significantly higher percentage of desmonemes (DS) than the Shoals population, and significantly fewer HME and holotrichous anisorhizas (HoA) (Fig. 5B). Con- versely, the control comparison from the Carcinus maenas experiment showed that nudibranchs from Shoals kept more DS (Fig. 5C). Discussion The animal world offers numerous illustrations of pred- ator incorporation of prey defense mechanisms. Some of the NTDIBRANCH RESPONSE TO PREDATORS 371 80 • 70- 60 50 40 30- 20 - 10 80 • 70 • „ 60- 01 S 50 • if 40 -30 20 10 0 0 Nubble • Shoals ST DS HoA HeA HME MM Nematocyst Type Bl HI DS HoA HeA HME MM Nematocyst Type MA 80- 70- g,60' O |50. S 40- * * 0 T °- 30 | I 20- 10 - 1 I 1 -B 0 - Si '•' $B Si _3 , , ST DS HoA HeA HME MM MA Bl HI Nematocyst Type Figure I. Initial distribution of nematocyst types in Flabellina \-er- rucosa collected from two sites in the southern Gulf of Maine. Results are pre-emed according to the predator experiment for which the nudibranchs were collected: (Ai Crossasrer papposus. (B) Tautogolabrus adspersus. and iCl Carcinus maenas. 'Indicates significant differences. P < 0.05. NematocNst t\pev ST. -tenoteles: DS. desmosomes: HoA. holotrichous anisorhizas: HeA. heterotrichous anisorhizas: HME. heterotrichous mi- crobasic euryteles; MM. microbasic mastigophores: MA. microbasic amastigophores: BI. basitrichous isorhizas: HI. holotrichous isorhizas. most specialized species store prey-deri\ed toxins and use them for their own chemical defenses against predators at the third trophic level (Rowell-Rahier and Pasteels. 1992). Among the many examples in insects and vascular plants (Rothschild. 1973: Rowell-Rahier and Pasteels. 1992). the most famous involves monarch butterflies utilizing carde- nolides from milkweed host plants (Brower et ai, 1972. 1975; Brower and Moffitt, 1974: Dixon et ai, 1978: Calvert etal, 1979:Seibereffl/.. 1986: Malcolm and Brower. 1989: Malcolm and Zalucki. 1996). In the marine realm, some dorid nudibranchs sequester chemical compounds from sponges for defense against predators (Karuso. 1987: Scheuer. 1990: Faulkner. 1992: Proksch. 1994). With both monarch butterflies and dorid nudibranchs. the uptake process is selective in that the predator incorporates specific compounds from those available in the host (monarchy Seiber et ai, 1986: nudibranchs: Proksch. 1994). However. \\ hile the process is discriminatory, there is no evidence that selectivity can be modified in response to environmental cues. In the case of Flabellina vermcosa and its incorpora- tion of cnidarian nematocysts. the animal not only has choices as to the specific defensive structure to sequester. but as shown in this study, the selection can also be based on chemical cues from specific predators. This is an instance of secondary induction, where the nudibranch responds to the presence of predators by altering incorporation of de- fensive organelles from the cnidarian prey. Flabellina vermcosa from the southern Gulf of Maine responded to both Crossaster papposus and Tautogolabrus adspersus by increasing incorporation of one type of pene- trating nematocyst. the microbasic mastigophore. In cnidar- ians. this nematocyst has been suggested to be an effective defense against predation by the nudibranch Cratena pilata (Kepner. 1943): when incorporated by a nudibranch. it may also be effective against a diverse suite of predators. For nudibranchs. as for cnidarians. use of a single type of nematocyst against many predators may be more efficient than needing a different nematocyst for each potential pred- ator. Given the experimental nudibranchs" lack of field expo- sure to some of the predators used in this study, the uptake of a single nematocyst type iMMi in response to multiple predators could reflect an inducible defensive selectivity. Such selectivity may be attributable to a variety of factors, including the long-term coevolution of nudibranchs with these predators, genetic control over nematocyst response to predator cues, or a propensity to take up a particularly noxious nematocyst when any potential predators are present. The ability of F. vermcosa to respond to C. pap- posus and T. adspersus can be explained in two ways: either nudibranchs in this generation have had prior experience with these predators, or they are demonstrating a coevolu- tionary response based on exposure to these predators over time. Individuals of F. vermcosa collected from the south- ern Gulf of Maine are likely to have been exposed to T. adspersus. and they responded strongly by increasing up- take of a potent nematocyst (Fig. 2B). Nudibranchs from the two collection sites showed the same trends when exposed to cunner. but the response was not significant for the specimens from Nubble (Fig. 4. Table 3). The ecological 372 K. FRICK Table 3 Significant clim<: < '.r.'cv.w incorporation between experimental and control Flabellina verrucosa when exposed to each predator for each individual < t< and the combined southern Gulf of Maine Significant changes Predator Population in nematocyst incorporation Crossaster papposus Tautogolahrus adspersus Ctirciiuis imienas Combined southern Gulf of Maine Increased MM(P = 0.004) MM (P = 0.001) Decreased HeA(/> = 0.006) HME = 0.033). ST (P = 0.003) Nubble Increased (NR> Decreased MA (P = 0.009) Decreased HeA(P = 0.003) HME (P = 0.000). ST (P = 0.000). HeA (P = 0.047) rtr»trrt ST DS HoA HeA HME MM MA Bl HI 60 ' l_J WrfWI III >-" 0 experimental Nematocyst Type 50 o 70 • 60 • j 0> o 2 40 • c 0) I30' ft i * 50 • a 1 20- i f % 1 i re" 40 • I & „ i §30. s. I ,„. o4 i \ fL u 20- 1 I T ST DS HoA HeA HME MM MA Bl HI 10 • \ I 1 I \ \ Nematocyst Type m pm L ^ Jh B 701 ST DS HoA HeA HME MM MA Bl HI gg . Nematocyst Type 50- Figure 2. Changes in nematocyst uptake by Flahellinu verrucosa o -• . collected in the southern Gulf of Maine in response to predator cues: (A) ra 40 • (.'rn>,\iiMi-rpiippi>!,u.\. (B) Tautogolabrus adspersus. (C) Carcinus maenas. a |j *Indicates significant differences. P < 0.05. Nematocyst types as in Figure 1. 5 3' ~\ have been affecting the cnidom 20 • though nematocysts in F. I I tL I f verrucosa are replaced quickly. with complete exchanges T I ^ I \ | occurring within 1 ~> davs of switching diets (Dav and Harris, F i i — 1 U ' \ X. 1978). These j _ results indicate that ingestive conditioning ST DS HOA HeA HME MM MA Bl HI may play a role in nematocyst preference in subsequent Nematocyst Type feeding trials, though directed responses to predator cues are , ,,, , . Figure 3. Site-dependent responses in nematocyst uptake by Flabel- possible despite such tendencies. /ina verrucosa caused by cues from Crossaster papposus for nudibranchs Preferential feeding on Obelia geniculata by F. verru- collected from (A) Isles of Shoals and (B) Nubble, indicates Mgnificant cosa in the presence of T. adspersus and C. papposus could differences, P < 0.05. Nematocyst types as in Figure 1. 374 K. FRICK A 8°- 70 - * ^ D control 0 experimental 60 - ' g.50, , 1 * £ 40- o £ 30 • * T| I 20 • X rh * M 10- n - 1 1 L ft i_ \ i ST OS HoA HeA HME MM Nematocyst Type MA Bl B 80 70 60 £ 40 u £ 30 20 • 10 • ST DS HoA HeA HME MM Nematocyst Type MA HI Figure 4. Modification of nematocyst uptake by nudibranchs exposed to Taiirogolabrus adspersus when collected from (A) Isles of Shoals and (B) Nubble, indicates significant differences. P < 0.05. Nematocyst types as in Figure 1. Work should continue on the mechanism of selection (prey choice vs. active cellular selection by the nudibranch), vari- ation in the types or proportions of nematocysts produced by the cnidarian prey in response to nudibranch predation. and differences in selective incorporation by other nudi- branch species in the face of changing predation pressures. The phenomenon of aeolid nudibranchs changing their defensive nematocyst regime may be widespread, though that has yet to be determined. Since the ability of a nudi- branch to choose noxious organelles depends upon the variety of nematocysts available in the prey species, then the nudibranch's ability to respond to external cues is influ- enced by whether it is a specialist with a definite nematocyst array contained within its prey or a generalist capable of acquiring a wider range of nematocyst types from a variety of prey species. It may also be that large or potent nema- tocysts can only be incorporated at certain life-history stages due to physical limitations. Obviously the hydroid prey does not passively provide its defenses, and the nudi- branch is not merely an adapted consumer that can choose from among an array of weapons without cost. The inter- action is dynamic in its intricacies and malleable from the perspective of both predator and prey. These concepts pro- vide avenues of further study to elucidate the dynamics that control nematocyst uptake and the potential for predator response by aeolid nudibranchs. Determining how such predilection operates and its efficacy in deterring predation will help us better understand ecological communities and 70 • 60 • 50 • 40 • 30- 20 • 10 • 60 • 50 n 40 • c O e 30 -i S. 20- 10 • 60 - 50 3 40- c a> 3. 20 - 10- ST ST HNubble B Shoals DS HoA HeA HME MM MA Nematocyst Type i DS HoA HeA HME MM MA Nematocyst Type HI DS HoA HeA HME MM Nematocyst Type MA HI Figure 5. Comparison of control groups of Flabellina vemicosa col- lected from two sites in the southern Gulf of Maine. Results for each predator treatment are presented separately: (A) Crossaster papposus. (B) Tautogolabrus adspersus, and (C) Carcinus maenas. "Indicates significant differences. P < 0.05. Nematocyst types as in Figure 1. NUDIBRANCH RESPONSE TO PREDATORS 375 the selective pressures that have brought about their forma- tion. Acknowledgments This project was possible thanks to support, space, and funding through the University of New Hampshire. Larry Harris and Rebecca Toppin helped with formulating ideas and collecting specimens for this work. Additional thanks go to Glen Rice for hydroid collection and Chris Neefus for statistical advice. Comments from Lauralyn Dyer and Jenn Dijkstra improved the experimental design, and Larry Har- ris. Anne Stork, and the members of the writing seminar made helpful suggestions on earlier drafts of this paper. This research was supported by funds from the University of New Hampshire Center for Marine Biology; the National Sea Grant College Program of the National Oceanic and Atmospheric Administration, Department of Commerce, under grant number N A56RGO 1 59 to the University of New Hampshire/University of Maine Sea Grant College Pro- gram: and by additional funds provided by the Open Ocean Aquaculture project component of the NOAA UNH Coop- erative Institute for New England Mariculture and Fisheries (CINEMAR), NOAA grant number NA16RP1718. Literature Cited Abrams, P. A. 1995. Implications of dynamically-variable traits for identifying, classifying, and measuring direct and indirect effects in ecological communities. Am. Nat. 146: 1 12-134. Abrams, P. A., B. A. Menge, G. G. Mittelbach, D. Spiller. and P. Vodzis. 1996. The role of indirect effects in food webs. Pp. 371-195 in Food Webs: Integration of Patterns and Dvnamics, G. Polis and K. Winemiller. eds. Chapman & Hall, New York. Ajeska. 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Fingerut, 1 10 Zinc, 209, 213 Zinc chelation enhances the sensitivity of the ERG b-wave in dark-adapted skate retina. 213 Zinc modulation of hemichannel currents in Xenopus oocytes, 209 Zooplankton. 93 Zooxanthella. 66. 339 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 telenceph- alon has no residual effects on physiological recordings of supramed- ullary /dorsal neurons of the cunner. Taulogolabrus adspersus, 21 1 THE BIOLOGICAL BULLETIN (www.biolbull.org) 2004 SUBSCRIPTION FORM (VOLUMES 206-207, 6 ISSUES) (please print) NAME: INSTITUTION: ADDRESS: CITY: STATE:. POSTAL CODE: COUNTRY:. TELEPHONE: FAX:. 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