Volume 201 THE Number 1 BIOLOGICAL BULLETIN AUGUST 2001 Published by the Marine Biological Laboratory http://www.biolbull.org Made to my exact Let's address my specs first, specifically lugh resolution, contrast and infinity-corrected optics. They've all reached Olympian standards thanks to Olympus. But even more to the point, here's how the BX2's modular design came through for me. First, the eight-position universal condenser offers the flexibility to choose from brightfield, darkfield and phase as well as DIC. Next, its assortment of prisms makes it possible to match the optical image shear to the specimen, achieving the optimal balance of contrast and resolution. Finally, the Plan APO objectives, with superb chromatic correction and contrast, provide extraordinary detail. Now let's move on. And yours Picture yourself sitting here, looking into your Olympus BX2 research microscope, your fluorescence requirements having been met. Specifically: The aspherical collector lens produces a fluorescence intensity that's twice as bright as others and more even across the field. The unique excitation balancers improve visualization of multiple labels by revealing details that would otherwise be unseen. The six-posi- tion filter turret makes single and multiband imaging faster and simpler. And the rectangular field stop, another Olympus exclusive, protects the specimen by exposing only the precise area being imaged in addi- tion to enhancing the S/N ratio. Time to see what's next. OLYMPUS FOCUS ON LIFE Visit us at www.olympusamerica.com or call 1-800-446-5967. specifications And yours. And yours. Here, imaging and automation is a must. And here, the BX2 responds as a high-performance, highly efficient, digital imaging machine. The motorized nosepiece, Z-drive, condenser, illuminator and filter wheels are fully integrated through the user-friendly software package. It's you who commands this automated imag- ing system with your PC, optional keypad or preset buttons located on the microscope frame itself. Digital images can now be acquired, processed and analyzed faster than before. And reports and documentation have never been this easy to generate. Which leaves one more set of specs. Now modularity really is in high gear as the Olympus FLUOVIEW 500 is added, resulting in a complete confo- cal laser scanning microscope system. It provides five imaging channels and has an intuitive operation that makes it readily available to everyone so that productivity is greatly enhanced. By the way, the BX2 is the only microscope that offers a Metal Matrix Composite frame the ultimate in static and thermal rigidity making it the optimal solution for frequent use of 3D microscopy, time-lapse observations and high-end digital imaging. So you see, with all this mod- ularity and flexibility, my BX2 microscope is also your BX2 microscope. Research Microscope Series 2001 Olympus Am Cover About 3.5 million years ago (Ma), rising sea levels opened the Bering Strait, and the North Atlantic Ocean was invaded by hundreds of taxa from the North Pacific. Among the invaders was the seastar genus Asterias. At present, two species of Asterias are recognized in the North Atlantic: A. forbesi on the west coast of the Atlantic, from Cape Cod south to Cape Hatteras, and A. rubens, a European species that ranges from southern France to Norway and Iceland, but also occurs in the northwestern Atlan- tic, mainly from Cape Cod north. Representatives of these species are shown on the cover, as is a specimen of A. amurensis, which inhabits the North Pacific from British Columbia to Japan. After entering the Atlantic, populations of Asterias were separated, and speciation subsequently occurred. The timing of the separation is critical, for it deter- mined, in part, the mechanism involved in the specia- tion, and it is the basis for the present geographic distribution of Asterias species in the North Atlantic. However, as the map on the cover illustrates, the timetable of these events was constrained by habitat and oceanographic instability during the Pleistocene glaciation. 1 In particular, most of the current North American habitat of Asterias rubens was repeatedly covered by a kilometer of ice and was unavailable to this seastar until about 15,000 years ago long after the opening of the Bering Strait. 1 The map on the cover is a polar view of the North Atlantic and Pacific Oceans during the Wisconsin glacial maximum, about 20.000 years ago. The solid blue line marks the average glacial margin; the dashed blue lines show the extent of sea ice in summer (upper) and winter (lower); the dotted black line illustrates how lower sea levels during glacial maxima altered the Atlantic coastline; and the shades of blue and green represent isotherms, highly compressed in the north- western Atlantic, and producing a strong temperature gradient. The speciation of Asterias in the Atlantic has been explained by two hypotheses. Either the event oc- curred recently, with strong natural selection pre- cluding hybridization; or the speciation into North American and European species occurred shortly after Asterias entered the North Atlantic, with a recolonization of the northwestern coast of the At- lantic by A. rubens taking place in recent times. The second hypothesis implies that speciation was due to prolonged isolation and was independent of ob- served adaptations to different water temperatures. As reported in this issue (p. 95), John P. Wares has collected genetic sequence data from populations of A. forbesi, A. rubens, and A. amurensis and used them in phylogenetic and population genetic analy- ses to test the two hypotheses. He concludes that, although changes in climate and ocean currents particularly the formation of the Labrador Cur- rent were concomitant with the separation of As- terias populations in the North Atlantic 3 Ma, permanent colonization of New England and the Canadian Maritimes by A. rubens occurred very recently. (Credits: map from B. Frenzel, M. Pecsi, and A.A. Velichko, eds., 1992, Atlas of Paleociimates and Paleoenvironments of the Northern Hemi- sphere, Geographical Research Institute, Hungarian Academy of Sciences, Budapest, p. 43; images of Asterias forbesi and A. rubens from the George M. Gray Museum collection, formerly administered by the Marine Biological Laboratory, now at the Peabody Museum of Natural History of Yale Uni- versity; image of A. amurensi from a photograph by Jan Haaga, provided online by the Alaska Fish- eries Science Center/National Marine Fisheries Ser- vice; cover design by Beth Liles, MBL.) THE BIOLOGICAL BULLETIN AUGUST 2001 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 The Whitney Laboratory, University of Florida Grice Marine Biological Laboratory, College of Charleston California Institute of Technology Georgia State University University of Chicago SHINYA INDUE, Imaging and Microscopy Marine Biological Laboratory JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL GREGORY HINKLE MAKOTO KOBAYASHI ESTHER M. LEISE DONAL T. MANAHAN MARGARET MCFALL-NGAI MARK W. MILLER TATSUO MOTOKAWA YOSHITAKA NAGAHAMA SHERRY D. PAINTER J. HERBERT WAFTE RICHARD K. ZIMMER ENSR Marine & Coastal Center, Woods Hole Hunter College, City University of New York University of California, Davis Bodega Marine Lab., University of California, Davis Louisiana State University Oregon Institute of Marine Biology, Univ. of Oregon Hopkins Marine Station, Stanford University Cereon Genomics, Cambridge, Massachusetts Hiroshima University of Economics, Japan University of North Carolina Greensboro University of Southern California Kewalo Marine Laboratory, University of Hawaii Institute of Neurobiology, University of Puerto Rico Tokyo Institute of Technology, Japan National Institute for Basic Biology, Japan Marine Biomed. Inst., Univ. of Texas Medical Branch University of California, Santa Barbara University of California, Los Angeles PAMELA CLAPP HINKLE VICTORIA R. GIBSON Managing Editor Staff Editor ~ <-, 'ceanoo _ ,. . , . CAROL SCHACHINGER Editorial Associate WENDY CHILD AUG 3 1 2001 Subscription & Advertising Secretary Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS http://www.biolbull.org Genomic Research Leaders Choose Microway Scalable Clusters E os Biotechnology, Marine Biological Laboratory, Millennium Pharmaceu- ticals, Mount Sinai Medical School, NIH, Pfizer, and Rockefeller University All Choose Microway Custom Clusters and Workstations for Reliability, Superior Technical Support and Great Pricing. 1.4 GHz Dual Athlon, 1.7 GHz Pentium 4, or 1 GHz Dual Pentium III in 1U or 2U Clusters Dual Alpha 833 MHz Clusters and Towers For maximum price/performance choose our Alpha 1U 833 MHz, 4 MB DDR Cache CS20, 4U UP2000+ or 4V 264DP RuggedRack Myrinet, Gigabit Ethernet or Dolphin Wulfkit High Speed Low Latency Interconnects RAID and Fibre Channel Storage Solutions Microway' 1 " Screamer Dual Alpha UP2000* 833 MHz. 4MB Cache in RuggedRack Chassis with RRR Redundant Power Supply Microway has earned an excellent reputation since 1982. 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Please call 508-746-7341 for a technical salesperson who speaks your language! Visit us at www.microway.com A Research Park Box 79, Kingston, MA 0236* 508-746-7341 info@microway.com AUG 3 1 2001 CONTENTS VOLUME 201. No. 1: AUGUST 2001 RESEARCH NOTE Seibel, Brad A., and David B. Carlini Metabolism of pelagic cephalopods as a function of habitat depth: a reanalysis using phylogenetically in- dependent contrasts NEUROBIOLOGY AND BEHAVIOR Herberholz, Jens, and Barbara Schmitz Signaling via water currents in behavioral interac- tions of snapping shrimp (Alpheus heterochaelis) .... PHYSIOLOGY AND BIOMECHANICS Reddy, P. Sreenivasula, and B. Kishori Methionine-enJkephalin induces hyperglycemia through evestalk homiones in the estuarine crab Stylla sermta . . . Mogami, Yoshihiro, Junko Ishii, and Shoji A. Baba Theoretical and experimental dissection of gravity- dependent mechanical orientation in gravi tactic micro- organisms 26 SYMBIOSIS AND PARASITOLOGY Hanten, Jeffrey J., and Sidney K. Pierce Synthesis of several light-harvesting complex I polypep- tides is blocked by cycloheximide in symbiotic chloro- plasts in the sea slug, Elysia chlorotica (Gould): A case for horizontal gene transfer between alga and animal?. . . McCurdy, Dean G. Asexual reproduction in Pygospio elegans Claparede (Annelida, Polychaeta) in relation to parasitism by Lepocreadium setiferoides (Miller and Northup) (Platy- helminthes, Trematoda) 17 34 DEVELOPMENT AND REPRODUCTION Stewart-Savage, J., Aimee Phillippi, and Philip O. Yund Delayed insemination results in embryo mortality in a brooding ascidian 52 CELL BIOLOGY Ballarin, Loriano, Antonella Franchini, Enzo Ottaviani, and Armando Sabbadin Momla cells as the major immunomodulatory hemo- cytes in ascidians: evidences from the colonial species Botnllm schlosseri 59 ECOLOGY AND EVOLUTION Halanych, Kenneth M.. Robert A. Feldman, and Robert C. Vrijenhoek Molecular evidence that Sclerolinum brattstromi is closely related to vestimentiferans, not to frenulate pogonophorans (Siboglinidae. Annelida) 65 Ponczek, Lawrence M., and Neil W. Blackstone Effect of cloning rate on fitness-related traits in two marine hydroids 76 Meidel, Susanne K., and Philip O. Yund Egg longevity and time-integrated fertilization in a tem- perate sea urchin (Stnmgylocenfrotus droebachiensis) .... 84 Wares, J. P. Biogeography of Astmas: North Atlantic climate change and speciation 95 SYSTEMATICS Gershwin, Lisa-ann Systematics and biogeography of the jellyfish Aurelia labiata (Cnidaria: Scyphozoa) 104 45 Annual Report of the Marine Biological Laboratory. ... Rl ANNOUNCEMENT The Marine Biological Laboratory is pleased to announce that it has entered into an agreement with HighWire Press of Stanford University to publish The Biological Bulletin electronically. The online journal will be launched on 23 August 2001. 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Other than these charges for au- thors' alterations, The Biological Bulletin does not have page charges. Reference: BiW. Bull. 201: I-?. (August 2001) Metabolism of Pelagic Cephalopods as a Function of Habitat Depth: A Reanalysis Using Phylogenetically Independent Contrasts BRAD A. SEIBEL 1 * AND DAVID B. CARLINI 2 1 Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, California 95039; and 'Department of Biology, 101 Hurst Hall, American University, 4400 Massachusetts Avenue, NW, Washington, DC 20016-8007 Metabolic rates of deep-living animals have been in- tensely studied (1 ). Within pelagic fishes, crustaceans, and cephalopods, a strong decline in rates of mass-specific metabolism with depth has been observed. Childress and Mickel (2) put/onward the visual interactions hypothesis to explain this general pattern. Their hypothesis states that reduced metabolic rates among manv deep-sea pelagic tax- onomic groups result from relaxed selection for strong locomotory abilities for visual predator-prey interactions in the light-limited deep sea. This pattern has, however, been tested using mean metabolic rates for species as individual data points. Felsenstein (3) warned that, because species are descended in a hierarchical fashion from common an- cestors, they generally cannot be considered as independent data points in statistical analyses. Statistical methods have recently been developed that incorporate phylogenetic in- formation into comparative studies to create phvlogeneti- cally independent values that can then be used in statistical analyses. Reliable independent phylogenetic information has only recently become available for some deep-sea or- ganisms. The present contribution reanalyzed the metabolic rates (4, 5) of pelagic cephalopods as a function of, for consistency with previous studies, MDO (minimum depth of occurrence) using phylogenetic independent contrasts de- rived from a recent molecular phytogeny (6). This analysis confirms the existence of a significant negative relationship benveen metabolism and minimum habitat depth in pelagic cephalopods but suggests that phylogenetic history also has Received 29 August 2000; accepted 12 April 2001. * To whom correspondence should be addressed. E-mail: bseibel@ mbari.org considerable influence on the metabolic rates of individual species. Childress ( 1 ) argued against a phylogenetic basis for the observed relationships between metabolism and depth. He based the argument on the identification of convergence of metabolic rates at a given depth among distantly related taxa (fishes, crustaceans, cephalopods) as well as divergence within closely related groups as a function of depth. This pattern strongly suggests that species experience similar selective regimes at any given depth and that rates of metabolism are evolved in response to that selection. Seibel e t al. ( 5 ) further argued, on the basis of an analysis of higher nodes, that most of the variation in metabolic rates among cephalopods is between families within an order, as opposed to between genera within a family or species within a genus. Therefore, families are more appropriate units for compar- ison. A decline in metabolic rates with increasing habitat depth was also observed when families were used as inde- pendent data points (5). Nevertheless, the degrees of free- dom used for statistical analyses in these studies are ele- vated, to varying degrees, due to phylogenetic non- independence of the data. Felsenstein (3) proposed computing weighted differences ("contrasts") between the character values of pairs of sister species nodes, or both, as indicated by phylogenetic topol- ogy, thereby estimating an ancestral character value (e.g., the ancestral states of log-transformed depth and metabolic data presented in Fig. 1 ). Insofar as the ancestral nodes are correctly determined, each of these contrasts is independent of the others in terms of the evolutionary changes that have occurred to produce differences between the two members of a single contrast (7). Felsenstein's (3) method requires knowledge of the cladistic relationships between the species B. A. SEIBEL AND D. B. CARLINI A. 1.85, -0.50 | 1.79. -0.40F 1.98, -0.36. 2.03, -0.28 2.02, -0.26_T 2.12, -0.28 2.10, -0.08. 1.91,0.10 1.72,0.27 2.14, -0.25 2.15, -0.27 1.00,0.73 1.00,0.75 1.32,0.63 2.74, -0.99 2.30, 0.07 | 2.82, -0.81 1 2.89, -0.78 2.71, -0.83 .Cranchia 1.00, -0.43 .Liocranchia 2.70, -0.57 .Leachia 1.70, -0.25 .Helicocranchia 2.48, -0.23 .Histioteuthis 2.18,0.01 .Octopoteuthis 2.00, -0.21 .Joubiniteuthis 2.70, -0.39 .Gonatus 2.00,0.82 Jllex 1.00,0.95 .L. pealei 1.00,0.81 ,L. opalescens 1.00,0.68 .Sepioteuthis 1.00,0.71 .Chtenopleryx 1.70,0.37 .Bathyteulhis 2.90, -0.23 .Heteroteuthis 2.04,0.63 .J. diaphana 2.85, -0.82 .J. heathi 2.78, -0.80 .Eledonella 2.99, -0.74 .Amphitretus 2.48, -0.89 .Vampyroteuthis 2.78, -1.22 .Nautilus 2.18, -0.30 B. 1.60, -0.38 1.85, -0.50 r 1.91, -0.31 1.88,0.23 2.09, -0.23 2.15, -0.28 1.35, -0.26 r 2.18, -0.25r 1.74, -0.27 r 1.00, 0.72r 1.00, 0.75 r 2.00, 0.33 1.70,0.27 1.70,0.47 2.76, -0.17r 2.48. -0.1 5 r 2.00, 0.68 r 2.00. -0.06 r 2.18, -0.07 r 0.00, 0.86 2.92, -0.77 2.82, -0.81 r 1.23, -0.17 2.35, -0.78 Cranchia 1.00, -0.43 Liocrancha 2.70, -0.57 L. dislocata 1.00, -0.26 L. pacifica 1.70, -0.25 Galliteuthis 2.48, -0.27 Megalocranchia 1.00, -0.27 Helicocranchia 2.48, -0.23 L. pealei 1.00,0.81 L. opalescens 1.00,0.68 Sepioteuthis 1.00,0.71 Chtenopteryx 1.70,0.37 Bathyteuthis 2.90, -0.23 Heteroteuthis 2.04, 0.63 A.felis 1.70,0.33 A. pacificus 1.70,0.21 Enoploteuthis 1.70, 0.70 Pterygioteuthis 1.70, 0.43 C. calyx 2.48, -0.17 C. imperator2A8,-Q.\2 Valbyteuthis 2.95, -0.18 G. om.'* 2.00, 0.82 G.pwos 2.00,0.53 O. deletron 2.00, 0.09 O. nielseni 2.00, -0.21 H. heteropsis 2.18, -0.14 //. hoy lei 2.18,0.01 llex 1.00,0.95 Todarodes 1.00,0.76 Onychoteuthis o.oo, 0.76 oubiniteuthis 2.70, -0.39 Mastigoteuthis 2.57, -0.23 . diaphana 2.85, -0.82 /. heathi 2.78, -0.80 Eledonella 2.99, -0.74 Amphitretus 2.48, -0.89 Oc\thoe 1.00, 0.44 Octopus 1.00, 0.44 Vampyroteuthis 2.78, -1.22 Nautilus 2.\&, -0.30 Figure 1. Phylogenetic trees used for calculating independent contrasts on metabolic rate data. Log- transformed minimum depth of occurrence (MDO) and metabolic rates, in that order, are shown to the right of taxon names. Ancestral states of log-transformed MDO and metabolic rate data (i.e.. weighted differences or "contrasts." see text), calculated using the CAIC software application ( 18), are also shown at the internal nodes. (A) A 21-taxa tree for which both COI sequences and metabolic rate data are available. Branch lengths INDEPENDENT CONTRASTS FOR CEPHALOPOD METABOLISM being analyzed. Several studies have attempted to construct phylogenies for cephalopods. However, only a single reli- able family-level phylogeny exists that includes deep-water fauna. One previous phylogenetic analysis relied exclu- sively on morphological characters that are associated with buoyancy and locomotion and are thus confounded with metabolism and depth (8). We therefore felt that analysis was unsuitable for use in the present study. Other analyses have been unable to obtain sufficient resolution for familial relationships (9) or have included only shallow-living taxa (10, 11). Carlini and Graves (6) recently analyzed the higher level phylogenetic relationships of extant cephalopods by using a 657-bp sequence of the mitochondrial cytochrome c oxidase (COI) gene. The molecular sequence data from Carlini and Graves (6) provide an opportunity to test the visual interactions hypothesis directly, using a more valid statistical approach. An additional analysis based on actin gene sequences (12) was not included, primarily because there was very little overlap between taxa for which actin gene sequences were available and those for which meta- bolic data are available. Furthermore, the actin study pro- vides a more accurate reconstruction of gene family evolu- tion within the cephalopods than of specific relationships among taxa. The phylogenetic trees presented here from which the independent contrasts were calculated include only those species for which metabolic data are available. Similar trees were constructed including species for which enzymatic data are available. Although it may have been preferable to "prune" the complete COI tree rather than reconstruct trees using only taxa for which metabolic data are available, we decided to calculate new trees so that we could include taxa for which COI sequences were obtained after the publica- tion of the COI paper (6). The species we added were Amphitretus pelagicus, Helicocranchia pfefferi, and Jape- tella heathi. Pruning the tree would have had only a small effect on the values of the standardized contrasts and would not have significantly altered our conclusions. A second requirement of Felsenstein's (3) method is knowledge of branch lengths in units of expected variance of change. Ideally, branch lengths should represent expected units of evolutionary change (gradual model). For this ap- proach to be valid, independent contrasts must be ade- quately standardized so that they will receive equal weight- ing in subsequent regression analyses. We plotted the absolute value of each standardized independent contrast, generated from the fully resolved tree (Fig. la), versus its standard deviation (7) and found no relationship between the two variates (data not shown). Thus, the contrasts were adequately standardized and properly weighted in regres- sion analysis. However, even if a particular phylogenetic tree is well resolved and well supported, branch lengths are always estimates and are thus subject to error. A less optimal approach, but one that involves fewer assumptions about the evolutionary relationships of the taxa in question, is to assume that every branch in the phylogeny is the same length (punctuated model). The advantage of this approach is that it can be used for poorly resolved trees or for data sets where branch lengths cannot be estimated, such as those derived from both molecular (6) and morphological (13, 14) data. This allows more contrasts to be performed, increasing the power of subsequent statistical tests. On the other hand, the punctuated model is unrealistic for most data sets, as there is likely to be significant heterogeneity with respect to the evolutionary rates of the taxa under study. In any case, use of a punctuated model is far superior to any method that treats species values as independent data points. In the present study we employed both gradual and punc- tuated models in constructing trees for comparison. The gradual model tree is depicted in Figure la (21 taxa, met- abolic rates as a function of MDO). A similar tree was constructed including species for which enzymatic data are available (not shown, 18 taxa, enzymatic activities as a function of MDO). A tree constructed using the punctuated model for contrasts involving all taxa for which data are available is depicted in Figure Ib (39 taxa, metabolic rates versus MDO). A similar tree was constructed including species for which enzymatic data are available (not shown. 32 taxa, enzymatic activities versus MDO). Independent contrasts for log-transformed, normalized mean oxygen consumption rates (4, 15-18) were produced, for both gradual and punctuated models, using CAIC v. 2.0.0 (19), and were regressed against those produced for (molecular clock enforced) were calculated from the strict consensus of two most-parsimonious trees (Tree Length = 1432 steps; Consistency Index = 0.348: Retention Index = 0.334) derived from parsimony analysis of the COI data in PAUP* (28). (B) Partially resolved 39-taxa tree representing relationships between all pelagic taxa for which metabolic rate data are available. The conservative tree topology is based on a consensus of molecular and morphological evidence. In this case, branch lengths are unknown and a punctuated model of change was assumed; that is. all branches are of equal length. For example, the ancestral character state for log-transformed metabolic rate for the Cranchia-Liocranchia node, assuming a punctuated model of change, is calculated assuming a branch length equal to one and taking an average of the two species (0.43 + 0.57/2 = 0.50. corresponding to a calculated ancestral oxygen consumption rate of 0.61 /j,m O ; g 'h *). Determination of ancestral character states, assuming a gradual model of change, requires calculation of branch length using the CAIC software. B. A. SEIBEL AND D. B. CARLINI c o a, E 0.2-r 0-- u -0.4- 00 6 -- 64 orj 2 -0.8- -I- -t- -r- H- 0.2 0.4 0.6 0.8 1 Contrast: Log (Minimum Depth of Occurrence) Figure 2. Standardized contrasts of log-transformed oxygen consump- tion data plotted as a function of standardized contrasts of log-transformed minimum depth of occurrence calculated from the 39-taxon tree (Fig. Ib; punctuated model ). Contrasts for the three sister-species groupings within the cranchiid family (Cranchia-Liocranchia; Leachia dislocata-L. paci- fica; Galliteuthis-Megalocranchia; Fig. Ib) are indicated with open sym- bols and are included in the plotted regression. The slope of the regression is significant (P < 0.01 1. See Table 1 and text for equation and related statistics. MDO (Fig. 2, Table 1). We produced similar regressions for contrasts of activities of citrate synthase (CS) and octopine dehydrogena.se (ODH) (5, 20-22), indicators of aerobic and anaerobic metabolic potential, respectively (Table 1). We tested the validity of log transformation by using a method suggested by Purvis and Rambaut (19). authors of the CAIC package. Regressions of the absolute values of the contrasts on the estimated nodal values were performed, and none had slopes significantly different from zero. We also performed regressions of the absolute values of the contrasts against the standard deviations of the contrasts and detected no relationship in any case. These two tests ensure that we did not violate any of the assumptions of Felsenstein's (3) model of evolution of continuous characters as a random walk process. Relationships between contrasts of metabolism and depth are summarized in Table 1. A significant decline in oxygen consumption rate with habitat depth was observed when all taxa were included and a punctuated model was assumed (Fig. 2: v = -0.36.Y -- 0.02, P = 0.01). A similar relationship was observed using the gradual model ( v = -0.59.V - 0.049. P = 0.03). but only when the Cranchia versus Liocranchia contrast was excluded (see below). CS and ODH activities were weakly correlated with habitat depth when a gradual model was assumed, even with the Cranchia versus Liocranchia contrast excluded from anal- ysis (Table 1;CS, v = -l.Ol.v + 0.46, P = 0.06; ODH, v = -1.26.x - 0.22, P = 0.099). Contrasts performed using the punctuated model for the CS and ODH data indicated a significant negative relationship between enzy- matic activity and habitat depth with the Cranchia versus Liocranchia contrast excluded from analysis (Table 1: CS, v = -0.64.V + 0.08, P = 0.01; ODH, y = -1.02* - 0.04. P = 0.005). Although these results suggest a negative trend in metab- olism with increasing depth independent of phylogeny, there are clearly phylogenetic influences on the data. For example, members of the family Cranchiidae (including Cranchia and Liocranchia, the contrast excluded from sev- eral of the analyses) have low metabolic rates regardless of Metabolism of pelagic cephalopods as a function of habitat depth Table 1 Parameter Model All contrasts included MO, Punctuated 39 -0.36 -0.02 0.29 0.01 Gradual 21 n.s. CS Punctuated 32 n.s. Gradual 18 n.s. ODH Punctuated 32 n.s. Gradual 18 n.s. Cranchia vs. Liocranchia contrast excluded M0 2 Punctuated not performed Gradual 21 -0.59 -0.05 0.27 0.03 CS Punctuated 32 -0.64 0.08 0.32 0.01 Gradual 18 -1.01 0.46 0.26 0.06 ODH Punctuated 32 -1.02 0.04 0.40 0.005 Gradual 18 -1.26 -0.22 0.21 0.099 Log-transformed contrasts (y) of oxygen consumption rates (MO 2 = /j.mole O, g 'h ') and enzymatic activities (citrate synthase, CS. and octopine dehydrogenase. ODH, units g~'l of pelagic cephalopods were regressed against minimum depth of occurrence (.v), expressed as y = mA" + b. Number of taxa (n). regression coefficients (R 2 ) and P values are also presented. INDEPENDENT CONTRASTS FOR CEPHALOPOD METABOLISM habitat depth. The Cranchiidae is a very diverse family, and our data set is slightly biased toward cranchiid species (/; = 7 out of 39. MO 2 . punctuated model. Fig. Ib). Although many cranchiid species undergo ontogenetic vertical migra- tions in which successive developmental stages occupy progressively greater depths (12, 23), some species appear to remain near the surface until sexual maturity (24. 25). Seibel ct ul. (4) argued that the use of transparency (26) by the cranchiids reduces detection distances (27) at all depths and therefore allows them to employ sit-and-wait predation strategies, facilitating low metabolic rates, even in well-lit epipelagic waters. With the Cranchia-Liocranchia contrast removed, we consistently observed a much stronger rela- tionship between metabolism and habitat depth. 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(August 2001) Signaling via Water Currents in Behavioral Interactions of Snapping Shrimp (Alpheus heterochaelis) JENS HERBERHOLZ 1 * AND BARBARA SCHMITZ 2 1 Georgia State University, Department of Biology, P.O. Box 4010, Atlanta, Georgia 30302; and 2 Lehrstuhl fur Zoologie, TU Miinchen, Lichtenbergstr. 4. 85747 Garching, Germany Abstract. The snappping shrimp Alpheus heterochaelis produces a variety of different water currents during in- traspecific encounters and interspecific interactions with small sympatric crabs (Eurypanopeus depressus). We stud- ied the mechanisms of current production in tethered shrimp and the use of the different currents in freely behaving animals. The beating of the pleopods results in strong pos- teriorly directed currents. Although they reach rather far, these currents show no distinctions when directed toward different opponents. Gill currents are produced by move- ments of the scaphognathites (the exopodites of the second maxillae) and can then be deflected laterally by movements of the exopodites of the first and second maxillipeds. These frequent but slow lateral gill currents are most probably used to enhance chemical odor perception. The fast and focused, anteriorly directed gill currents, however, represent a powerful tool in intraspecific signaling, because they reach the chemo- and mechanosensory antennules of the opponent more often than any other currents and also because they are produced soon after previous contacts between the animals. They may carry chemical information about the social status of their producers since dominant shrimp release more anterior gill currents and more water jets than subordinate animals in intrasexual interactions. Introduction Alpheus heterochaelis of the family Alpheidae (Deca- poda, Caridea) is one of the largest snapping shrimp, reach- ing a body length of up to 55 mm. It shows a large, modified Received 27 November 2000; accepted 10 April 2001. * To whom correspondence should be addressed. E-mail: biojhh@ panther.gsu.edu snapper claw on one (left or right) side and a small pincer claw on the other side in both sexes (Williams, 1984). The snapper claw allows the animals to produce an extremely fast water jet (of up to 25 m/s; Versluis et al., 2000) by rapid claw closure after cocking the claw in the open position (Ritzmann. 1974). The high velocity of the water jet results in a pressure drop below vapor pressure that causes a cavitation bubble to grow to a size of about 3.5 mm in front of the snapper claw. The collapse of this bubble (and not as previously supposed the mechanical contact of both claw surfaces) causes the extremely loud (up to 215 dB re 1 ;u,Pa at 1 m distance; Schmitz, 2001) and short (about 500 ns) snapping sound (Versluis et al., 2000). The strong effect of the water jet and the cavitation bubble collapse can be seen during interspecific encounters. Small prey (e.g., worms, goby fish, or shrimp) can be stunned or even killed by the jet (MacGinitie, 1937; MacGinitie and MacGinitie, 1949; Mor- ris et al., 1980; Suzuki, 1986; Downer, 1989), and interspe- cific opponents (e.g., small sympatric crabs, Eurypanopeus depressus) can be injured at interaction distances of on average 3 mm (Schultz et al., 1998). Toward conspecifics the water jet was not observed to cause any damage but functions as a communicative signal (Herberholz and Schmitz, 1999), both opponents ensuring an interaction distance of on average 9 mm (Schmitz and Herberholz, 1998), which is far enough away from danger caused by implosion of the cavitation bubble. This hydrodynamic sig- nal is analyzed by the receiving shrimp predominantly with the help of mechanosensory hairs on the snapper claw, and may contain information about the strength, motivation, and sex of the snapper (Herberholz and Schmitz, 1998; Herber- holz, 1999). The still rather small interaction distance of less than 1 cm in agonistic encounters between two snapping shrimp WATER CURRENTS IN SNAPPING SHRIMP 7 also favors the exchange of chemical signals between the opponents. The literature on chemical orientation and com- munication in snapping shrimp is limited: Hazlett and Winn ( 1962) tested aggressive and defensive responses of Svnal- pheus lu'inphilli to crushed male or female extract, and Schein (1975) and Hughes ( 1996) investigated the choice of Alpheus heterochaelis toward extracts of male or female water in Y-maze experiments without clear-cut results. On the other hand, ablation of the chemosensitive antennules in Alpheus edwardsii strongly reduced pair formation and sex recognition, which may be due to impeded distant or contact chemoreception since the pairing frequency remained high when only the antennae were ablated (Jeng, 1994). The importance of olfactory signals during hierarchy formation was shown in male American lobsters (Karavan- ich and Atema. 1998a). In these experiments, the recogni- tion of urine-carried chemical signals, which were received by the antennules, allowed the subordinate animal to avoid the familiar dominant shrimp, and therefore reduced the duration and aggression of fights. The exchange of chemical signals is also assumed to play a major role in individual recognition and memory in male and female Homarus americamts (Karavanich and Atema, 1998b; Berkey and Atema, 1999). In lobsters, urine is released through a paired set of nephropores on the ventral sides of the basal segments of the second antennae (Parry, 1960). Agonistic behavior in lobsters causes an increase in the probability and volume of urine release (Breithaupt et al., 1999). The released urine is then carried by the powerful anteriorly directed gill currents and may therefore transfer chemical information from one animal to another (Atema, 1985). In recent studies (Zulandt Schneider et al., 1999; Zulandt Schneider and Moore. 2000), chemical cues were also described as an important source for recognition of the dominance status or stress condition of conspecifics in another crustacean, the red swamp crayfish (Procambarus clarkii). In light of these examples, a similar mechanism of chem- ical signal exchange via gill currents in snapping shrimp seems likely. We cannot, however, exclude the possibility that the animals also exchange hydrodynamic signals. In fact, it has been shown that the antennules of crayfish (Mellon, 1996) and lobsters (Guenther and Atema, 1998; Weaver and Atema, 1998) are equipped with both chemical and mechanosensory receptors, and detailed morphological studies of antennule sensory hairs favor the same situation in snapping shrimp (Schmitz, unpubl. obs.). Therefore, snapping shrimp may also perceive hydrodynamic stimuli as well as chemical stimuli with their antennules. Previous studies (Herberholz and Schmitz, 1998. 1999) have shown that the transfer of hydrodynamic signals is realized by the powerful water jet that is formed by rapid closure of the large claw. In contrast, the much weaker gill currents appear to be more suitable for transferring chemical information. Suspended plastic particles were successfully used to visualize and quantify biological flow fields in lobsters and crayfish in a series of experiments by Breithaupt and Ayers ( 1996, 1998). Small floating particles of the same density as seawater were added to the aquarium water and illuminated in a horizontal or vertical plane in the vicinity of a tethered animal. Flow fields were then analyzed by tracking individ- ual particles. It was shown that both lobsters and crayfish produce a great variety of flow fields by using the exopo- dites of the maxillipeds and by fanning the pleopods. The latter was also discussed with respect to chemical commu- nication: male American lobsters commonly fan their pleo- pods at the second entrance of their shelter, thus creating a strong current that may contain chemical information about the female positioned at the first entrance (Atema, 1985, 1988). The pleopod fanning frequencies in males correlate with the frequencies of females checking the shelter. The existence of pheromones that control female choice and molting as well as male aggression was therefore assumed (Cowan and Atema, 1990; Atema, 1995; Bushman and Atema. 1997). The possible exchange and use of different water currents during agonistic encounters has rarely been studied; but see Rohleder and Breithaupt (2000) for a preliminary study in the crayfish Astacus leptodactylus. To test the possibility that snapping shrimp use guided water currents as signals, we visualized and analyzed all water currents that the shrimp produced during their encounters with conspecifics of the same or different sex and in encounters with sympa- trically living mud flat crabs (Eurypanopeus depressus). Materials and Methods We analyzed the behavior of 12 adult specimens of Alpheus heterochaelis. a species of snapping shrimp (6 males, 6 females; body size: 3.9 0.4 cm. mean SD). Each animal was tested in an encounter with a conspecific of equal size of either the same or different sex, as well as in an encounter with a small crab (Eurypanopeus depressus; mean length and width of carapace: 1.6 0.2 X 1.2 0.2 cm, mean SD). All animals were caught in waters of the Gulf coast of Florida at the Florida State University Marine Laboratory near Panacea. Prior to the experiments the ani- mals were labeled with small numbers designated for mark- ing queen bees and were kept individually in perforated plastic containers ( 1 1 X 11 X 15 cm) containing gravel and oyster shells for shelter. The containers were placed within a large tank (90 X 195 X 33 cm) with 330 1 of circulating filtered seawater (salinity: 23%c^28%o; temperature: 22- 23C). Proteins were removed from the water, and pH. carbonate, oxygen, CO 2 . and NO 3 were regularly con- trolled. The shrimp were exposed to an illumination cycle of 12 h light/ 12 h dark and fed frozen shrimp, fish, or mussels three times a week. For visualization of the different water currents, we pre- J. HERBERHOLZ AND B. SCHMITZ pared the aquarium water (temperature: 22-24"C, water level: 5 cm) with small, floating plastic panicles (ABS- particles, Bayer, Leverkusen, diameter: 500-710 jum; spe- cific weight: 1.03 kg/1). The aquarium (30 X 24 X 24 cm; floor covered with black cloth to facilitate walking) was positioned on a platform isolated from vibrations (Breit- haupt et at., 1995). At the level of the interacting animals, the seawater was illuminated from one side by a slide projector holding a slide with a thin horizontal slit. Before each experiment fresh seawater and particles were added, and two animals (two snapping shrimp or one snapping shrimp and a crab) were placed in the aquarium for 10 min for acclimatization: the animals were separated by an opaque divider to prevent visual, tactile, and directed-chem- ical contact. After the partition was removed, all interac- tions between the animals during the following 20 min were videotaped from above (camera: Panasonic AG 455; video recorder; Panasonic AG 7355; monitor: Sony Trinitron). The reflexive characteristics of the suspended particles then allowed a precise tracking using standard video-frame anal- ysis. Each experiment (interactions between two snapping shrimp of the same or different sex or between a snapping shrimp and a crab) was characterized by the number of physical contacts between the opponents, regardless of their duration and strength, as well as by the number of water jets. Three different water currents were characterized, in- cluding a lateral gill current, an anterior gill current, and a pleopod current (Fig. la). The pleopod current was mea- sured only when the shrimp was not in locomotion, because this current is also likely to be used in supporting the animal's walking. Moreover, no current was included in our analysis unless the single-frame video analysis gave clear evidence that it had moved two or more plastic particles. The following parameters were evaluated for all visualized water currents: frequency, duration (time between onset of movement of the first floating particle and end of movement of the last particle), range (total distance covered by an identified particle due to a certain current: possibly under- estimated when the current hit an opponent or an aquarium wall), velocity and target of the currents, their potential to transfer chemical information (i.e.. entering the area of chemical perception at the receiver's side), the temporal correlation between currents and previous physical contacts, and the correlation between produced currents and water jets in winners and losers during intrasexual interactions. To determine a winner or loser, we counted the number of aggressive acts and the number of submissive acts after each physical contact between the conspecitic opponents throughout the encounter. Aggressive acts include behav- iors such as approach, aggressive stance, and grasping and opening of the claws. Submissive acts include moving back- wards and turning and tail flipping away from the opponent. These definitions are largely adopted from Nolan and Salmon (1970). In 11 out of 12 experiments, one animal produced more aggressive acts and fewer submissive ones than its opponent and was therefore determined to be the winner while the opponent was determined to be the loser. Statgraphics Plus 6.0 (Manugistics Group, Inc.) and SPSS 6.0.1. (SPSS Science Software GmbH) were used for statistics. Mean and standard deviation were calculated for each variable of interest for each tested individual, and only one value per individual (grand mean) is included in each statistical test. The behavior of the respective opponents (male and female snapping shrimp, and crabs) was not analyzed and is not included in our results (exception: data presented in Fig. 7). If not otherwise stated, the Friedman rank test for repeated measurements (sample size >2) or the Wilcoxon rank test (sample size = 2) were used, and values with P < 0.01 and P < 0.05 are indicated in the text. We used nonparametric statistical tests because most of the data did not fulfill the requirements for the use of parametric tests i.e., normality or equal variance. To gain more insight into the mechanism of gill current production and redirection, two snapping shrimp were teth- ered upside down in a small petri dish filled with seawater and floating plastic particles, and the activity of the different mouth parts, which produced or deflected the currents, was videotaped using a CCD camera (Sony XC-77CE) mounted on a binocular microscope with high magnification. In ad- dition, small drops of black ink (Brilliant Black 4001. Pelikan) were placed between the third and fourth walking legs of these shrimp as well as of animals tethered dorsal side up to a vertical holder and standing on a platform so that the gill currents could be visualized. (Fig. Ib). Results Visualization of water currents in tethered shrimp A unique feature of snapping shrimp is the production of an extremely rapid water jet by fast closure of a specialized snapper claw. Apart from this water jet. the snapping shrimp Alpheus heterochaelis is able to produce four kinds of water currents (Fig. 1), which can be subdivided into two main categories. Fanning of the pleopods causes a strong, poste- riorly directed pleopod current, and a gill current is pro- duced by rhythmically beating the scaphognathites as re- vealed by our visualization experiments in two tethered shrimp. Beating of the scaphognathites produces a depres- sion in the gill chamber; water is therefore sucked into this chamber and subsequently released anteriorly through two small openings in the carapace. This "normal" gill current can be visualized with ink in tethered animals, but it is too slow and weak to move floating particles and was therefore not analyzed during encounters of snapping shrimp and their opponents. It can, however, be accelerated and de- flected into a lateral gill current (see Fig. IB) by the exopodites of the second and third maxillipeds. The exopo- WATER CURRENTS IN SNAPPING SHRIMP 'normal" gill current pleopod current lateral gill current antennule anterior gill current Figure 1. (A) Schematized drawing (lateral view) of a snapping shrimp modified after Kim and Abele ( 1988) showing four different water currents (gray arrows): the "normal" gill current, the lateral gill current, the anterior gill current, and the pleopod current. Black arrows show the direction of water entering the gill chamber. (B) Frontal view of an A/pheiis helerochaelis snapping shrimp, tethered to a vertical holder by means of a plastic nut glued to the carapace and standing on a textile platform. Black ink was placed with a syringe between the third and fourth left pereiopods (see ink trace) to visualize the gill currents. The shrimp is fanning the exopodites of the right second and third maxillipeds. thus producing an ink-stained lateral gill current to the right. dites of the first maxilliped do not participate in this process. Fanning of the left exopodites results in acceleration and deflection of the released gill current to the left side, and fanning of the right exopodites results in deflection to the right side. Tethered snapping shrimp never beat the exopo- dites of both sides simultaneously, and this was also never observed during interactions in which the illuminated par- ticles were directed to only one side at a time. Interestingly, 10 J. HERBERHOLZ AND B. SCHMITZ D 1-gc a-gc homo hetero type of interaction inter Figure 2. Frequency of three different water currents (1-gc, lateral gill current, a-gc, anterior gill current, pc, pleopod current) produced by Al- pliens heterochaelis snapping shrimp in interactions with another shrimp of the same sex (homo), of different sex (hetero), and with a Eurypanopeus depressus crab (inter). Grand means and standard deviations for 12 snap- ping shrimp each are shown. Significant differences within interaction types with P < 0.01 are indicated by two asterisks (**). a (fast) anterior gill current was restricted to encounters of freely moving animals; it could not be elicited in tethered shrimp. Its production obviously requires physical, chemi- cal, or visual contact between the animals. As a result, we were not able to analyze the producing mechanism; that is, we did not identify the involved mouth parts. General characteristics of released water currents Encounters between two snapping shrimp of different sex (hetero) are characterized by a significantly higher number of physical contacts (23.9 8.3, /; = 287; P < 0.01) than seen in encounters between two shrimp of the same sex (homo; 13.8 6, n = 165), or between a snapping shrimp and a crab (Eurypanopeus depressus) (interspecific; 12.7 5.3. n = 157). On the other hand, snapping (water jet production) of the tested shrimp is significantly increased after a contact with a crab (38% 16<7r; P < 0.01) when compared to snapping after hetero and homo contacts (5% 4% and 11% 11%, respectively). These differences in mind, we first evaluated the number of water currents (lateral gill currents, anterior gill currents, and pleopod currents) in each experiment. Figure 2 shows that there are no essential differences between interaction types (homo, hetero, or interspecific). Within each interac- tion type, however, the number of lateral gill currents sig- nificantly (P < 0.01 ) exceeds that of anterior gill currents as well as that of pleopod currents. In addition, in interspecific encounters with a crab, the frequency of anterior gill cur- rents is significantly lower than the frequency of pleopod currents (P < 0.01). The duration of the different water currents (Fig. 3A) tends to be longest for lateral gill currents, with no signif- icant differences regarding the type of the opponent. The duration of anterior gill currents is generally shorter, with similar values in intraspecific interactions, yet almost twice as long as in interactions with a small crab. Anterior gill currents in interspecific encounters are significantly shorter in duration than lateral gill currents (P < 0.05). Pleopod currents, in contrast, reveal very consistent values for all types of interactions. Figure 3B shows the range of the different currents in all interaction types. Regardless of the opponent, the snapping shrimp tend to produce lateral gill currents with small ranges. Anterior gill currents generally cover larger dis- tances in intraspecific interactions, whereas the mean value is reduced in interactions with a crab. The most powerful current is the pleopod current, which covers long distances in all interaction types. Range differences within interaction types are significant at P < 0.05 and P < 0.01, respectively. The velocity of the water currents during the first 120 ms (6 video frames) was evaluated for 10 examples for each current and interaction type (Fig. 3C). There are no signif- icant differences in the velocities within and between dif- ferent types of interactions. The lateral gill current shows the slowest velocities in all encounters. The anterior gill current and the pleopod current show similar values and are both more powerful than the lateral gill current. Initial velocities are higher, but their analysis has not proved satisfactory because of the standard video time resolution of 20 ms (50 frame/s). Temporal relation of water currents to physical contact Figure 4 compares the frequency of water currents that were elicited within 10 s after a physical contact between the opponents with those that were "spontaneously" pro- duced that is, emitted more than 10 s after a preceding contact. As shown in Figure 4A. in all interaction types the lateral gill current is significantly more often produced spontaneously than following a physical contact (P < 0.01 ). In homo interactions it occurs in only 6.2% of all cases (n = 10 of 162) shortly after a contact. During hetero interactions this current is elicited by a contact in 11.5% of all cases (n = 2\ of 183); in interactions with a crab, the lateral gill currents occur within 10 s after a contact in only 8.5% of all cases (n = 13 of 153). The analysis of the anterior gill current reveals a com- pletely different frequency pattern, with more elicited cur- rents than spontaneous ones (Fig. 4B). In homo interactions the anterior gill current is produced in 65.5% of all cases (/; = 19 of 29) within 10 s after a preceding contact. Similarly, in hetero interactions this gill current is elicited by a contact in 62.5% of all cases (n = 15 of 24). Finally, during interactions with a crab, anterior gill currents are WATER CURRENTS IN SNAPPING SHRIMP II 1 u -a 25 20 15 10 B 1 U M I CJ 10s) homo hetero type of interaction inter 25 i/i 1 20 -i C ^ o 1 5 JO B B I o D 40 3 C 1-gc (ha) D 1-gc (ot) ** ** 1 homo hetero inter type of interaction a-gc (ha) D a-gc (ot) homo hetero inter type of interaction pc(<10s) D pc(>10s) urrents s DC C ** ** ** O 4-1 O 1 A 4 JO 1 9 c 2 J. hoi , - no hetero inter type of interaction 4. Mean number ol' lulcriil gill currents (A), anterior gill cur- rcnls (If), and |iloo|ioil cuiu-nls (C'l \\ithin 10 s (lilack tolunin-.) cu more than 10 s (white columns) alter a physical contact between the opponents in inlet, K lions ol Iwo siuppmg sin imp ol ilie same sex (homol, ot tlitTercnt se\ (heleio). anil ol a snapping shrimp ami a crab (inter), (iraiul means and standard deviations for 12 shrimp are shown. Significant differences with /' < 0.01 are indicated In luo asterisks ('*). V) *- o u- OJ I 3 C pc (ha) D pc (ot) homo hetero type of interaction inter ' 5. Mean number of lateral gill currents (A), anterior gill currents (B). and pleopod currents (C) hitting the antennules of the opponent (black columns, ha) or reaching other targets (white columns, ot) in interactions of two snapping shrimp ol the same se\ (homo), of different sex (hetero), and of a snapping shrimp and a crab (inter). Grand means and standard deviations for 12 shrimp are shown. A significant difference with P < 0.05 is indicated by tine asterisk (*) and with P < 0.01 by two asterisks (**). WATER CURRENTS IN SNAPPING SHRIMP 13 homo hetero number of a-gc to ^ O^ 00 C 1 1 / "/i 1 2 4 6 8 10 number of jets 2 4 6 8 10 number of jets 1 1 1 3246 number ot jets i i Figure 6. Correlation between the number of water jets and the num- ber of anterior gill currents produced in interactions of two snapping shrimp (A) of the same sex (homo; Spearman's coefficient of rank corre- lation r, = 0.9, P < 0.01). (B) of different sex (hetero). and (C) of a snapping shrimp and a crab (inter). Data of \2 shrimp each some data points overlap. that of lateral gill currents: the undirected currents signifi- cantly exceed the antennule-directed ones in each interac- tion type (P < 0.05 or 0.01, respectively; Fig. 5C). In homo interactions an average of only 1 1.5% (n = 6 of 52) of all pleopod currents are projected towards the chemoreceptive antennules. and during hetero interactions 16.7% (/; = 8 of 48) of all pleopod currents reach the antennule area. Finally. in interspecific interactions no pleopod current is aimed towards the antennules of the crab, but all (;i = 54) are directed elsewhere. Anterior gill currents and water jets In view of the prominent role of the anterior gill current with respect to its timing after a physical contact and the increased possibility of chemosensory information transfer, we tested the correlation between these gill currents and emitted water jets (Fig. 6). As mentioned before, in com- parison to intraspecific interactions, encounters with crabs are characterized by an increased number of water jets and a reduced number of anterior gill currents (Fig. 6C). In addition, more water jets are emitted in homo interactions between snapping shrimp (Fig. 6A) than in hetero encoun- ters (Fig. 6B). Thus, the number of anterior gill currents significantly increases with an increasing number of water jets only in interactions between two snapping shrimp of the same sex (Spearman rank correlation coefficient: >\ = 0.9, P < 0.01: Fig. 6A). This is not the case in interactions between two shrimp of different sex d\ = 0.5, P > 0.05), though a noticeable trend is shown and the overall low number of water jets may have prevented a significant result. An even lower degree of correlation is seen in interactions with a crab (r v = 0.4, P > 0. 1 ). As shown in Figure 7, winners of homo interactions (as defined by aggressive and submissive acts see Materials and Methods) not only produce a significantly higher mean number of water jets (N = II, P < 0.01) but also a significantly higher mean number of anterior gill currents than losers produce (N = 1 1; P < 0.01 ). Discussion Snapping shrimp (Alpheus hetemcluielis) produce two main water currents, a strong posteriorly directed pleopod current and an anteriorly directed gill current. We show that the "normal" anteriorly directed gill current can be modified and redirected into a lateral and a fast anterior gill current. The production of the latter is restricted to social interac- tions, in which it represents a powerful tool for chemical signaling. Moreover, the use of the fast anterior gill currents varies for the winners and losers of individual encounters. Mechanisms of gill current production Our experiments in tethered snapping shrimp show that water is sucked into the gill chamber due to a depression elicited by the beating scaphognathites (Fig. 1A). A "nor- mal" gill current is then released anteriorly with low veloc- ity through two small openings of the carapace. Once the left or right expodites of the second and third maxillipeds start fanning, the current is accelerated and deflected later- ally to that side (Fig. IB). As previously described in winner loser Figure 7. Frequency of water jets (jets, black columns) and anterior gill currents (a-gc, white columns I lor winners and losers in interactions of two snapping shrimp of the same sex. The significant differences between winners and losers with P ^ 0.01 are indicated by two asterisks (**). 14 J. HERBERHOLZ AND B. SCHMITZ lobsters (Homarus americanus), the exopodites of the first maxillipeds do not contribute to these lateral gill currents in snapping shrimp, whereas in crayfish (Procambarus clarkii) these appendages are also involved (Breithaupt. 1998). The production mechanism of the fast anterior gill current remains unclear, since this behavior obviously requires physical, chemical, or visual contact during intra- or inter- specific encounters of snapping shrimp, and thus was never seen in tethered animals. From our knowledge about the lateral gill current, we assume that the fast anterior gill current is created by high-frequency beating of the scapho- gnathites without contribution of the exopodites of the sec- ond and third maxillipeds. Since it is difficult to video- record the mouth parts with high magnification during social interactions, we are currently testing other methods of monitoring scaphognathite beating frequencies during en- counters to verify this hypothesis. Role of the fast anterior gill current during social interactions The analysis of the fast anterior gill current revealed the most surprising and interesting results. Although anterior gill currents were observed and well described in lobsters (Atema, 1985. 1995) and crayfish (Breithaupt, 1998), we found decisive differences in snapping shrimp. First of all, Alpheus heterochaelis produces different types of anterior gill currents. The "normal" anterior current is a slow, weak release of water, which was sucked through the gill cham- ber, as opposed to the fast, strong, anteriorly directed gill current, which occurs during social interactions. The pro- duction of the fast anterior gill current is rare (Fig. 2) but strongly linked to previous contacts with a conspecific or a crab (Fig. 4B). Among the observed currents, only the fast anterior current is created shortly after a preceding contact, regardless of the type of opponent. In fact, this current never occurred before the first contact. Moreover, we show that only this current is suited to transfer chemical information towards the other animal (Fig. 5B): it reaches the antennules of the opponent in nearly 50% of all cases. Of all analyzed currents, only the fast anterior gill current shows some peculiarities with respect to the shrimps' op- ponent. The number, duration, and range is smaller in en- counters with a crab than in interactions with conspecifics (Figs. 2, 3). We assume that the shrimp collect information about the genus of their opponent and reduce the effort to communicate accordingly, if it is a crab. Role of lateral gill currents during social interactions During social interactions between snapping shrimp and conspecifics of the same or different sex as well as during interactions with small crabs, the lateral gill currents are most prominent and significantly outnumber all other ob- served currents (i.e., pleopod currents and fast anterior gill currents; Fig. 2). Moreover, they are produced for long intervals but have a short range and a low velocity (Fig. 3). They are barely elicited by physical contact (Fig. 4A) and hardly ever reach the antennules of their opponents (Fig. 5A). These properties of the lateral gill currents do not change with different opponents but appear to result from a stereotyped form of production. Thus, obviously lateral gill currents are not predestinated to play a prominent role in active (chemical) signaling between the animals. Still, their function needs explanation. From our obser- vations we conclude that the lateral gill current is used to improve the shrimps' ability to sense possible odor signals that occur at close distance. By redirecting the "normal" gill current, the shrimp refreshes the area around its chemical receptors from its own smell (released by the slow and permanent gill current) and thereby improves the detection of the chemical surrounding. This idea is supported by our knowledge that Alpheus heterocliaelis naturally inhabits small, oyster-shell-covered areas with little water flow and that individuals of the species appear to be rather stationary within that area (Herberholz and Schmitz, pers. obs.). The lateral gill current produced by snapping shrimp seems to be used to remove water from the area around the antennules and to a much lesser extent to draw water toward that region as proposed for the posteriorly or laterally redirected gill currents of lobsters and crayfish (Atema, 1995; Breithaupt. 1998). In contrast to lobsters and crayfish, snapping shrimp were never observed to fan simultaneously with appendages on both sides. Instead, they beat the exopodites of one side at a time, and there are no obvious movements of particles from the opposite side toward the animal's anterior region. Role of pleopod currents during social interactions In lobsters (Homarus americanus), pleopod currents are used for chemical (possibly pheromonal) communication during courtship at a shelter (Atema. 1985. 1988. 1995; Cowan and Atema, 1990: Bushman and Atema, 1997). The snapping shrimp Alpheus heterocliaelis, in addition to using its pleopods for locomotion and to provide an oxygen sup- ply for attached eggs, uses them for shelter digging, fanning the substrate (sand or muddy-sand) backward behind it (Nolan and Salmon, 1970). These authors also mention (pleopod) fanning as an aggressive act, with a shrimp vig- orously beating its pleopods and directing a water current posteriorly quite close to another shrimp. The frequency of pleopod fanning is not noted by Nolan and Salmon (1970), but the behavior was described to occur between two fe- males at the entrance of a shelter. In our experiments, we did not provide a shelter, and all shrimp were in the middle of their molt cycle. In view of the finding that the actual impact of pleopod currents in lobsters depends to a high degree on the molt state of the animals as well as on their readiness to mate (Cowan and Atema, 1990), these condi- WATER CURRENTS IN SNAPPING SHRIMP 15 tions may have affected our results. Though pleopod cur- rents were rather often produced (Fig. 2) and (in comparison to gill currents) show an average duration, a large range, and high velocity (Fig. 3). there is a lack of correlation with previous contacts (Fig. 4C) and a low precision in hitting the antennules of the opponent (Fig. 5C). There are hardly any differences in the characteristics of these currents towards different opponents. All this indicates that pleopod currents are of little relevance for (chemical) signaling or commu- nication among snapping shrimp and between shrimp and sympatric crabs under our conditions. A specialized gill current for chemical .signaling and communication ? The transfer of chemical signals between interacting lob- sters (see e.g., Atema. 1995; Bushmann and Atema. 1997) and crayfish (Breithaupt et al., 1999) has been described in detail. In lobsters these signals can evoke long-term indi- vidual recognition (Karavanich and Atema, 1998a, b), and in crayfish they communicate dominance status or stress condition (Zulandt Schneider et al., 1999; Zulandt Schnei- der and Moore, 2000). In all cases, urine-borne signals were assumed to be the source of chemical signaling (Breithaupt et al., 1999; Breithaupt, pers. comm.). Since the urine is released through a paired set of nephropores on the ventral sides of the basal segments of the second antennae (Parry. 1960). it can be carried toward an opponent by the anterior gill current. Moreover, agonistic behavior in catheterized lobsters increases the probability and volume of urine re- lease (Breithaupt et al., 1999). In the present study we show for the first time that the pattern of water current production actually changes with respect to the social situation of an aquatic animal. Although snapping shrimp have the ability to produce "normal" an- terior gill currents, they create different, more powerful, anteriorly directed gill currents shortly after contacting their interaction partner. These elicited currents are then more likely to reach the opponents' area of chemical perception. The same may hold true for lobsters and crayfish, but their currents have not yet been quantified during social interac- tions. On the other hand, we still have to prove that the fast anterior gill current in snapping shrimp actually carries chemical signals toward the opponent. Although the data presented favor this assumption, we cannot exclude the possibility that hydrodynamic signals transferred by the gill currents participate in the communication between the ani- mals. Judging by their sensory equipment, snapping shrimp like crayfish (Mellon. 1996) and lobsters (Guen- ther and Atema, 1998; Weaver and Atema. 1998) are most likely to perceive hydrodynamic stimuli as well as chemical stimuli with their antennules (Schmitz, unpubl.). We plan to test this possibility by deactivating the chemical receptors only. In any case, the production of the fast anterior gill current may play a critical role during hierarchy formation in snap- ping shrimp. We show that in intrasexual encounters the numbers of water jets and anterior gill currents are posi- tively correlated (Fig. 6) and that both are significantly higher in the winner than in the loser (Fig. 7). In the present study, winner and loser met in only a single 20-min exper- iment. Preliminary experiments show that repetitive pairing of winners and losers reduces the number of water jets and anterior gill currents (Obermeier and Schmitz. unpubl.). 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Williams, A. B. 1984. Shrimps, Lobsters, and Crabs of the Atlantic Coast of the Eastern United States. Maine to Florida. Smithsonian Institution Press, Washington. DC. / nl. inch Schneider, R. A., and P. A. Moore. 2000. Urine as a source of conspecific disturbance signals in the crayfish Procambarus clarkii. J. E\p. Biol. 203: 765-771. Zulandt Schneider, R. A., R. W. S. Schneider, and P. A. Moore. 1999. Recognition of dominance status by chemoreception in the red swamp crayfish, Procambarus clarkii. J. Chem. Ecol. 25(4): 781-794. Reference: Biol. Bull. 201: 17-25. (August 201)1) Methionine-Enkephalin Induces Hyperglycemia Through Eyestalk Hormones in the Estuarine Crab Scylla serrata P. SREENIVASULA REDDY* AND B. KISHORI Department of Biotechnology, Sri Venkateswara University, TIRUPAT! - 517 502, India Abstract. The hypothesis is tested that methionine-en- kephalin. a hormone produced in and released from eyestalk of crustaceans, produces hyperglycemia indirectly by stim- ulating the release of hyperglycemic hormone from the eyestalks. Injection of methionine-enkephalin leads to hy- perglycemia and hyperglucosemia in the estuarine crab Scylla serrata in a dose-dependent manner. Decreases in total carbohydrate (TCHO) and glycogen levels of hepato- pancreas and muscle with an increase in phosphorylase activity were also observed in intact crabs after methionine- enkephalin injection. Eyestalk ablation depressed hemo- lymph glucose (19<7r) and TCHO levels (22%), with an elevation of levels of TCHO and glycogen of hepatopan- creas and muscle. Tissue phosphorylase activity decreased significantly during bilateral eyestalk ablation. Administra- tion of methionine-enkephalin into eyestalkless crabs caused no significant alterations in these parameters when compared to eyestalk ablated crabs. These results support the hypothesis that methionine-enkephalin produces hyper- glycemia in crustaceans by triggering release of hypergly- cemic hormone from the eyestalks. Introduction In decapod crustaceans, hemolymph sugar level is regu- lated by hyperglycemic hormone. Abramowitz et al. ( 1944) were the first to demonstrate that injection of eyestalk extract induced hyperglycemia in Callinectes. Since then, hyperglycemia as a response to injection of eyestalk extract Received 14 July 2000; accepted 6 March 2001. * To whom correspondence should be addressed. E-mail: psreddy@vsnl.com has been observed in almost all groups of crustaceans (see review by Keller, 1992). This neurohormone is stored in and released from the sinus gland. The chemical nature, mode, and site of action of hyperglycemic hormone has been extensively studied in a number of crustaceans (see reviews by Keller et al., 1985; Sedlmeier, 1985). The amino acid sequence of hyperglycemic hormones has been determined from a large number of crustaceans (see La Combe et al., 1999, for review). The gene for hyperglycemic hormone was also cloned from crabs (Kegel et al., 1989), lobster (Tensen et al., 1991), prawn (Ohira et al., 1997). isopod (Martin et al., 1993). and crayfish (Kegel et al., 1991; Huberman et al., 1993; Yasuda et al., 1994). Recently, we reported the expression of hyperglycemic hormone gene at different molt stages in Homarus americanus, the American lobster (Reddy et al.. 1997). Since the discovery of opioid peptides in decapod crus- taceans by Mancillas et al. (1981). several workers have attempted to determine the physiological function of these peptides, but the results are fragmentary. Sarojini et al. (1995. 1996. 1997) provided evidence that methionine- enkephalin slowed ovarian maturation in the fiddler crab Uca pugilator and the crayfish Procanibarus clarkii, and suggested that methionine-enkephalin produces this effect indirectly by stimulating the release of gonad-inhibiting hormone from eyestalks. In Uca pugilator, methionine- enkephalin appears to stimulate release of the concentrating hormones for black and red pigment cells (Quackenbush and Fingerman. 1984) and the dark-adapting hormone for distal retinal pigment cells (Kulkarni and Fingerman. 1987). We reported a neurotransmitter role for methionine-en- kephalin in regulating the hemolymph sugar level of the freshwater crab O-ioielphusa senex senex, and hypothesized that methionine-enkephalin produces hyperglycemia indi- 17 18 P. S. REDDY AND B. KISHORI rectly by stimulating release of hyperglycemic hormone (Reddy, 1999). The objectives of the present study were threefold: (a) by extending our studies to the estuarine crab Scylla serrata, to test our hypothesis, generated by the study of Oziotelphusa senex senex, that methionine-enkephalin produces hyper- glycemia in decapod crustaceans; (b) to determine the changes in levels of tissue carbohydrates and phosphorylase activity during methionine-enkephalin treatment; and (c) to provide evidence that supports the triggering of release of hyperglycemic hormone during methionine-enkephalin treatment. Materials and Methods Individuals of Scylla serrata (15 2 cm in carapace width; 110 5 g wet weight) were collected from the Chennai coast, India. They were kept in large aquaria with continuous aeration and acclimatized to laboratory condi- tions for one week under constant salinity (25 1 ppt), pH (7.2 0.1 ), and temperature (23 2C). During this period the crabs were fed fish flesh. Feeding was stopped 24 h before the beginning of the experiments, and no food was given during experimentation. Only intermolt (Stage C 4 ), intact, male crabs were used in the present study. Methionine-enkephalin (Sigma Chemical Co.) was dis- solved in physiological saline (Pantin, 1934). In these ex- periments, each of the 10 groups of crabs used consisted of 10 individuals. The first group served as normal and re- ceived no treatment. A second group served as control, with each crab in this group receiving an injection of 10 /il of physiological saline (Pantin, 1934) through the base of the coxa of the 3rd pair of the walking legs. In groups 3-5 respectively, each crab received an injection of 10~ 7 , 10~ s , and 10~ y mole methionine-enkephalin in 10 jal volume. Both eyestalks were ablated from all the crabs in groups 6-10. The eyestalks were extirpated by cutting them off at the base, without prior ligation but with cautery of the wound after operation. Twenty-four hours after eyestalk ablation, these groups were used for experimentation. Crabs in group 6 served simply as eyestalkless animals, and crabs in group 7 received 10 ju,l crustacean Ringer solution and served as eyestalkless controls. In groups 8-10 respectively, each crab was injected with 10~ 7 , 10~ x , and 10~ 9 mole methionine-enkephalin in 10 /xl volume. Based on prelim- inary kinetic studies, the crabs were sacrificed for analysis 2 h after injection (Figs. 1. 2). Hemolymph (500 jul) was aspirated by syringe, through the arthrodial membrane of the coxa of the 4th pair of walking legs. The other tissues (hepatopancreas and muscle from chela propodus) were then quickly dissected out. weighed, and analyzed by the procedures outlined below. Hemolvmph total carbohydrate level. Hemolymph total carbohydrate (TCHO) levels were estimated in trichloroace- tic acid supernatant (10% TCA w/v) according to the method of Carroll et al. ( 1956). Hemolymph glucose level. For measurement of glucose, 100 /u,l of hemolymph was mixed with 300 ju.1 of 95% ethanol. After deproteinization (4 C, 14,000 X g, 10 min), the sample was combined with a mixture of glucose enzyme reagent (glucose-6-phosphate dehydrogenase and NADP) and color reagents (phenazine methosulfate and iodo- nitrotetrazolium chloride) (kit from Sigma). After 30 min, the intensity of the color was measured at 490 nm and quantified with standards. Tissue TCHO and glycogen levels. TCHO levels in the tissues (hepatopancreas and muscle) were estimated in the 10% TCA supernatant (5% w/v), and glycogen was esti- mated in the ethanolic precipitate of TCA supernatant, ac- cording to the method of Carroll et al. ( 1956). To 0.5 ml of clear supernatant was added 5.0 ml of anthrone reagent, and the combination was boiled for 10 min in a water bath. The samples were then immediately cooled. A standard sample containing a known quantity of glucose solution was always tested along with the experi- mental samples. Absorbance was measured at 620 nm against a reagent blank. Tissue phosphorylase activity. Phosphorylase activity was assayed in hepatopancreas and muscle by colorimetric determination of inorganic phosphate released from glu- cose- 1 -phosphate by the method of Cori et al. (1955). First, 0.4 ml of the enzyme was incubated with 2.0 mg of glyco- gen for 20 min at 35 C, then the reaction was initiated by the addition of 0.2 ml of 0.016 M glucose- 1 -phosphate (G-l-P) to one tube (phosphorylase a) and a mixture of 0.2 ml of G-l-P and 0.004 M adenosine-5-monophosphate (phosphorylase ah) to another tube. The reaction was incubated for 15 min for determining total phosphorylase and for 30 min for active phosphor- ylase. The reaction was terminated by the addition of 5.0 ml of 5 N sulfuric acid. Released inorganic phosphate was estimated by the method of Taussky and Shorr (1953). Protein determination. Total protein levels in the enzyme source were estimated following the method of Lowry et al. (1951) using bovine serum albumin as standard. MET-ENKEPHALIN-INDUCED HYPERGLYCEMIA IN CRAB 19 Table 1 Effect of eyestalk ablation fESX) (24-h post-ablation) and injection of methionine-enkephalin into intact and ablated crabs on hemolymph total sugar aiui glucose levels in Scylla serrata No treatment Ringer injection 10~ 7 mol/crab 10 " mol/crab 10~ 9 mol/crab Dunnet's comparison test Total Sugar (mg/100 ml) Intact (Group 1 ) ESX (Group 2) 12.11 1.01 9.41 1.13" (-22.22) 12.73 1.84 a (5.12) 9.34 1.03 b ' c (-0.74) 28.8 2.18 h (126.23) 9.41 1.13 b ' c (0.74) 19.64 I.41 h (54.28) 9.43 1.01 b - c (0.96) 16.52 1.94 h (29.77) 9.21 1.08 b ' c (-1.39) F (4-45) = 137.160 F (4 . 45 , = 0.099 Intact (Group 1 ) ESX (Group 2) Two-way ANOVA: F, w (Between groups) = 772.002, P < 0.001; F 4 9n (Among treatments) = 98.747, P < 0.001; F 490 (Interaction) = 94.552, P < 0.001. Glucose (mg/100 ml) 6.55 0.76 a 12.07 1.34" 11.44 1.28" 9.13 0.78" F (4 45l = 75.613 (2.16) (84.27) (74.65) (39.38) 5.52 0.81 Kc 5.19 1.01 b - c 5.21 0.91 bc 5.44 0.77 Kc F, 445 , = 0.387 (6.35) (-5.97) (-5.61) (-1.44) 5.19 1.01 h (-19.03) Two-way ANOVA: F l 90 (Between groups) = 440.810. P < 0.001; F 4 90 (Among treatments) = 40.092, P < 0.001: F.,,,,, (Interaction) = 44.753, P < 0.001. Values are mean SD of 10 individual crabs. Values in parentheses are percent change from control. For calculation of percent change for eyestalk-ablated (ESX) crabs and Ringer-injected intact crabs, intact crabs served as control; for met-injected crabs. Ringer-injected crabs served as control. a Not significant compared with intact crabs. b /> < 0.001 compared to intact crabs. L Not significant compared io eyestalkless crabs. Statistical analysis. Statistical analysis of the results was made using a two-way ANOVA test followed by Dunnet's multiple range test (preceded by one-way ANOVA), using SPSS version 10.0 (SPSS Inc., Chicago, ID. the possible mobilization of glucose molecules from hepa- topancreas and muscle to hemolymph. Phosphorylase (both total and active) activity levels were significantly increased in both hepatopancreas and muscle Results Effects of methionine-enkephalin on carbohvdrate metabolism of intact crabs Injection of methionine-enkephalin into intact crabs re- sulted in significant hyperglycemia and hyperglucosemia in a dose-dependent manner (Table 1 ). whereas injection of physiological saline had no effect on hemolymph carbohy- drate levels. At doses between 10~ 9 mol/crab (36.41%) and lO" 6 mol/crab (147.81%), the effect of methionine-en- kephalin was statistically significant. For doses lower than 10~ 9 mol/crab, however, methionine-enkephalin did not elicit a hyperglycemic response (Fig. 1 ). A time course for methionine-enkephalin-induced hyperglycemia is shown in Figure 2 for a IO" 7 mol/crab dose, which is a nearly saturating dose. The hemolymph glucose level increased significantly within 30 min of methionine-enkephalin injec- tion, reached a peak at 2 h, then declined gradually. Hepatopancreas glycogen and TCHO levels in crabs that received methionine-enkephalin were significantly lower than those of control crabs (Table 2). Decreases in muscle glycogen and TCHO levels were also significant after the injection of methionine-enkephalin (Table 3), suggesting 36 ~ 30 IIS MS SALINE- _1Q INJECTED 10 -9 '0 10 10 [Methioninc -Enkcphatin] (mol/crab) -6 10 10* Figure 1. Dose-dependent effect of methionine-enkephalin on the hemolymph glucose levels in intact Scylla serrata. Two hours after injec- tion of saline (10 /nl/animal) or methionine-enkephalin at the doses indi- cated, hemolymph was withdrawn from crabs for glucose determination. Each bar represents a mean SD (n = 10). Numbers in parentheses indicates the percent increase from the normal values. * Significant differ- ence from normal crabs at P < 0.001. NS Not significant. 20 P. S. REDDY AND B. KISHORI Time after injection (h) Figure 2. Time course of methionine-enkephalin-induced hyperglyce- mia. After injection of methionine-enkephalin (10~ 7 moL/crab). hemo- lymph was withdrawn from intact crabs at the time points indicated for glucose determination. Each point represents a mean SD (n = 10). Numbers in parentheses represent percent change from zero time controls. * Significant difference from zero time control at P < 0.001. ** Signif- icant difference from zero time control at P < 0.001. NS Not significant from zero time control. kephalin, indicating conversion of inactive to active phos- phorylase. Effects of bilateral e\estalk ablation and injection of methionine-enkephalin into ablated crabs on carbohydrate metabolism Bilateral eyestalk removal caused a significant decrease in hemolymph carbohydrate level (Table 1 ). Enhancement of TCHO level of hepatopancreas and muscle was also significant in eyestalk-ablated crabs (Tables 2, 3). The in- crease was greater in muscle. Glycogen level in hepatopan- creas increased significantly in eyestalkless crabs. A similar pattern was observed in muscle. Tissue phosphorylase ac- tivity levels decreased significantly in eyestalk-ablated crabs (Tables 4, 5). Injection of methionine-enkephalin into eyestalkless crabs did not significantly change hemolymph carbohydrate levels compared to Ringer-injected eyestalkless crabs (Ta- ble 1 ). The levels of tissue TCHO and glycogen and activity levels of total and active phosphorylase were also not sig- nificantly altered in eyestalkless crabs after methionine- enkephalin injection (Tables 2-5). after the injection of methionine-enkephalin (Tables 4. 5). The ratio of active to total phosphorylase also increased in the tissues of crabs after the injection of methionine-en- Discussion The effect of eyestalk hormones on tissue carbohydrate levels and phosphorylase activity has been extensively stud- Table 2 Effect of eyestalk ablation (ESX) (24-h post-ablation) and injection of methionine-enkephalin into intact ami ablated crabs on hepatopancreas total carbohydrate (TCHOl and glycogen levels in Scylla serrata Intact (Group 1) ESX (Group 2) No treatment Ringer injection 10 7 mol/crab 10~ s mol/crab 10 9 mol/crab Dunnet's comparison test TCHO (mg/g) 13.66 1.54 13.84 1.6T' 8.47 0.97 b 9.01 1.51 h 9.47 1.49 b ^,4.45, = 38.033 (1.32) (-38.80) (-34.89) (-31.57) P < 0.001 17.87 1.94 h 18.01 \.91 h -' 17.44 1.43 hx 17.X1 1.62 bx 17.93 1.59 b ' c F I44S , = 0.229 (30.96) (0.67) (-0.74) (-0.96) (-1.39) Two-way ANOVA: F, ,, (Between groups) = 566.317. P < 0.001; F 4 , m (Among treatments) = 19.027. P < 0.001; F 49(1 (Interaction) = 14.896. P < 0.001. Glycogen (mg/g) Intact 1.22 : t 0.10 1.23 0.09 a 0.58 ().14 h 0.61 0.13 h 0.64 0.2 l h F, 4 . 45 , = 148.477 (Group 1 ) (0.82) (-52.84) (-50.40) (-47.96) P < 0.001 ESX 2.04 0.29 h 2.06 0.31 h.c 2.11 1.1 8"^ 2.09 0.21 ht 2.07 0.28 b ' c F, 44 ,, = 0.230 (Group 2) (67. 21) (0.98) (2.42) (1.45) (0.48) Two-way ANOVA: F, g,, (Between groups) = 1658.593, P < 0.001; F 490 (Among treatments) = 24.964, P < 0.001; ^4.90 (Interaction) = 27.016. P < 0.001. Values are mean SD of 10 individual crabs. Values in parentheses are percent change from control. For calculation of percent change for ESX crabs and Ringer-injected intact crabs, intact crabs served as control; for met-injected crabs. Ringer-injected crabs served as control. J Not significant compared with intact crabs. * P < 0.001 compared to intact crabs. c Not significant compared to eyestalkless crabs. MET-ENKEPHALIN-INDUCED HYPERGLYCEMIA IN CRAB Table 3 Effect of eyestalk ablation treatment Ring er injection 10~ 7 mol/crab 10~ K mol/crab 10"' mol/crab Dunnet's comparison test TCHO (mg/g) Intact (Group 1 ) ESX (Group 2) 4.39 6.26 (4 0.53 0.71 h 2.59) 4.41 0.49 a (0.46) 6.31 0.8 l bx (0.80) 2.94 0.3 l h (-33.33) 3.01 0.37 h (-31.74) 6.25 O.S4 1 " (-0.95) 3.12 0.92 h (-29.25) 6.33 0.92 b - c (0.31) M4.45) P < ^14.45) ~ 30.829 .001 0.045 6.31 0.76 b ' c (0) Intact (Group 1) ESX (Group 2) Two-way ANOVA: F, go (Between groups) = 579.612. P < 0.001; F 4 (Among treatments) = 8.707, P < 0.001; F 4 QO (Interaction) = 9.1 14, P < 0.001. Glycogen (mg/g) 0.66 0.06 F<4.45> = 45.114 P < 0.001 F t 4.45)= 0.188 0.64 0.09" 0.34 0.09 b 0.37 0.06" 0.41 0.08 h (-3.03) (-46.87) (-42.18) (-35.31) 1.01 0.09 b 1.02 O.ll"- c 0.99 0.14 b ' c 1.07 0.33 bx 1.03 0.2 l hx (53.03) (0.99) (-2.94) (4.90) (0.98) Two-way ANOVA: F, gn (Between groups) = 422.031. P < 0.001; F 4 9() (Among treatments) = 6.391, P < 0.001; F 41 ,,, (Interaction) = 6.713. P < 0.001. Values are mean (mg glucose/g tissue) SD of 10 individual crabs. Values in parentheses are percent change from control. For calculation of percent change for ESX crabs and Ringer-injected intact crabs, intact crabs served as control; for met-injected crabs. Ringer-injected crabs served as control. a Not significant compared with intact crabs. b /> < 0.001 compared to intact crabs. 1 Not significant compared to eyestalkless crabs. led (Keller, 1965; Ramamurthi et al.. 1968; Sagardia, 1969). Eyestalk removal inactivates the phosphorylase system and activates uridine-diphosphate-glucose glycogen transglu- cosylase (glycogen synthetase) (Keller, 1965; Ramamurthi ct nl.. 1968). Ramamurthi et al. ( 1968) also observed stim- ulation of uptake and incorporation of glucose I4 C into the glycogen fraction of muscle tissue after eyestalk removal; this stimulation was accompanied by a decrease in hemo- lymph sugar level. Injection of eyestalk extract reversed these changes. The hyperglycemic hormone of eyestalks of the crab Oziotelphusa senex sene.x and the prawn Penaeus monodon enhances the activity of the phosphorylase system (Reddy et al.. 1982, 1984; Reddy, 1992). An increase in phosphorylase activity and a decrease in glycogen and TCHO levels in hepatopancreas and muscle of Scylla serrata, followed by hyperglycemia after the injec- tion of methionine-enkephalin, indicate glycogenolysis and mobilization of sugar molecules from tissues to hemo- lymph. This is in agreement with other findings (see review by Reddy and Ramamurthi, 1999). Though the hormone that elevates hemolymph sugar is conventionally called crustacean hyperglycemic hormone (CHH). Hohnke and Scheer (1970) suggested that the primary function of the CHH is not to elevate hemolymph sugar level, but to elevate intracellular glucose through the degradation of glycogen by activating the enzyme phosphorylase. The conversion of phosphorylase from its inactive to active form results in glycogenolysis, and the resultant glucose molecules leak into the hemolymph, causing hyperglycemia. This view has been supported by Telford (1975). Our results clearly demonstrate that methionine-enkepha- lin is involved in the regulation of carbohydrate metabolism in the crab Scylla serrata. In the present study, we show that methionine-enkephalin elicited a hyperglycemic response in S. serrata in a dose-dependent manner (Fig. 1 ). Methionine- enkephalin-induced hyperglycemia has been similarly dem- onstrated in the freshwater crab Oziotelplutsa senex senex (Reddy, 1999) and the brackish-water prawns Penaeus in- dicus and Metapenaeits monocerus (Kishori et al., 2001). The doses of methionine-enkephalin that induced hypergly- cemia ranged from 10 9 to 10~ 6 mol/animal (Fig. 1 ), which is comparable to those reported for O. senex senex (Reddy, 1999). Our observation that methionine-enkephalin was in- effective in inducing hyperglycemia in eyestalk-ablated S. serrata (Table 1) is also consistent with those obtained in crabs (Reddy, 1999) and prawns (Kishori et al., 2001 ) and suggests that the hyperglycemic effect of methionine-en- kephalin results from an enhanced release of CHH (Keller, 1992; Soyez. 1997). Injection of methionine-enkephalin into intact S. serrata also has two other effects. It activates the phosphorylase system, which causes degradation of glycogen. It also re- sults in accumulation of sugar molecules in the tissues; these molecules are ultimately mobilized to hemolymph. 22 P. S. REDDY AND B. KISHORI Table 4 Effect of evestalk ablation I ESX) (24 h post-ablation) and injection of methionine-enkephalin into intact and ablated crabs on hepatopancreas phosphorylase activity levels in Scylla serrata No treatment Ringer injection 10~ 7 mol/crab 10~ 8 mol/crab 10 " mol/crab Dunnet's comparison test Phosphorylase a 3.63 0.34 h 3.60 0.42" F, 4 45 , = 28.430 (35.95) (34.83) P < 0.001 1.81 0.22 b - c 1.84 0.31 b ' c F, 445 , = 1.473 (8.38) (10.17) Two-way ANOVA: F, w , (Between groups) = 716.848. P < 0.001; F 4 91 , (Among treatments) = 23.852, P < 0.001; F 4 , (Interaction) = 18.208, P < 0.001. Intact 2.62 0.29 2.67 0.33" 3.87 0.46 b (Group 1) ESX 1.72 0.3 l h (1.91) 1.67 0.29 b - c (44.94) 1.69 O.ll"- 1 - (Group 2) (-34.35) (-2.33) (1.19) Phosphorylase ab Intact (Group 1) ESX (Group 2) 4.52 0.41 4.56 0.44 a 5.81 0.67 b (0.89) (27.41) 4.06 0.44 b 4.08 0.41 hL 4.10 0.39"-' (-10.18) (0.49) (0.49) 5.69 0.52" (24.78) 4.12 0.34 h - c (0.98) 5.56 0.73" (21.92) 4.09 0.51 b ' c (0.24) F (4 . 45) = 15.846 P < 0.001 F, 44 ,, = 0.044 Two-way ANOVA: F, uo (Between groups) = 169.103, P < 0.001; F 4 w (Among treatments) = 11.291, P < 0.001: F 4 ,,,, (Interaction) = 9.985. P < 0.001. Values are mean (iP released/mg protein/h) SD of 10 individual crabs. Values in parentheses are percent change from control. For calculation of percent change for ESX crabs and Ringer-injected intact crabs, intact crabs served as control; for met-injected crabs. Ringer-injected crabs served as control. a Not significant compared with intact crabs. b P < 0.001 compared to intact crabs. c Not significant compared to eyestalkless crabs. causing hyperglycemia. Methionine-enkephalin might have elevated the phosphorylase system in intact crabs in several different ways for example, by triggering release of hy- perglycemic hormone or by mimicking the action of this hormone. However, because methionine-enkephalin was not able to produce these changes in eyestalkless crabs, it seems most likely that methionine-enkephalin exerted its hyperglycemic effect by triggering release of hyperglyce- mic hormone from the sinus gland of eyestalks. This sup- ports our earlier results that sinus glands in the eyestalks of crabs are the main release site for hyperglycemic hormone (Reddy and Ramamurthi, 1982). The mechanisms whereby methionine-enkephalin causes release of neurohormones are still uncertain. In mammals, endogenous opioid peptides are involved in regulating the release of neurohypophysial peptides (Bicknell et al.. 1988; Yamada et al., 1988; Sasaki et ai, 2000). In crustaceans, opioid-peptide-like (methionine-enkephalin-like, leucine- enkephalin-like and /?-endorphin-like) hormones were iso- lated and characterized from X-organ sinus gland com- plexes of eyestalks (Fingerman et ai, 1983, 1985). However, there is little information about the effect of opioid peptides on release of neurohormones in crustaceans. Sarojini et al. (1995, 1996). using highly selective opioid antagonists, provided evidence that methionine-enkephalin exerts its effect by acting through delta-type opioid recep- tors in regulating ovarian maturation in Procambarus clarkii. In vivo studies with tissues of P. clarkii showed that methionine-enkephalin exerted its effect by at least modu- lating the release of eyestalk peptide hormone (Sarojini et al., 1997). Recently, we provided evidence for a neurotrans- mitter role for methionine-enkephalin in causing hypergly- cemia in the crab O. senex senex (Reddy, 1999). Methio- nine-enkephalin also triggers the release of red-pigment- concentrating hormone, black-pigment-dispersing hormone (Quackenbush and Fingerman, 1984). and dark-pigment- adapting hormone (Kulkarni and Fingerman, 1987). Three facts make it seem likely that this hyperglycemic action of methionine-enkephalin in the present study on S. serrata is also indirect and involves stimulation of release of CHH. Methionine-enkephalin-like material is present in the neu- roendocrine complex of the eyestalk of crustaceans (Finger- man et al., 1983. 1985). Methionine-enkephalin mediation of release of neurohormones has been demonstrated (Reddy, 1999). In cases where methionine-enkephalin has been found to stimulate neurohormone release, it does not act in the absence of neuroendocrine organs. As further support for the conclusion, eyestalk extract from methio- nine-enkephalin injected prawns showed significantly less activity than the normal eyestalk extract in inducing hyper- glycemia (Kishori et al., 2001 ). Although the mechanisms that trigger release of CHH are still unknown, it is noteworthy that 5-hydroxytryptamine (5-HT), or serotonin, triggers CHH release in the crayfish MET-ENKEPHALIN-INDUCED HYPERGLYCEMIA IN CRAB 23 Table 5 Effect of evesta/k ablation I ESX) (24 h post-ablation) and injection of methinine-enkephalin into intact and ablated crabs on muscle phosphor/lose activin levels in Scylla serrala No treatment Ringer injection 10~ 7 mol/crab 10 ~ s mol/crab 10~" mol/crab Dunnet's comparison test Phosphorylase a Intact 1 .92 0.09 1.94 0.1 4 a 3.26 0.36 b 3.01 0.12' 1 3.02 0.26 h F, 4 . 451 = 84.853 (Group 1 ) (1.04) (68.04) (55.15) (55.67) P < 0.001 ESX 0.99 0.08 h 1.02 O.ll hl 1.01 0.09 Kc 1.04 O.I3 hc 1.06 0.2 1 KC F |44 ,, = 0.368 (Group 2) (-48.44) (3.03) (-0.74) (1.96) (3.92) Two-way ANOVA: F, 91 , (Between groups) = 1711.188. P < 0.001; F 4 M1 , (Among treatments) = 58.745. P < 0.001; F 4 w (Interaction) = 52.927, P < 0.001. Phosphorylase ab Intact 2.49 0.45 2.52 0.49 a 3.49 0.4 l h 3.46 0.44 h 3.44 0.51 h F ( 44S) = 16.086 (Group 1) (1.21) (38.49) (37 .30) (36.50) P < .001 ESX 2.22 * 0.32 b 2.18 0.31 bc 2.22 0.34 b - c 2.24 0.42 hc 2.25 0.41 b.t F, 4.45) = 0.061 (Group 2) (-11 .65) (-0.91) (1.83) (2. 75) (3.21) Two-way ANOVA: F, ,, (Between groups) = 136.048, P < 0.001; F 4 , m (Among treatments) = 10.259, P < 0. 001; " 4, MO (Interaction) = 8.734, P < 0.001. Values are mean (iP released/mg protein/h) SD of 10 individual crabs. Values in parentheses are percent change from control. For calculation of percent change for ESX crabs and Ringer-injected intact crabs, intact crabs served as control; for met-injected crabs. Ringer-injected crabs served as control. a Not significant compared with intact crabs. * P < 0.001 compared to intact crabs. c Not significant compared to eyestalkless crabs. Orconectes limosus (Keller and Bayer, 1968), Astacus lep- todactylus (Strolenberg and Van Herp, 1977), and Procam- barus clarkii (Lee et ai. 2000). Strolenberg and Van Herp (1977). working with A. leptodactylus, and Martin (1978). working with Porcellio dilatatits, found that the sinus glands of specimens injected with 5-HT show increased numbers of exocytotic profiles, suggestive of increased CHH release. Exocytosis in A. leptodactylus was maximal 2 h after 5-HT was injected, and the hemolymph glucose concentration peaked 4 h after the injection (Strolenberg and Van Herp. 1977). In P. dilatatus, hyperglycemia in- duced by 5-HT is mediated by 5-HT,- and 5-HT : -like receptors in triggering release of CHH (Lee et ai, 2000). In summary, we have shown that methionine-enkephalin is a potent hyperglycemic regulator in the crab Scylla ser- nita. The most likely site of action of methionine-enkepha- lin is the eyestalks. where the X-organ-sinus glands may respond to methionine-enkephalin stimulation by releasing CHH. Based on these results, experiments are being con- ducted to determine whether methionine-enkephalin en- hances the release of CHH in crustaceans. Acknowledgments We thank Prof. Armugam, University of Madras. Chen- nai, for supplying Sc\lla serrata and providing necessary laboratory facilities, and Dr. K. V. S. Sharma, Professor, Department of Statistics, Sri Venkateswara University, for analyzing the data. We also thank the anonymous reviewers whose comments improved our manuscript. We are grateful to Prof. R. Ramamurthi. Department of Zoology, for his encouragement. Mr. S. Umasankar and Miss B. Prema Sheela provided skilled technical assistance. This work was carried out with the financial assistance from Department of Science and Technology research grant (SP/SO/CO4/96) to Dr. PSR. We also thank the staff. 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One results from the differential density within an organism (the grav- ity-buoyancy model) and the other from the geometrical asymmetry of an organism (the drag-gravity model). We first introduced a simple theory that distinguishes between these models by measuring sedimentation of immobilized organisms in a medium of higher density than that of the origanisms. Nr + -immobilized cells of Paramecium caitda- tuin oriented downwards while floating upwards in the Percoll-containing hyper-density medium but oriented up- wards while sinking in the hypo-density control medium. This means that the orientation of Paramecium is mechan- ically biased by the torque generated mainly due to the anterior location of the reaction center of hydrodynamic stress relative to those of buoyancy and gravity; thus the torque results from the geometrical fore-aft asymmetry and is described by the drag-gravity model. The same mechan- ical property was demonstrated in gastrula larvae of the sea urchin by observing the orientation during sedimentation of the KCN-immobilized larvae in media of different density: like the paramecia, the gastrulae oriented upwards in hypo- density medium and downwards in hyper-density medium. Immobilized pluteus larvae, however, oriented upwards re- gardless of the density of the medium. This indicates that the orientation of the pluteus is biased by the torque gen- erated mainly due to the posterior location of the reaction center of gravity relative to those of buoyancy and hydro- Received 14 July 2000; accepted 30 March 2001 * To whom correspondence should he addressed. E-mail: mogami@ cc.ocha.ac.jp t Died on 1 March 1999. dynamic stress; thus the torque results from the fore-aft asymmetry of the density distribution and is described by the gravity-buoyancy model. These observations indicate that, during development, sea urchin larvae change the mechanical mechanism for the gravitactic orientation. Evi- dence presented in the present paper demonstrates a definite relationship between the morphology and the gravitactic behavior of microorganisms. Introduction Many swimming microorganisms, including ciliate and flagellate protozoa and the planktonic larvae of some inver- tebrates, are negatively gravitactic; that is, they tend to swim preferentially upwards in water columns despite being heavier than water. This behavior requires the organism to orient upwards in relation to the gravity vector. Several mechanisms have been postulated for the gravitactic orien- tation of aquatic microorganisms (Chia el al., 1983; Bean, 1984; Machemer and Braucker. 1992). From a physical point of view and taking account of the mechanical prop- erties of these microorganisms, it has been postulated that the interaction of gravitational and hydrodynamic forces may cause them to orient with fore end upward. In addition to the mechanical basis, gravitactic orientation might also be explained on the physiological basis of gravity percep- tion. To modulate the propulsive activity, some mechano- sensitive devices that sense gravity (for example, statocysts) might be needed. Although functional statocysts have been found in some unicellular organisms (Fenchel and Finlay, 1984, 1986), a line of evidence for gravity-dependent mod- ulation of propulsion has been accumulated for Paramecium (Machemer et al., 1991; Ooya et al., 1992) and Eitglenu (Machemer-Rohnisch el al., 1999), which have no stato- cyst-like structure. The present paper focuses on the mechanical properties of 26 MECHANICAL BIAS OF MICROBIAL GRAVITAXIS 27 microorganisms, which, irrespective of propulsion, generate the torque to orient the organisms either upwards or down- wards. This mechanical torque should bias the gravitactic orientation, even if the organisms have active physiological mechanisms of gravitaxis. According to Roberts (1970), two mechanical mechanisms have been considered as possible sources of the orientation torque. These are reconsidered, in the present paper, as two mechanical models, the gravity-buoy- ancy model and the drag-gravity model. The gravity-buoyancy model was first postulated by Ver- worn ( 1 889. cited in Machemer and Braucker. 1992) for the negative gravitaxis of Paramecium. This model is based on the differential density within an organism. If the internal density of the organism is not homogeneous, the center of mass (the center of gravity) does not necessarily coincide with the centroid (the center of buoyancy). Posterior accu- mulation of the mass would result in the upward orientation of the organisms, and anterior accumulation would result in the downward orientation. The drag-gravity model was postulated by Roberts (1970) on the basis of the low Reynolds number hydrodynamics of the swimming of microorganisms that have a geometrical fore-aft asymmetry. This model is characterized by a dumb- bell with two spheres of unequal diameter but homogeneous density, which could mimic the fore-aft asymmetry of the microorganisms. According to Stokes' drag formula, the larger sphere of the dumbbell can sink faster than the smaller, at the rate of the square of the ratio of diameters. The applicability of this model has been confirmed by scale-model experiments (Roberts, 1970). Organisms, in general, possess some asymmetry both in internal density and in external geometry. It is therefore pos- sible that these two mechanical models operate independently to generate the gravity-induced orientation torque. Since the mechanical properties for gravitactic orientation are indepen- dent of propulsive thrust, we can assess the mechanical influ- ence by measuring the orientation of immobilized organisms sinking under gravity. Both models predict that, when immo- bilized, an organism orients upwards when sinking in a me- dium with a density lower than its own. In the present paper, we show that the above two models can be distinguished by observing what happens to an organism placed in a medium whose density is higher than its own. We show the results of the experiments on the gravitactic orientation of Paramecium and sea urchin lar- vae, both of which are known to perform typical negative gravitaxis (Mogami et /., 1988: Ooya et al.. 1992). Theory The external forces acting on the body of an aquatic micro- organism due to gravity acceleration are gravitational (F c ) and buoyant forces (F B ), each of which is generated as the product of the volume and density of the body or of the external fluid. The vector sum of the forces encounters the hydrodynamic force (F H ). Since the Reynolds number of an aquatic micro- organism in translational motion is significantly less than unity (of the order of 10 2 ). F H is generated in proportion to the velocity (Happel and Brenner, 1973; Vogel, 1994). F c , F B , and F H act on the center of mass (G), the centroid (B), and the reaction center of hydrodynamic stress (//), respectively. For an immobilized microorganism sinking in the fluid, these three forces are balanced as B + FH = 0. (1) Each term in the equation (positive in upward direction) is described as F G = - Vp,g, F B = Vpg. and (2) (3) (4) where V and p, are the total volume and the average density of the organism, p and g the density of the external fluid and the acceleration due to gravity, and K and 5 the coefficient of hydrodynamic drag and the sinking velocity. We assume in the present paper that a microorganism has a body of rotating symmetry on its fore-aft axis. The sim- plest case of this approximation is that the body has fore-aft symmetry, such as a prolate spheroid. When a prolate spher- oid with uniform density is sinking in the fluid, the three forces act on the same point and therefore do not generate any torque to rotate the body (Fig. la). If. however, the body of a prolate spheroid has a region of higher density in the rear half of the body, as postulated in the gravity-buoyancy model, G is located posterior to B and H (Fig. Ib). This generates the torque (7\,; subscript V is after Verworn) which is given by TV = sin 0, (5) where L G is the distance between G and B (and/or H), and 6 is the orientation angle of the fore-aft axis of the body to the vertical. The fore-aft asymmetry of the external geometry, as postulated in the drag-gravity model, also separates the reaction centers of the forces. If a microorganism of homo- geneous density has a larger radius of revolution around the fore-aft axis in the posterior part (Fig. Ic). H is located anterior to B and G, according to the analogy of a fore-aft asymmetrical dumbbell of homogeneous density (Happel and Brenner. 1973). The torque (T R : subscript R is after Roberts) by the anterior shift of the center of hydrodynamic force is given by T R = -F H L H s'm = (F c + F B )L H sin 6, (6) where L H is the distance between H and G (and/or B). Provided that the Reynolds number of rotational motion is sufficiently small, all torques should be proportional to 28 Y. MOGAMI ET AL. Figure 1. Schematic drawings illustrating the mechanical (physical) basis for the generation of gravity- dependent orientation torque. Gravity (F G ), buoyancy (F B ), and hvdrodynamic force (F H ) are balanced in sinking microorganisms; these forces act at the center of mass (G), the centroid (B). and the reaction center of hydrodynamic stress (//), respectively, (a) Three forces act at the same point in the body of prolate spheroid with uniform density, (b) The center of mass is deviated to the rear end of the body of prolate spheroid, which generates the torque in proportion to F a and the sine of the orientation angle to the gravity vector (W). (c) The reaction center of hydrodynamic stress is deviated to the front end of the body with fore-aft asymmetry but with uniform density, which generates the torque in proportion to the vector sum of F Cl and F H and the sine of the orientation angle. the first power of rotational velocity (dQIdt). In such cases equations of rotational motion are given by -flTj~ = T v orT K , (7) where R is the coefficient of resistance for rotational motion and T) is the viscosity of the external fluid. From these equations the rotational velocity of each model is given as a common form of dO -=3sin0. (8) where the proportional factor is the instantaneous rate at = 90 degrees, and given by (9) (10) Rj] V(p,-p)gL H Rr, for the gravity-buoyancy and drag-gravity models, respec- tively. Equations 9 and 10 indicate that /3 r is insensitive to changes in the density of the external medium (p), whereas f3 K reverses the sign as p exceeds the density of organisms (p,-). This means that the two models can be distinguished by increasing p greater than p,. When im- mobilized organisms are immersed in the hyper-density medium (p > p,), they would orient upwards during floating upwards if they obeyed the gravity-buoyancy model, whereas they would orient downwards if they obeyed the drag-gravity model. The gravity-buoyancy and drag-gravity models are the two extremes of these conditions that can generate the orientation torque depending on the different physical mechanisms. Passive orientation of the organisms (Eq. 8), in fact, would be explained as a result of combining the two models, because none of three forces would necessarily have a common reaction center. In order to extract the origin of the mechanical bias of the orientation. Equation 8 should be examined by measuring |3 by the sedimentation experi- ment using media of different p. If j3 is constant independent of p, the gravity-buoyancy model is the only mechanism for generating the orientation torque. Otherwise, the drag-grav- ity model may play a part in the generation of the torque. A negative value of in the hyper-density medium indicates MECHANICAL BIAS OF MICROBIAL GRAVITAXIS 29 that the drag-gravity model is the major mechanism in passive gravitactic orientation. Materials and Methods Microorganisms and experimental solutions Paramecium caudatum was grown at 24 C in a hay infusion in Dryl's solution (2 mM sodium citrate, 1.2 mM Na,HPO 4 . 1 .0 mM NaH 2 PO 4 , 1 .5 mM CaCU, pH 7.2). Cells grown to the early stationary phase (14-20 d after incuba- tion) were collected and adapted in the experimental solu- tion (KCM; 1.0 mM KC1, 1.0 mM CaCU, 1.0 mM MOPS, pH 7.2). After the adaptation, cells gravitactically accumu- lating beneath the water surface were collected and immo- bilized in the KCM containing 5 mM NiCU. Hyper-density KCM (P-KCM) was prepared by substituting a colloidal solution of Percoll (Sigma) for water up to 60% (v/v) in KCM. At 24 C, the specific gravity and relative viscosity of KCM were 1.00 and 1.02, respectively; those of P-KCM were 1.06 and 1.57. Specific gravity of the experimental solutions was determined by weighing the known volume, and viscosity was measured by means of an Ostwald vis- cometer. Larvae of the sea urchin Hemicentrotus pulcherrimus were grown in the laboratory at 17 C (Degawa et ai, 1986). Larvae at the mid- to late gastrula stage and the early pluteus stage (ca. 24 and 48 h after insemination, respec- tively) were collected by hand centrifuge and washed once with artificial seawater (ASW; 450 mM NaCl. 10 mM KC1, 10 mM CaCl 2 , 25 mM MgCl 2 . 28 mM MgSO 4 , 10 mM Tris-HCl, pH 8.0). For immobilization, larvae were im- mersed in ASW containing 2 mM KCN. Hyper-density ASW (P-ASW) was prepared by substituting Percoll for water up to 22% (v/v) in ASW. At 25 C, the specific gravity and relative viscosity of ASW were 1.01 and 1.07, respectively; those of P-ASW were 1.04 and 1.14. Recordings and analyses of gravity-dependent orientation Ni 2+ -immobilized Paramechtm cells and KCN-immobi- lized sea urchin larvae were transferred, with experimental solutions to be tested, into a chamber made of a slide and coverslip and silicone rubber spacer (inner dimension 12 X 24 X 1 mm for Paramecium and 1 6 X 1 6 X 1 mm for sea urchin larvae) and kept air bubble-free without any partic- ular sealant. The chamber was set on a horizontal micro- scope equipped with a rotating stage. After trapping immo- bilized specimens at the bottom or the top of the chamber (depending on the density of the medium), the chamber was rotated upside down, and the orientation motion during vertical movement due to gravity was recorded with a video camera (XC-77, Sony, Tokyo) and a videotape recorder. To avoid the hydrodynamic interactions between nearby mov- ing objects, we chose organisms moving down (or up) far from neighbors (>1 mm, about 5 body lengths, apart). For measuring the orientation angle, we selected recordings in which the orientation motion was observed in a single focal plane. The orientation angle as a function of time (0, t) was measured directly on the video monitor. The rotational velocity as a function of orientation angle (dOldt, 6) was obtained as an average velocity ((fl,+ i - 0,)/A?) at the angle of geometrical average ((0, + 0, + ,)/2) between every successive datum of inclination angle versus time. /3 in Equation 8 was obtained by nonlinear least-squares re- gression of the velocity data (dO/dt, 0) to the equation d9 ~dt = /3 sin (9 + a). (ID where a is a factor to adjust the angle between the morpho- logically defined fore-aft axis and the mechanically defined axis. Results The drag-gravity model is the major mechanism of Paramecium When Paramecium was immobilized by Ni 2 + , it main- tained an anterior-thinner cell shape. This shape was pre- served in P-KCM as well as in KCM; cells showed no significant changes in axial length (162 17 jam [/; = 30] and 163 16 jim [n = 21]. P = 0.64, for cells in KCM and P-KCM, respectively) or in maximum width (47.2 6.9 p.m and 46.5 4.7 p.m, P = 0.69). Thus it is highly likely that rotational motion of the immobilized cell occurs with the same coefficient of resistance in both media. Typical recordings of gravity-dependent orientation of immobilized paramecia in the hypo- and hyper-density me- dia are shown in Figure 2a and b. In KCM (p < p,.), paramecia oriented upwards during sinking due to gravity, whereas in P-KCM (p > p,) they oriented downwards during floating up. As shown in Figure 2c. plots of orien- tation rates (d6/dt) against orientation angle (0) fit well to the sinusoidal function of Equation 1 1 . Values for |3 ob- tained by least-square regression were positive in the con- trol hypo-density medium and negative in the hyper-density medium (Table 1 ). Negative values of (3 in the hyper- density medium indicate that the drag-gravity model is the major mechanism of mechanical gravitactic orientation in Paramecium. Sea urchin lamie change the mechanical mechanism of gravitactic orientation during development When sea urchin larvae were treated with KCN, their cilia ceased beating and stood nearly perpendicular to the larval surface. The outer morphology of the larvae was observed to be well preserved in P-ASW as well as in ASW: for gastrulae, axial length was 151 7.6 p_m (n =~- 16) and 145 6.1 jum (n = 13), P = 0.19, in ASW and P-ASW, 30 Y. MOGAMI ET AL. a T3 0.15 0.10 0.05 -0.05 -0.10 -0.15 9 (rad) Figure 2. Typical examples of gravity-dependent orientation of Ni 2 + immobilized Paramecium caudutiim. (a, h) Sequential images of gravity- dependent orientation of a cell in KCM (a) and of another in P-K.CM (h), in which recorded images are superimposed at l-s intervals and the time sequence of the motion is illustrated by cyclic change in tone (dark medium > light). In each figure the anterior end of the cell is located to the right, and the gravity vector is towards the bottom of the figure. Scale bar. 0.1 mm. (c) Orientation rates (iltt/ilo as a function of the inclination angle (0). Data from the cells shown in a (KCM) and b (P-KCM) are plotted with open and closed circles, respectively. Sinusoidal curves were obtained by the least-squares fitting to Equation 1 1. respectively, and the maximum width was 135 3.7 JLUTI and 132 5.7 p,m, P = 0.06; for plutei, axial length was 235 19 ju.m (H = 26) and 240 13 /am <;i = 18), P = 0.29, in ASW and P-ASW, respectively, and the maximum width was 175 13 jam and 175 12 juni, P = 0.98. This may justify the common basis for drag coefficients in rota- tion in the different density media, as in Parumeciiim. The gravity-dependent orientation of immobilized larvae is shown in Figure 3a to d, which demonstrates the clear difference between gastrula and pluteus. In ASW (p < p,). both gastrula and pluteus oriented upwards while sinking; in hyper-density P-ASW, however, gastrula oriented down- wards but pluteus upwards while floating up. As shown in Figure 3e and f, the orientation rate appears to be a sinu- soidal function of the orientation angle; although data from larvae fitted less closely to Equation 1 1 than did those from Paramecium, this was probably due to the uncertainty in measuring the orientation angle of the larvae. We some- times observed that larvae rotated slowly around the fore-aft axis during sedimentation. This slow axial rotation made it difficult to determine the fore-aft axis of the larvae. As shown in Table 1. values of |3 obtained from gastrula larvae were positive in the control medium and negative in the hyper-density medium. Thus, in gastrulae as in Para- mecium, the drag-gravity model is the major mechanism of passive gravitactic orientation. However, pluteus larvae have positive values of |3 both in the control and in the hyper-density medium (Table 1). The relatively weak de- pendency of |3 of plutei on the density of the external medium indicates that the gravity-buoyancy model is the major mechanism of passive gravitactic orientation in these larvae. These results indicate that sea urchin larvae change the mechanical mechanism of gravitactic orientation during development. Discussion Estimation of the contribution of the mechanical models in the gravitactic orientation The Reynolds number of rotational motion (Re,) of the microorganisms is defined as Re, = / : o>p (12) where / is a characteristic body length and w is the angular velocity of rotation (Happel and Brenner. 1973). From the maximum velocity of rotation (cu. 0.2 rad s~'. Table 1), Re, of Paramecium or sea urchin larvae is calculated to be about 2 X 10~\ which is sufficiently smaller than unity. This means that the linear assumption of Equation 7 (see the Theory section) is valid to formulate the rotational motion of these microorganisms. The orientation torque generated as a result of the com- bination of the torque originating from different mechanical sources causes the passive orientation of the immobilized organisms. It is difficult to formulate the combination, be- cause we know little about the density distribution within an organism and its geometrical asymmetry. The simplest as- sumption for the combination of the rotational torque is that G. B. and H are located on the geometrical fore-aft axis of the organisms. This gives a sinusoidal function as a linear summation of the sinusoidal equations, each of which is deduced from the gravity-buoyancy and drag-gravity model, respectively. As a result, the orientation rate is given as MECHANICAL BIAS OF MICROBIAL GRAVITAXIS Table 1 Orientation rate t{5), in rad ' s . measured in different densitv media 31 Normal medium Percoll-containing medium Organism Mean SD Range n Mean SD Range Paramecium 0.090 0.033 0.043 - 0. 183 23 -0.104 0.058 -0.257 - -0.041 14 Sea urchin larvae Gastrula 0.140 0.032 0.107 - 0. 197 8 -0.120 0.020 -0.150- -0.090 7 Pluteus 0.157 0.03 1 0.105 - 0. 190 9 0.1 10 0.013 0.097 - 0.137 7 sintf. 13) This simple linear assumption seems to be supported by the fact that a in Equation 1 1 was calculated on average as nearly zero (0.00 0.26 rad (n = 37) for Paramecium, 0.03 0.18 (;i = 15) for gastrula, and 0.06 0.21 (// = 16) for pluteus). Therefore, it is likely that the morpholog- ically defined fore-aft axis almost coincides with the me- chanically defined axis. According to the assumption above, |3 5 obtained in the different density media are given by Vp ig L V(p, - p N )gL H ~ ~~^ ~ _ PP V(p,-p P )gL H (15) where /3 ;V is the maximum orientation velocity measured in the normal density (p N ) medium (KCM or ASW) of the viscosity of TJ^, and fB P is that measured in the hyper- density (p p ) medium (P-KCM or P-ASW) of the viscosity of T] P . Equations 14 and 15 give L H . the distance from B to H, as PP - Ps Vg ' and. thus, f3 R and j8 v are given by: = /3. v - For Paramecium, p v = 1.00, p p = 1.06 and p, = 1.03 g cm" 3 (Ooya et ai, 1992), and T) P lr\ N = 1.53. For sea urchin larvae, p N = 1.01. p p = 1.04, and p, = 1.03 and 1.03 g cm~ 3 , for gastrula and pluteus, respectively (values were obtained by sedimentation equilibrium experiments: data not shown), and TJ/./TJ^. = 1.07. Using these values and /3 V and P P in Table 1. Equations 17 and 18 can be used to obtain values for the contribution of the two mechanisms to negative gravitaxis in normal-density medium. The up- ward orientation of Paramecium in KCM, corresponding to f3 N = 0.09 rad s~ ', is the result of an upward drag-gravity component ( J3 R = 0. 1 2 rad s ' ) combined with a smaller downward gravity-buoyancy component (/3 V = -0.03 rad s '). The situation is similar for sea urchin gastrulae. The upward orientation with |3 A , = 0.14 rad s~' results from an upward drag-gravity component ({$ R = 0.18 rad s" 1 ) combined with a small downward gravity-buoyancy component (j8 v = -0.04 rad s~'). However, the upward orientation of pluteus larvae with fi N = 0.16 rad s" 1 reflects a very different situation. The gravity-buoyancy component has reversed direction from downward to up- ward, and has increased to j8 v - = 0.13 rad s~ ' . The upward drag-gravity component has diminished greatly, to f3 K = ( 14) 0.03 rad s , so that it now makes only a small contribu- tion to the upward orientation. The mechanical property of 'Paramecium There have been several investigations on the mechanical basis of the passive upward orientation of Paramecium. Most of them favored the gravity-buoyancy model as a major mechanism of gravitactic orientation. Fukui and Asai (1980) reported that Triton-treated immobilized cells ori- ented mostly upwards at the sedimentation equilibrium in sucrose density gradient. This upward orientation was evi- dent in well-fed cells but not in starved cells. The upward- orienting posture was found under centrifugal forces in Ni 2 + -immobilized cells in the isodensity medium (Taneda et ai, 1987) and also in the cells swimming at isopycnic level in the density gradient with Ficoll or Percoll (Kuroda and Kamiya. 1989). It was also reported that upward orien- tation was induced by centrifugal force effectively in the cells at the early culture phase but not in those at the late phase, which showed little or no gravitaxis. These results appear to conform with the conclusion that the upward orientation of Paramecium is strongly biased by the torque resulting from the higher density of the posterior part of the organism: the increased density is mainly due to the accu- mulation of food vacuoles (Fukui and Asai, 1985). It should be noted, however, that the results of the sedi- mentation equilibrium experiments were ascribed only to the function of the gravity-buoyancy model and not to the contribution of the drag-gravity model, since F H = with buoyancy artificially balanced with gravity. Furthermore, it (16) 17) 32 Y. MOGAMI ET AL. 1/5 TO 0.20 0.15 010 0.05 o -0.05 -0.10 -0.15 -0.20 0.25 0.20 0.15 0.10 0.05 -0.05 n/2 6 (rad) O n/2 6 (rad) Figure 3. Typical examples of gravity-dependent orientation of KCN- immobilized sea urchin (Hemicentrotus pulcherrimus) larvae, (a-d) Se- quential images of gravity-dependent orientation of the single different larvae at the gastrula (a and b) and the pluteus (c and d) stages. Movements of a larva in ASW (a and c) and of another in P-ASW (b and d) are shown at 3-s intervals in the same way as in Fig. 2a and b. In each figure the animal pole of the larva (leading end in forward swimming) is located to the right, and the gravity vector is towards the bottom of the figure. Scale bar. 0.1 mm (e. f) orientation rates (ilti/dt) as a function of the inclination angle (D). measured from gastrula (e) and pluteus (f). In e. data from the gastrulea shown in a (ASW) and b (P-ASW) are plotted with open and closed circles, respectively. In f, data from the plutei shown in c (ASW) and d (P-ASW) are plotted with open and closed circles, respectively. Sinusoidal curves were obtained by the least-squares fitting to Equation 1 1. seems likely that the gravity-buoyancy component of the orientation torque might be enhanced in these experiments. Since the center of gravity would shift in relation to the content and the distribution of organelles such as food vacuoles, it is probable that in the sedimentation equilib- rium experiments, the intracellular distribution of the or- ganelle was reorganized by gravity during long-lasting sed- imentation of Triton-permeabilized cells through the sucrose density gradient (Fukui and Asai, 1980), or by a large centrifugal acceleration ( 100 X g, Taneda et al., 1987; 300-400 x g, Kuroda and Kamiya, 1989). This may result in accumulation of organelles in the rear part of the cell, and may cause upward orientation, even if the cells originally have a slightly top-heavy organelle distribution that gives a negative j3 v / as estimated above. These facts suggest that the results of previous experiments are still equivocal for the contribution of the drag-gravity model in the gravitactic orientation of Parameciiini. The evidence presented in the Results, on the contrary, indicate that the drag-gravity model makes a major contri- bution to generating a torque for the gravitactic orientation. Although the possibility of a minimal contribution cannot be ruled out, it is clear that the gravity-buoyancy model cannot solely explain the alteration of the sign of the rota- tional torque in the hyper-density medium. In addition, paramecia were observed in P-KCM to swim mostly down- wards (data not shown). Swimming cells changed the net direction of their helical swimming trajectory gradually downwards and accumulated at the bottom of the chamber against the strong floating bias. Positive gravitaxis of Par- ameciitm in the hyper-density medium can be explained by the drag-gravity model, not by the gravity-buoyancy model. Developmental clmnges in the mechanical property in sea urchin lan'ae In the present paper we demonstrated a change in the mechanical basis for gravitactic orientation during the de- velopment of sea urchin larvae: from the drag-gravity model in gastrulae to the gravity-buoyancy model in plutei. Gas- trulae have a thicker posterior part, similar to that of Par- ciiiieciiiin. which is required for the drag-gravity model to function. Plutei. on the other hand, have a thicker anterior part. Therefore they may orient the rear end upwards if the rotational torque is generated according to the drag-gravity model. This was not the case for plutei. Regardless of the remarkable fore-aft asymmetry in morphology, plutei obeyed the gravity-buoyancy model. Gravitactic orientation by different mechanisms was also revealed in the gravitactic swimming behavior of the larvae in P-ASW. In spite of the strong floating bias, gastrulae swam preferentially down- wards (positive gravitaxis) and accumulated at the bottom of the chamber, whereas plutei swam upwards (negative gravitaxis) and accumulated at the top of the chamber (data not shown). Mogami et al. ( 1 988) found that sea urchin larvae change their gravitactic behavior during development. Larvae at the blastula stage to the early gastrula stage swim preferentially MECHANICAL BIAS OF MICROBIAL GRAVITAXIS 33 upwards. This may be explained by a major upward drag- gravity component of orientation torque. The negative gravitatic behavior becomes less remarkable in prism lar- vae: they tend to swim in random directions independent of the gravity vector. This transient disappearance of gravi- taxis may correspond to the alteration of the orientation mechanism revealed in the present paper. At the pluteus stage, larvae again show negative gravitaxis as they acquire the orientation mechanism with a major upward gravity- buoyancy component. A strong separation between the cen- ters of gravity and buoyancy may develop in association with the growth of skeletal structures. Rudiments of spicules initiated in the early gastrula fully extend to give rise to the specific shape of the pluteus larva. The spicule is made of magnesian calcite with a density about three times higher than the average density (Okazaki and Inoue, 1976). As spicules grow, they may change the density distribution to shift the center of gravity toward the rear of the cell. If plutei hereafter maintained the rear-end-heavy mass distribution, they could maintain negative gravitactic behavior irrespec- tive of pronounced morphological changes during the late larval stages. Although the functional role of the drag-gravity model has been accepted in theory, it was not experimentally demonstrated in the orientation movement of organisms. In the present paper we present the first evidence that external geometry is actually important to the gravitactic behavior of aquatic microorganisms. The morphology-dependent inter- action of the organisms with the external fluid seems to be more complicated than hypothesized in the Theory section of this paper. The slow axial rotation observed in sediment- ing sea urchin larvae indicates a hydrodynamic coupling between translational and rotational motion (Happel and Brenner, 1973). Therefore, it is probable that the hydrody- namic coupling secondarily functions to drift the swimming direction upwards, as argued in previous researches (Winet and Jahn. 1974; Nowakowska and Grebecki, 1977). In conclusion, the present study on the mechanical prop- erties of gravitactic orientation in the gravity field demon- strates a relation between the morphology of microorgan- isms and their gravitactic behavior. This relationship might be instructive in researching cases of microbial gravitaxis whose mechanism is still disputed. Acknowledgments This study was carried out as a part of "Ground Research Announcement for Space Utilization" promoted by Japan Space Forum. Literature Cited Bean, B. 1984. Microhial geotaxis. Pp. 163-198 in Membrane and Sensory Transduction, G. Colombetti and F. Lenci, eds. Plenum Press, New York. Chia. F-S.. J. Buckland-Nicks, and C. M. Young. 1983. Locomotion of marine invertebrate larvae: a review. Can. J. Zool. 62: 1205-1222. Degawa, M., Y. Mogami, and S. A. Baba. 1986. Developmental changes in Ca"^ sensitivity of sea-urchin embryo cilia. Comp. Bio- chem. Physiol. 82A: 83-90. Fenchel, T., and B. Finlay. 1984. Geotaxis in the ciliated protozoan Loxodes. J. Exp. Biol. 110: 17-33. Fenchel, T., and B. Finlay. 1986. The structure and function of Muller vesicles in loxodid ciliates. J. Protozool. 33: 68-76. Fukui, K., and H. Asai. 1980. The most probable mechanism of the negative geotaxis of Paramecium caudatum. Pmc. Jpn. AcuJ. 56(B): 172-177. Fukui, K., and H. Asai. 1985. Negative geotactic behavior of Parame- ciiim caudatum is completely described by the mechanism of buoyan- cy-oriented upward swimming. Bioph\s. J. 47: 479-482. Happel, J., and H. Brenner. 1973. Low Reynolds Number Hydrody- namics. Noordhoff International Publishing, Leyden. Kuroda, K., and N. Kamiya. 1989. Propulsive force of Paramecium as revealed by the video centrifuge microscope. Exp. Cell Res. 184: 268-272. Machemer, H., and R. Braucker. 1992. Gravireception and gravire- sponses in ciliates. Acta Protozool. 31: 185-214. Machemer, H., S. Machemer-Riinisch, R. Braucker, and K. Takahashi. 1991. 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(August 2001) Synthesis of Several Light-Harvesting Complex I Polypeptides Is Blocked by Cycloheximide in Symbiotic Chloroplasts in the Sea Slug, Elysia chlorotica (Gould): A Case for Horizontal Gene Transfer Between Alga and Animal? JEFFREY J. HANTEN 1 ' 2 AND SIDNEY K. PIERCE 2 * 1 Department of Biology. University of Man-land, College Park, Maryland 20742; and 2 Department of Biology, University of South Florida. Tampa, Florida 3362G Abstract. The chloroplast symbiosis between the asco- glossan (=Sacoglossa) sea slug Elysia chlorotica and plas- tids from the chromophytic alga Vaitcheria litorea is the longest-lived relationship of its kind known, lasting up to 9 months. During this time, the plastids continue to photosyn- thesize in the absence of the algal nucleus at rates sufficient to meet the nutritional needs of the slugs. We have previ- ously demonstrated that the synthesis of photosynthetic proteins occurs while the plastids reside within the diver- ticular cells of the slug. Here, we have identified several of these synthesized proteins as belonging to the nuclear- encoded family of polypeptides known as light-harvesting complex I (LHCI). The synthesis of LHCI is blocked by the cytosolic ribosomal inhibitor cycloheximide and proceeds in the presence of chloramphenicol, a plastid ribosome inhibitor, indicating that the gene encoding LHCI resides in the nuclear DNA of the slug. These results suggest that a horizontal transfer of the LHCI gene from the alga to the slug has taken place. Introduction Most alga-animal symbioses are extracellular associa- tions between two genetically distinct organisms. The alga is usually located extracellularly or enclosed within vacu- Received 22 September 2000; accepted 19 April 2001. * To whom correspondence should be addressed. E-mail: pierce@chumal.cas.usf.edu Abbreviations: CAP, chloramphenicol: CHX. cycloheximide; FCPC, fucoxanthin chlorophyll ale binding proteins; LHC. light-harvesting com- plex; PSI; photosystem I. oles inside the animal's cells. Rarer, but not uncommon, are intracellular symbioses occurring with intact algal chloro- plasts that are captured by specialized cells within the animal. In particular, several species of ascoglossan ( = Sacoglossa) (Opistobranchia) sea slugs capture intact, functional plastids from their algal food source and retain them within specialized cells lining the mollusc's digestive diverticula. This phenomenon has been termed chloroplast symbiosis (Taylor. 1970) or kleptoplasty (Clark et al.. 1990). The sequestered plastids continue to photosynthesize for periods ranging from a few days to a few months, depending on the species (Greene, 1970: Hinde and Smith, 1974; Graves et al., 1979; Clark et al., 1990). The longest such association, lasting as long as 9 months, is found in Elysia chlorotica (Gould), which obtains sym- biotic plastids from the chromophytic alga Vaucheria lito- rea (C. Agardh) (West, 1979; Pierce et al.. 1996). The association begins at metamorphosis of the slug from plank- tonic veliger to juvenile. In laboratory cultures, filaments of V. litorea must be present for metamorphosis to take place (West et al.. 1984). Veligers home in, attach to the fila- ments, and metamorphose into juvenile slugs over the next 24 h. The juveniles eat the algal filaments and sequester the chloroplasts within one of at least two morphologically distinct types of epithelial cells lining the walls of the digestive diverticula (West et al., 1984). Once the plastids are sequestered, the slugs can sustain photosynthesis at rates sufficient to satisfy the nutritional needs for the complete life cycle of the slug, when provided with direct light and carbon dioxide (Mujer et al., 1996; Pierce et al., 1996). 34 SYMBIOTIC PLASTID GENES IN SLUGS 35 Even in nature the slugs obtain most of their energy from photosynthesis (West. 1979). The longevity of this relationship in E. chlorotica makes it especially interesting. Photosynthesis requires the contin- uous synthesis of a variety of chloroplast proteins because many of them, including those used in light harvesting, are rapidly degraded and must be replaced (Greenberg et cil.. 1989; Mattoo et ai, 1989; Barber and Andersson, 1992; Wollman et ai. 1999). Furthermore, photosynthesis re- quires the interaction of as many as 1000 proteins, only about 13% of which are coded in the plastid genome (Mar- tin and Herrmann, 1998). In the plant cell, substantial nu- clear input is required to sustain photosynthetic function, in the form of direct coding of the proteins as well as providing the means for their intracellular transport and regulation (Berry-Lowe and Schmidt. 1991; Wollman et ui, 1999). Considering the level of nuclear and extra-plastid input required, it is not surprising that the longevity of the plastids in most kleptoplastic slugs is relatively short. However, several photosynthetic proteins are synthesized in the se- questered plastids of E. chlorotica (Pierce et ai, 1996), including the large subunit of RuBisCO, Dl, D2, CP43, cyt /and others (Pierce et ai, 1996; Mujer et ai, 1996; Green et ai, 2000). Although all of the synthesized plastid proteins identified to date are plastid encoded (Mujer et ai, 1996; Pierce et ai, 1996; Green et ai, 2000). two groups of synthesized plastid proteins can be distinguished pharma- cologically: those inhibited by cycloheximide (CHX). an SOS cytosolic ribosome inhibitor (Obrig et ai, 1971). and those inhibited by chloramphenicol (CAP), which inhibits protein synthesis on 70S plastid and mitochondrial ribo- somes (Lamb et ai, 1968: Stone and Wilke. 1975). Because the inhibition by CHX suggests that the genes for several plastid proteins must reside in the nuclear DNA, we have done some experiments to identify these proteins and test that possibility. Our present study reports the iden- tification of several of the CHX-blocked proteins as mem- bers of the light-harvesting complex 1 (LHCI). a family of pigment-binding proteins responsible for collecting radia- tion energy from sunlight and transferring it to photosystem I (PSI). LHCI proteins are encoded by the Lhca genes in the nuclear genome of all the plants and algae whose genomes have been examined (Jansson, 1994. 1999; Green and Durn- ford, 1996; Durnford et ai, 1999; Wollman et ai, 1999). This result suggests that the LHCI genes have been some- how transferred from the algal nucleus to the slug's DNA. Materials and Methods Animals and alga Specimens of Elysici chlorotica were collected in both the spring and fall from an intertidal marsh near Menemsha Pond on the island of Martha's Vineyard, Massachusetts. The slugs were maintained in 10-gallon aquaria at 10 C in aerated, artificial seawater (ASW: Instant Ocean. 925-1000 mosm) on a 16/8-h light/dark cycle (GE cool-white fluores- cent tubes. 15 W). Sterile cultures of Vaucheria litorea were maintained in enriched ASW (400 mosm) [modified from the F/2 medium (Bidwell and Spotte. 1985)]. The alga was grown at 20 C on a 16/8-h light/dark cycle (GE cool-white fluorescent tubes; 40 W). and the medium was changed weekly. Inhibitor treatments and plastid protein labeling All reagents used were molecular bio-grade (DNase-, RNase-. and protease-free) purchased from Sigma unless otherwise noted. Effective concentrations of CHX and CAP were determined empirically with initial dose-response curves (Pierce et ai, 1996). CHX (2 mg ml" 1 ) was used to inhibit protein synthesis on SOS cytosolic ribosomes; CAP (160 /o,g ml , stock concentration 50 mg ml" 1 in absolute ethanol) was used to inhibit translation on 70S plastid and mitochondrial ribosomes. Two to four slugs, total wet weight about 1.25 g, were placed into glass scintillation vials containing ASW (1000 mosm) and the appropriate inhibitor, and incubated under intense light (150 W, GE Cool Beam incandescent indoor flood lamp) at 20 C in a gently agitating water bath. After 1 h. 20 /iCi ml" 1 [ 35 S]- methionine (0.7 MBq ml" 1 , trans-[ 35 S]-methionine, ICN) was added, and the slugs were incubated for an additional 6 h. previously demonstrated to provide ample time to incorporate radioactive label into the plastid proteins (Pierce et ai, 1996). Additional slugs were incubated in 0.025% ethanol/ASW (v/v) solution plus [ 35 S]-methionine to serve as a control for the carrier in CAP treatments. Chloroplast isolation and protein separation Chloroplasts were isolated from slugs by using a centrif- ugation protocol. The slugs were homogenized in the pres- ence of the mucolytic agent N-acetyl-cysteine (500 mM). and the homogenate was filtered successively through cheesecloth, Miracloth (Calbiochem), and then nylon mesh (60 ;um to 10 jitm) to remove large debris and the copious amount of mucus the animals produce. The plastids were purified on a pre-formed. 25% Percoll (v/v) gradient, which provides a very pure fraction containing large numbers of intact plastids (Pierce et ai, 1996). In this experiment, the lowest green band containing labeled plastids was isolated from the gradient by using a flamed Pasteur pipette, and residual Percoll was removed by centrifugation. The puri- fied chloroplast pellets were resuspended, lysed by freeze- thawing. and stored at 20C until use. The incorporation of radioactive label was determined by a liquid scintillation counter (Beckman LS60001C). and the protein content was determined using the modified Lowry assay (Peterson, 1977). The resulting specific activity was calculated as counts per minute (cpm) (jug protein)^'. Chlorophyll con- 36 J. J. HANTEN AND S. K. PIERCE tent was determined by extracting the pigment in 80% acetone, then measuring the extract absorbance spectropho- tometrically at 652 nm. The results were calculated as micrograms per microliter according to standard equations (Joyard et al., 1987). Sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) autoradiography was used to assess the effects of CHX and CAP on the pattern of protein synthesis. The plastid lysates obtained from the above pro- cedure were boiled for 2 min in Tris-HCl (pH 6.8)-10% SDS (w/v) buffer containing 5% /3-mercaptoethanol (|3- ME) (v/v). The solubilized proteins were loaded in equal amounts onto 15% SDS-polyacrylamide gels and separated by electrophoresis (Laemlli, 1970). The gels were stained with Coomassie brilliant blue, dried, and exposed to film (Kodak Biomax MR) for 2 to 30 days at -80C, depending on the level of radioactive label present. Approximate mo- lecular masses of the proteins were determined by compar- ison to the migration distances of known molecular weight standards (BioRad, broad-range kaleidoscope) run in adja- cent lanes on each gel. Immunoblot identification of plastid proteins After the plastid isolation and protein separation via SDS-PAGE as described above, the proteins were electro- phoretically transferred (30 V, 4 C, overnight) to PVDF membranes (Immobilon-P; Millipore) (Towbin et til.. 1979). As additional controls, V. litorea chloroplasts [iso- lated and purified using a 30% to 75% Percoll step gradient as previously described (Pierce et al., 1996)] and thylakoids from the red alga Porphyridium cmentum (generously do- nated by Professor Elisabeth Gantt, University of Mary- land), were lysed. and the proteins were separated electro- phoretically and transferred to membranes as above. The membranes were blocked with 5% (w/v) dehydrated milk dissolved in Tris-buffered saline (TBS) (Tris-base 50 mM. NaCl 0.9%, pH 7.5) for 1 h at room temperature, washed twice in TBS for 10 min, and treated with primary antibody for 1 h. In this case, the primary antibody was a polyclonal antibody to LHCI which was produced in a rabbit using a 22-kDa, recombinant LHCI polypeptide produced from a clone of the LhcaRI gene of P. cmentum (Grabowski et al., 2000) (also provided by Professor Gantt) ["/?/" indicating it is a rhodophyte gene (Tan et al., 1997a)] as the antigen combined with Freund's adjuvant in a standard immuniza- tion procedure. After binding of the primary antibody, the membranes were washed twice as above and incubated with secondary antibody, anti-rabbit conjugated hydrogen perox- idase, for 1 h. After washing, the bands were visualized with a 4-chloro-l-napthol and hydrogen peroxide reaction ac- cording to manufacturer's instructions. The immunolabeled western blots were exposed to film as described above to identify the coincidence of antibody binding and radioactive incorporation in the presence of each inhibitor. As a control to confirm that the CAP was blocking plastid-directed protein synthesis and that CHX was not, parallel measurements were run to monitor cytochrome / (cyt/ ) synthesis. Earlier experiments conducted on E. clilo- rotica have demonstrated that cyt / is synthesized in the slugs and is encoded in the plastid DNA (Green et al., 2000). Thus, if CHX and CAP are working as expected, their effect on cyt / and any nuclear-encoded proteins should be opposite. Anti-cyt/, raised to P. cmentum cyt/, was also a gift of Professor Gantt. Immunoprecipitations Immunoprecipitations were conducted to confirm the identity of the radioactive immunolabeled bands on the western blots, using a modified version of the protocol previously used to precipitate proteins from isolated E. chlorotica plastids (Pierce et al., 1996). Plastid proteins were solubilized in lysing buffer ( 10 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1 mM PMSF, 1% (v/v) Nonidet P-40, pH 8.0), using equal amounts of chlorophyll per sample, mixed with a small amount of Protein-A Sepharose beads to eliminate nonspecific binding, and incubated on ice with occasional agitation. The beads were removed by cen- trifugation and discarded, the supernatant was saved, and the appropriate antibody was added to the lysate and rotated overnight (4 C). Protein-A beads, swelled in washing buffer (50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1 mM PMSF, 0.1% (v/v) Nonidet P-40, pH 8.0), were added the following morning and rotated (3 h, room temperature). The antigen-antibody-protein-A Sepharose bead com- plexes were washed several times in washing buffer and removed by centrifugation. In the case of cyt/, the antigen- antibody-protein-A Sepharose bead complexes were resus- pended in 10.0 M urea. 10% SDS (w/v), 5% |3-ME (v/v), pH 12.5, and boiled for 10 min to liberate the cyt/ antigen. The solution was centrifuged, the supernatant was removed, and the beads were discarded. The supernatant proteins were separated by SDS-PAGE as described above, and the gel was autoradiographed. The LHCI antibody-antigen complex could not be broken efficiently with any treatment, which prevented the visual- ization of the labeled LHCI proteins via SDS-PAGE. Al- though this was unexpected, it is not unusual and may have been caused by a number of factors. The presence of several different LHCI polypeptides with varying isoelectric points, ranging between 4.5 and 9.5 (De Martino et al., 2000). makes it very difficult to create optimal reaction conditions for each one. The polyclonal antibody molecules bind to all the LHCI polypeptides as well as to each other, creating a large antigen-antibody complex with a core inaccessible to the chemicals necessary to liberate the antigen. Very few SYMBIOTIC PLASTID GENES IN SLUGS 37 researchers have attempted LHC immunoprecipitations be- cause of the pitfalls involved in precipitating inner-mem- brane proteins (Anderson and Blobel, 1983). Instead, other protocols have been designed using mild detergents to ex- tract intact photosystem holocomplexes from the thyla- koids. followed by protein separation on sucrose density gradients (Fawley and Grossman, 1986; Buchel and Wil- helm, 1993: Wolfe end., 1994; Schmid et id.. 1997). These isolations require large amounts of starting material (Schmid et id.. 1997) that greatly exceed what is available to us in the slugs. So, instead, we used the LHCI antibody to demonstrate that LHCI had incorporated radioactivity. Following the procedure described above, the protein A Sepharose beads were reacted with anti-LHCI and then with a radiolabeled plastid protein extract. The antigen-anti- body-protein-A Sepharose bead complexes were repeatedly washed by centrifugation until the radioactivity in the su- pernatant was reduced to background. The washed antigen- antibody-protein-A Sepharose bead complexes were resus- pended in optifluor (Packard), and radioactivity was determined by a scintillation counter. Controls for nonspe- cific binding to protein-A Sepharose beads were conducted with the same procedure, but without the addition of the LHCI antibody. Counts per minute resulting from nonspe- cific binding were subtracted from experimental values for each inhibitor treatment and controls, and the final data were converted to cpm (jug chlorophyll)" ' (ju,g protein)" 1 . The normalized data were averaged and expressed in terms of percent of control for each inhibitor. Results Plastid protein synthesis and identification The Coomassie-stained SDS-PAGE gels of protein ex- tracts from isolated slug plastids were similar to controls regardless of the inhibitor present, either CHX or CAP, indicating no difference in the protein composition of the plastids after treatment (Fig. 1). However, autoradiograms of SDS-PAGE gels of plastid proteins extracted from slugs incubated in the presence of [ 35 S]-methionine indicate that very different patterns of protein synthesis occur in the slugs between controls and inhibitors as well as between inhibi- tors (Fig. 2). CHX has a profound effect on protein synthe- sis, preventing synthesis of the majority of the protein bands labeled in the absence of inhibitor (Fig. 2, CON), whereas the synthesis of many more labeled bands occurs in the presence of CAP. Furthermore, these protein bands differ from those visualized in the CHX treatments (Fig. 2). Verification of inhibitor effects Cyt/ antibodies reacted with a protein band synthesized in the presence of CHX on western blots at approximately 36 kDa (Fig. 3). Immunoprecipitations using anti-cyt / (kDa) CON CHX CAP 218_l 43.5 33.9 _ 17.4_ 7.6_ Figure 1. Coomassie brilliant blue-stained 15% SDS-PAGE gel of proteins extracted from isolated Elysia chloroplasts. The protein bands visualized are identical regardless of the inhibitor treatment, CHX or CAP (CON refers to control). Approximate molecular weights are indicated to the left. identify a band with a molecular weight corresponding to cyt/, confirming its identity (Fig. 4). Autoradiograms of the same gels show [ 15 S]-methionine incorporation into cyt/ in the presence of CHX. but not in the presence of CAP (Fig. 5). The anti-LHCI we made to Porphyridium cruentum re- combinant LHCI recognized both the recombinant LHCI antigen (Fig. 5 A, lane 1 ) and the LHCI polypeptides from P. cruentum thylakoids (Fig. 5B, lane 2). Six polypeptide bands were identified in P. cruentum, ranging in approxi- mate molecular weights from 19 to 24 kDa (Fig. 5B, lane 2), sizes consistent with those previously described for the LHCI polypeptides in this species (Tan et al, 1995). The antibody bound onto western blots of plastid proteins from Vciucheria litorea and Elysia chlorotica, with or without the CHX and CAP treatments (Fig. 5C, lanes V. lit.. CON, 38 J. J. HANTEN AND S. K. PIERCE (kDa) CON CHX CAP 126- 90. 43.5 Discussion LHCI, a family of plastid polypeptides essential for pho- tosynthesis, is synthesized while Vaucheria litorea chloro- plasts reside within the cells of the digestive diverticula of Elysia chlorotica. In addition, our data indicate the LHCI polypeptides are probably the products of genes located in the host-cell nuclear genome because their synthesis is inhibited by the cytosolic ribosome inhibitor, CHX, but not by the presence of the plastid ribosome inhibitor, CAP. This remarkable result would not be surprising in a plant or algal species since the LHCI polypeptide family's genes, Lhcal- Lhca6, reside in the nuclear DNA of all plants and algae examined to date (Jansson, 1994; Green and Durnford, 33.9_ (kDa) 126_ 90 B 17.4 7.6 Figure 2. Autoradiograph of plastid proteins separated by SDS-PAGE gel run under the same conditions as those depicted in Figure 2. The plastid proteins incorporating [ 35 S]-methionine label differ following treatment with CHX or CAP. The control (CON) represents chloroplast proteins isolated from slugs without inhibitor treatment. Arrows identify the ap- proximate positions of cyt/ (large arrow) and the LHCI (small arrows) proteins. CHX, CAP). As expected, the six polypeptide bands bound by the anti-LHCI in V. litomi and E. chlorotica plastids have a slightly greater size range 18 to 32 kDa than those identified in P. cnicntiiin. These same antibody-la- beled bands from E. chlorotica plastid proteins incorporate radioactive label in the presence of CAP. but incorporation is blocked by the presence of CHX (Fig. 6). The amount of radiolabel precipitated by anti-LHCI from the slug plastid extracts following CHX treatment is only 2% of the control level, indicating a reduction in LHCI synthesis (Fig. 7). In contrast, the LHCI proteins in CAP- treated slugs incorporated [ 35 S]-methionine at 92% of con- trol rates, more than 40-fold higher than the level found in CHX treated animals (Fig. 7). 43. 5 _ 33.9 _ 17.4_ 7.6 _ Figure 3. Immunoblot labeled with antibody to cyt / (A), and its corresponding autoradiograph (B). The slugs were exposed to CHX and the proteins were labeled as described in the methods. Anti-cyt / binds at approximately 36 kDa, coincident with a radiolabeled protein. The arrow indicates the autorudiograph band corresponding to the position of cyt /. SYMBIOTIC PLASTID GENES IN SLUGS 39 (kDa) CONTROL^ CHX CAP 16.8_ CBB Auto CBB Auto CBB Auto Figure 4. Immunoprecipitation of cyt/. Coomassie brilliant blue (CBB)-stained gels of proteins precipitated with anti-cyt / from chloroplast extracts from slugs subjected to no inhibitor (Control), to CHX. or to CAP. and their corresponding autoradiographs (Auto). The arrow indicates the position of cyt/. Large bands above and below cyt/ are the heavy and light chains of the antibody, respectively. The radioactivity corresponding to the antibody bands in control and CHX is probably undissociated cyt /. 1996: Durnford et at., 1999: Jansson. 1999; Wollman et a/.. 1999). However, the synthesis of LHCI directed by an animal's genome indicates that genes have been transferred into the slug DNA. Although surprising, the site of synthesis and the identi- fication of LHCI seem to be without question as long as inhibitor and antibody specificity are not problems. Both CHX and CAP have been used in a wide array of studies, and their sites of action are well established. In fact, they have been used, exactly as we have done here, to establish that the site of synthesis of the "light harvesting chlorophyll protein" (=LHCI) occurs on 80s cytoplasmic ribosomes in Phaeodactyliini tricomutum (Fawley and Grossman, 1986). There are several reasons to conclude that our antibody is specific. We raised the antibody against the red alga LHCI not only because it was available, but also because the chromophytes, the taxonomic group of V. litorea, probably arose through a secondary symbiosis from a red alga (Rieth, 1995; Green and Durnford, 1996; Palmer and Delwiche, 1996; Martin and Herrmann, 1998: Delwiche, 1999). Fur- thermore. Porphyridium cnientum LHCI possesses both sequence homologies and immunological relatedness to the chromophytic light-harvesting proteins (Wolfe et ai, 1994; Rieth, 1995; Tan et al.. 1997b). Thus, a polyclonal antibody raised to a rhodophyte LHCI should have a good chance of specifically recognizing the LHCI polypeptides in V. lito- rea. Our results indicate that the anti-LHCI binds the P. cnientum recombinant LHCI, the antieenic source for the antibody, as well as all six of the native P. cnientum LHCI proteins (Tan et al., 1995; Grabowski et al., 2000) in control immunoblots of extracted thylakoids. The anti-LHCI immu- noblots of E. chlorotica and V. litorea also identified six protein bands with a greater size range than the LHCI proteins identified in P. cnientum. Those bands are consis- tent with the sizes of LHCI polypeptides from many species (Gantt. 1996; Jansson. 1999: Wollman et ai, 1999), and no other bands were labeled by the antibody. Seeing six LHCI proteins is not surprising, because LHCI is typically found in multiple homologs in algae, ranging from two in one species of Xanthophyceae (Buchel and Wilhelm, 1993) to at least six paralogs in some rhodophytes (Tan et al., 1995), and as many as eight in the chromophyte Heterosigma carterae (Durnford and Green, 1994). With few exceptions [such as in Euglena gracilis (Jansson, 1994)], each is en- coded by a separate, nuclear gene belonging to the Lhc super-gene family (Jansson, 1999). Thus, location of the gene aside, the presence of six LHCI proteins in the endo- symbiotic plastids in E. chlorotica is not surprising. It seems clear that each of the bands immunodecorated by anti-LHCI corresponds to a single LHCI polypeptide and not a dimer. LHCI dimers can result from their association with other LHC proteins and their respective photosystems /;; situ, and they do not always readily dissociate under the denaturing conditions of SDS-PAGE (Tan et al., 1995). If LHCI dimers were present here, they should have minimum molecular weights of about 36 kDa, corresponding to dou- 40 J. J. HANTEN AND S. K. PIERCE A B C (kDa) 1 (kDa) 2 (kDa) V. lit CON CHX CAP 33.9 29.0 33.9 17.4 18.2 17.4 Figure 5. Immunoblots testing the antibody raised to Porphyridium inientum LHCI. (A) Anti-LHCI binds the recombinant 22 kDa Llica RI product from P. cruentwn (lane i ). Its appearance as a 28-30 kDa protein in SDS-PAGE and subsequent immunoblots results from the addition of a 33 amino acid N-termina! fusion in the recombinant protein (Grabowski el at., 2000). (B) Anti-LHCI binds LHCI polypeptides extracted from P. cruentum thylakoids (lane 2). (C) Vauclieria litorea (lane V. lit.) and Ely\ia chlorotica plastid proteins have six bands binding the anti-LHCI identical in size to each other. All six proteins are present in the slugs regardless of the inhibitor treatment [lanes CON (control). CHX and CAP]. Molecular weights are indicated to the left of (A). (B). and (C). ble the molecular weight of the smallest immunolabeled band. However, the largest of the six immunolabeled bands present in the gels is about 32 kDa, seemingly too small to be an LHCI dimer. Other dimers might form with a number of photosystem I (PSD proteins due to the close association of LHCI with the PSI subunits that compose the PSI-LHCI holocomplex (Wollman et al.. 1999; Jansson. 1999). This also does not seem to be the case here. Anti-PSI. raised against the cyanobacteria PSI holocomplex (again, courtesy of Profes- sor Gantt), binds a single 10-kDa protein band on western blots of E. chlorotica plastid proteins (data not shown). The combination of this PSI polypeptide with any of the three smaller bands (18-20 kDa) that react with the anti-LHCI could form a dimer with molecular weights comparable to each of the three larger polypeptides (28-32 kDa). How- ever, since anti-PSI and anti-LHCI do not co-label any bands, an LHCI-PSI dimer is unlikely. An additional possibility might be that one of the bands could be another LHC-type protein possessing immunolog- ical similarities to LHCI, such as the fucoxanthin chloro- phyll ale binding proteins (FCPC) found in chromophytes or light-harvesting complex II (LHCII) proteins. In fact, our previous work has demonstrated the presence of FCPC in plastids of both E. chlorotica and V. litorea. However, the size of the FCPC protein identified there does not corre- spond to the weights of the proteins bound by the anti-LHCI used here (Pierce et al.. 1996; Green et til., 2000). Further- more, previous attempts to demonstrate FCPC synthesis with radioactive labels in the slugs have not yielded positive results (Pierce et ai. 1996). The LHCII family of polypeptides is closely related to LHCI, performing similar functions in photosystem II to those performed by LHCI in PSI. The LHC II genes are in the same nuclear-encoded Lhc super-gene family (Jansson, 1999) and share sequence homologies with those genes encoding LHCI (Durnford et al., 1999; Jansson. 1999; Wollman et al., 1999). There is, however, a clear separation in the phylogenies of LHCI and LHCII (Durnford et al.. 1999), indicating some degree of dissimilarity between the two proteins. Nevertheless, the possibility seems to remain that the proteins bound by our antibody could be from LHCII. Of the LHCII components, CP24. CP26, and CP29 con- tain the most sequence similarities to the LHCIs (Green and Durnford. 1996) and have molecular weights. 25-30 kDa (Wollman et al., 1999). that roughly correspond to these of the three largest polypeptides identified in our anti-LHCI immunoblots of E. chlorotica and V. litorea plastid proteins (28-32 kDa), which appear to be slightly larger than most LHC proteins in chromophytes (Green and Durnford, 1996). An LHCII antibody derived from pea (generously donated by Dr. Kenneth Cline, University of Florida) was unreactive in our iinmunoblotting protocol (data not shown). This SYMBIOTIC PLASTID GENES IN SLUGS 41 CAP CHX Figure 6. Immunoblot (IB) of LHCI synthesized in the presence of CAP and 35 [S]-methionine, and its corresponding autoradiograph (CAP). The arrows indicate radiolabeled bands coinciding to LHCI immunola- beled bands shown in (IB). The bands in (CAP) are not labeled in the presence of CHX (CHX). result seems to indicate that the polypeptides are not LHCII, but since the similarity between the green plant and chro- mophyte LHC proteins is relatively low (Green and Durn- ford, 1996; Durnford et al., 1999), we probably cannot completely eliminate the possibility that the anti-LHCI is binding LHCII polypeptides. However, just like LHCI, all of the LHCII genes are nuclear encoded in the plants and algae where they have been found (Jansson, 1994, 1999; Wollman et al.. 1999), and even if we have identified LHCII, the conclusion is still the same: that an algal LHC gene has been transferred to the DNA of the slug. The immunoprecipitations provide additional evidence that the LHCI polypeptides are being synthesized on the cytoplasmic ribosomes in the slug. The high amount of radioactivity precipitated by the antibody in the presence of CAP compared to that precipitated in the presence of CHX demonstrates that the proteins recognized by the anti-LHCI are indeed synthesized in the slugs. Since the amount of radioactivity incorporated varied from slug to slug and from experiment to experiment, we had to normalize the immu- noprecipitation data as percent of control in order to com- pare them. However, in a typical experiment, the values for the amount of radioactive material incorporated into the precipitate in the presence of CAP ranged from 5000 to 25,000 cpm, whereas those in the presence of CHX ran from 150 to 400 cpm, which may give a clearer picture of the level of material bound by the antibody. The results of the pharmacological experiments, the im- munoblots, and the immunoprecipitations. taken together, provide substantial evidence that LHCI is the identity of some of the plastid proteins that are synthesized in the presence of CAP. The inhibition of LHCI synthesis by CHX suggests that the algal Lhca genes have somehow been transferred to the slug. To be certain that a gene transfer has occurred, direct evidence of the gene in the genomic DNA of the slug must be found, and we are pursuing this confirmation. However, in addition to the results presented here, other circumstantial evidence for the transfer of the LHCI genes between alga and slug is available in several characteristics of the asso- ciation. First, although the turnover rate of LHCI in E. chlomtica is unknown, the fact that it is synthesized indi- cates that it is not an unusually robust protein LHCI replacement is necessary for plastid function to proceed. Second, Lhca genes have not been found in the plastid genomes of any organism (Durnford et /., 1999), including other Vaucheria species (Linne von Berg and Kowallik, 1992). Of course, if LHCI were present in the plastid genome, it would be synthesized with CHX present, as is the case with the cyt / controls; but it is not. Third, the V. litorea plastid genome is 119.1 kb (Green et al., 2000), which is similar in size to those of other algae, including V. 125- o 75 -i O c 0) 50- 0) - 25- , i CONTROL CHX CAP Inhibitor-Treatment Figure 7. CHX inhibits synthesis of LHCI. In the presence of CHX. anti-LHCI precipitated only 2% of control radioactivity incorporated into LHCI compared to 92% of control in the presence of CAP. Control rates were defined as 100%. and inhibitor rates were calculated as a mean percent of control (>i = 6). 42 J. J. HANTEN AND S. K. PIERCE sessilis and V. hursata (Linne von Berg and Kowallik, 1988, 1992), hut small relative to those of other plants (Martin and Herrmann. 1998). Even though the plastid genomes of chro- mophytic algae have a greater coding capacity, relative to their size, than other algae because of fewer introns and inverted repeats (Rieth, 1995). they are too small to carry sufficient genetic information to encode all of the enzymes required for photosynthesis and plastid protein targeting. Fourth, transfer of algal DNA remnants or a nucleomorph- type structure during plastid capture seems unlikely. To date, nucleomorphs have been found only in the Crypto- phyta and Chlorarachniophyta (Delwiche. 1999; Zauner et til.. 2000) and have not been identified in any chromophyte (Maier et a!., 1991; Delwiche, 1999). Although DNA of this type would probably be transcribed on nucleomorph SOS ribosomes (Douglas et al, 1991) and blocked by CHX. neither substantial electron microscopy (Kawaguti and Ya- masu, 1965; Graves et til., 1979; Mujer et al., 1996) nor molecular testing (Green et al., 2000) has so far produced evidence for either nucleomorphs or algal nuclear remnants in E. chlorotica. Furthermore, if algal DNA remnants were present somewhere in the slug cells, the likelihood is remote of their containing the correct genes and being present in all of the plastid-containing cells in all of the slugs in the populations year after year. Finally, others have suggested that some of the proteins necessary to maintain photosyn- thesis may be encoded in the mitochondria! genome and are redirected to the chloroplast (Rumpho et al.. 2000). Al- though dual targeting of proteins has been demonstrated in Arabidopsis (Chow et al.. 1997: Menand et al.. 1998), it seems highly unlikely with LHC1. LHCI has never been found associated with mitochondria in any organism; and CAP, which inhibits the mitochondria! ribosomes in addi- tion to those associated with the plastids. would prevent its synthesis anyway. The horizontal transfer of DNA from the endosymbiont to the nucleus of the host cell provides the basis for the theory of the endosymbiotic origin of eukaryotic organelles. This movement of the symbiont's genes to the host enabled the host to incorporate the organelle's function into its own biochemistry and to faithfully replicate it in subsequent generations. The remnants of eubacterial genes in the mi- tochondria! and plastid genomes of modern eukaryotes probably resulted from such events (Martin and Herrmann. 1998). Most of the discussions regarding the evolution of plastids focus on the horizontal gene transfer resulting from the primary endosymbiotic event in which a primitive pro- karyote engulfed a cyanobacteria (Palmer. 1993; Reith. 1995; Palmer and Delwiche, 1996; Martin et al., 1998; Tengs et al., 2000). Other hypotheses propose a secondary endosymbiosis, probably involving a eukaryote that en- gulfed a red or green alga (Gibbs, 1981; Palmer and Del- wiche, 1996; Martin et al.. 1998: Zhang et al., 1999; Del- wiche, 1999; Tengs et al.. 2000), that produced the plastids of the chromophytic algae and their relatives. In many of these cases, the identity of the initial host, symbiont, or both is unknown. In the case of E. chlorotica and V. litorea, the origin of LHCI is known; if the gene has been transferred, the transfer occurred between two multicellular eukaryotes and represents a case of tertiary endosymbiosis. Finally, the mechanism by which such a gene transfer could occur may be found in the viruses that appear in each generation of the slugs at the end of their life cycle. The viruses have several features in common with Retroviridae and seem to be endogenous (Pierce et al.. 1999). Retrovi- ruses are capable of transferring genes between organisms; if they are incorporated in the germ cells, they are trans- ferred to the subsequent generations as Mendelian genes (Scharfman et al.. 1991). Thus, resolving the relationships between the slugs, alga, plastids, and viruses may have profound implications for both cell and evolutionary biol- ogy. Acknowledgments Research support was provided by a National Science Foundation award (IBN-9604679) to SKP. 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McCURDY* Coastal Studies Center, 6775 College Station, Bowdoin College. Brunswick, Maine 04011-8465 Abstract. Life-history theory predicts that parasitized hosts should alter their investment in reproduction in ways that maximize host reproductive success. I examined the timing of asexual reproduction (fragmentation and regeneration) in the polychaete annelid Pygospio elegans experimentally exposed to cercariae of the trematode Lepocreadium setiferoides. Con- sistent with adaptive host response, polychaetes that became infected by metacercariae of trematodes fragmented sooner than unexposed controls. Parasites were not directly associated with fission in that exposed polychaetes that did not become infected also fragmented earlier than controls. For specimens of P. elegans that were not exposed to trematodes, new frag- ments that contained original heads were larger than those that contained original tails, whereas original head and tail frag- ments did not differ in size for infected polychaetes. In infected specimens, metacercariae were equally represented in original head and tail fragments and were more likely to be found in whichever fragment was larger. Despite early reproduction, parasitism was still costly because populations of P. elegans exposed to parasites were smaller than controls when mea- sured 8 weeks later and because exposure to cercariae reduced survivorship of newly divided polychaetes. Taken together, my results suggest that early fragmentation is a host response to minimize costs associated with parasitism. Introduction Hosts respond to parasitism in a number of ways, which include avoidance of parasites in space or time (e.g.. mi- Received 19 October 2000; accepted 10 April 2001. * Current address: Department of Biology, Albion College, Albion, Michigan 49224. E-mail: dmccurdy20,000 m~ 2 ), but Ilyanassa obsoleta and its associated cercarial parasites were rare (<0.25 snails m" 2 ), minimiz- ing the likelihood that polychaetes used in experiments were already infected. I collected polychaete tubes in the mid- intertidal zone by sieving the top 5 cm of mud (500-jum mesh) and transported tubes to the nearby running-seawater laboratory at the Coastal Studies Center of Bowdoin Col- lege for sorting. I retained only undamaged, entire adult polychaetes (>2 mm) that were not about to fragment (detectable because P. elegans constricts just prior to fis- sion; Gibson and Harvey. 2000). To obtain cercarial trematodes for experiments. I col- PARASITISM AND ASEXUAL REPRODUCTION 47 lected specimens of /. obsoleta from throughout the inter- tidal zone at Strawberry Creek. Great Island, Maine (4349'N. 6958'W). This mudflat is located 2.5 km from the Wyer-Orr's mudflat and supports high densities of /. obsoleta (>10 m~ 2 ). In the laboratory. I housed 550 mud snails in separate 9-oz plastic cups with 125 ml of filtered seawater (55 ^im, 31 ppt, 23 C). I retained only large snails (>15 mm. tip of apex to lip of siphonal canal) because previous studies have shown that the prevalence of Lep o- creadium setiferoides increases with shell height of snails (Curtis. 1997; McCurdy el ai. 2000c). After 30 h. I exam- ined each cup for cercariae of L. setiferoides (identified using McDermott, 1951). combined cercarial-infested sea- water from cups of six snails that had shed cercariae, and pipetted 20 ml of the solution into each dish that contained a polychaete that was to be exposed. Unexposed polychaetes each received 20 ml of seawater from six cups that contained snails that did not shed cercariae (confirmed by dissection, as cercarial release is a poor indicator of infection status; Curtis and Hubbard. 1990). Experiments To investigate the impact of parasites on the timing of asexual reproduction, I individually housed 52 adult speci- mens of P. elegans in 150-ml custard dishes filled with unfiltered seawater with or without cercariae (18 C. 16 h light day" 1 ). After 24 h. I transferred each polychaete to a new dish filled with seawater and lined with defaunated mud (prepared by passing mud through a 425-ju.m sieve and heating it to 70 C). Every 24 h, I suspended each dish from a harness and determined the status of each polychaete by observing its tube (or tubes) through the bottom of its dish with the aid of a fiber-optic illuminator and 10X magnifying loupe. Polychaetes could easily be observed because they constructed tubes that opened against the bottoms of their dishes. Polychaetes were fed the pea-flower- based supple- ment Liquifry Marine (Interpet Inc.; Brown el ai, 1999) every 3 days (concentration = 1 drop 1 ~ ' ) following a complete change of water. I removed polychaetes from the experiment when they died or fragmented, and I measured the relaxed length of all fragments with an ocular microme- ter (nearest 0.1 mm; Gudmundsson. 1985). I then dissected each fragment to determine if it was infected by trematode metacercariae and compared median time-to-fragmentation among exposed but uninfected. exposed and infected, and unexposed polychaetes. In making this comparison. I sepa- rated exposed but uninfected polychaetes from unexposed ones because of the possibility that host response might be associated with indirect cues associated with parasitism (i.e., response might not require an actual infection to oc- cur). To compare time-to-fragmentation. I applied a non- parametric Kruskal-Wallis ANOVA because the residuals for all groups were non-normal. I then applied Dunn's method to compare differences among medians (Zar, 1996). To investigate how exposure to parasites affected the reproductive success of P. elegans, I randomly housed 18 sets of 10 polychaetes (hereafter referred to as populations of polychaetes) in separate dishes and exposed half of the sets to cercariae of trematodes (housing conditions for polychaetes were as described above). Because the infection status of polychaetes that died during this experiment could not be determined without disturbing surviving polychaetes, I assessed rates of experimental and background infection by randomly removing two sentinel populations after 3 days: a population of polychaetes that had been exposed to cercariae, and a population of unexposed polychaetes. Rates of infection at that time represented maximum levels that could occur because cercariae of L. setiferoides survive for less than 48 h outside a host (Stunkard, 1972). After 8 weeks. I removed the remaining dishes and processed each population by counting the number of polychaetes retained after sieving (425-jam mesh) and dissecting each polychaete to determine its infection status. To assess survivorship and regenerative ability of newly divided polychaetes in relation to parasitism. I cut 59 polychaetes into two fragments and exposed 30 pairs of fragments to cercariae. Cutting each polychaete resulted in a smooth, clean blastema similar to that resulting from sublethal predation or asexual fragmentation (Gibson and Harvey. 2000; pers. obs.). To mimic conditions in nature, where newly fragmented polychaetes generally remain in the same burrow during regeneration (Gudmundsson, 1985; Gibson and Harvey, 2000). I individually housed original head and tail fragments together in a dish with seawater and mud (housing conditions as described above). To avoid disturbing fragments (as above), I assessed initial rates of infection at 3 days after exposure or non-exposure by re- moving and dissecting randomly chosen sentinel pairs of exposed fragments (/; = : 10 polychaetes) and unexposed fragments (n = 10 polychaetes). At 10 days after exposure or non-exposure. I removed all remaining fragments, mea- sured their lengths, and determined their infection status. Results Parasitism and host fragmentation In the experiment investigating the impact of trematodes on the timing of asexual reproduction in Pygospio elegans, parasite prevalence was low (42.3% of polychaetes exposed became infected; n = 26). Asexual fragmentation always yielded two fragments; one containing the original head and thorax and a second containing the original tail (see Gibson and Harvey, 2000, for a description of body components). In all cases, polychaetes fragmented within 24 h of observable constrictions. Time-to-fragmentation differed between ex- posed and infected, exposed but uninfected. and unexposed 48 D. G. McCURDY 30 a -o F 2-1 S l b -= 1 < c b tr 1 6 , i i s i i Unexposed !x posed/ Exposed/ LtninlcLlcd Infected Treatment Figure 1. Median ( quartiles) numbers of days for asexual reproduc- tion to occur in individuals of Pygospio elegans that were experimentally infected, exposed but not infected, and not exposed to cercariae of the trematode Lepocreadiwn setiferoides. Polychaetes and parasites were col- lected from mudflats in Harpswell, Maine, and housed in the laboratory. Median-, with the same letter do not differ significantly from each other. polychaetes (// |2 . 52) = 10.56. P < 0.01: Fig. 1). Specif- ically, polychaetes that were exposed to cercariae but did not become infected fragmented earlier than unexposed polychaetes (Q = 2.99, P < 0.005), as did polychaetes that were exposed and became infected (Q = 2.16, P 0.05). Of all polychaetes that were exposed to cercariae, however, infection status did not affect time-to-fragmenta- tion (Q = 0.49, NS). For unexposed polychaetes and exposed polychaetes that remained uninfected. fragments that contained original heads were larger than those that contained original tails, whereas lengths of original head and tail fragments did not differ for infected polychaetes (Table 1 ). In infected polychaetes. parasites were just as likely to be found in fragments that contained original heads (n = 5) as those that contained original tails (n 5) (an additional polychaete harbored a metacercaria in each new fragment). For infected polychaetes, infected fragments were signifi- cantly larger than uninfected fragments (infected fragments: x s = 2.0 0.2 mm; uninfected fragments: x s = 1.4 0.2 mm; paired r (l)) = 2.28. P < 0.05). and in 9 of 10 cases, metacercariae were found in the larger fragment (Xf, > = 6.4, P = 0.01). Cercariae were not observed to penetrate segments that comprised, or were adjacent to, planes of fission. Parasitism and host asexual reproductive success At 3 days post exposure, 17 of 20 fragments (8.5 of the original 10 polychaetes) were alive in the sentinel popula- tion that was exposed to cercariae. Only one fragment in this population was infected by trematodes a living tail frag- ment infected with a single metacercaria. In the sentinel population that was not exposed to cercariae, 18 of 20 fragments were alive after 3 days and no parasites were found (one fragment, containing an original head, was lost during processing). At 8 weeks after exposure or non- exposure, I saw no evidence of recent fission in polychaetes as all fragments had complete or nearly complete heads and tails. Therefore. I considered all fragments equally when measuring population sizes at that time. Populations of polychaetes that were exposed to cercariae were smaller than those that were not exposed (exposed populations: A 5 = 17.3 2.4 polychaetes; unexposed populations: x s = 29.8 3.7 polychaetes; / (14) = 2.84, P = 0.01). When dissected, only seven polychaetes in exposed popu- lations were infected (one polychaete in each of three pop- ulations and two polychaetes in each of two populations), and none of the polychaetes in any of the unexposed pop- ulations was infected. Considering sentinel polychaetes that had been cut into two pieces, 2 of 10 polychaetes exposed to cercariae were infected at 3 days post-exposure. In each case, the infection was in the original head fragment and by a single metacer- caria. None of the 10 unexposed polychaetes was infected. When examining the remaining polychaetes 7 days later, I found that both head and tail fragments of exposed polychaetes were less likely to be alive than the respective fragments of unexposed polychaetes (head fragments: = 8.07, P < 0.005; tail fragments: , , = 12.22, P < 0.001 : Fig. 2). Only two exposed polychaetes were infected by metacercariae (one polychaete had an infected tail frag- ment and another an infected head fragment; /; = 20). and no unexposed polychaetes were infected (n == 19). In all cases, regeneration of "lost" components was nearly com- plete by 10 days, and lengths of original head and tail fragments did not differ in relation to exposure (unexposed heads: A SE = 2.65 0.15; exposed heads: x SE = 2.56 0.26; r (26) = 0.32. NS: unexposed tails: x SE = Table 1 Si;cs af fraxiiicnt* produced hy uxe.\iial fission of Pygospio elegans in relation to panisitism Fragment length (mm| Heads Tails Paired / test Unexposed 2.1 0.2 1.57 0.1 /, 2 ,, = 2.7. P = 0.01 Exposed but uninfected 2.4 0.2 1.57 0.2 f,, 4l = 2.3, P = 0.04 Exposed and infected 1.9 0.2 1.71 0.2 ?,,, = 0.8. P = 0.44 Data are means and standard errors for lengths of fragments containing original heads and those containing original tails of polychaetes that were experimentally infected, exposed but not infected, and not exposed to cercariae of the trematode Lepocreadiwn setiferoides. The last column shows results from paired t tests for lengths of original head versm, tail fragments. PARASITISM AND ASEXUAL REPRODUCTION 49 I cC A Heads Tails Original fragments Figure 2. Proportions (95% confidence intervals) of original head and tail fragments of individuals of Pygospio elegans that survived for 10 days in the laboratory following exposure or non-exposure to cercariae of the trematode Lepocreatl/iuii .fciiti'i-niilfs. Sample sizes are shown above the bars. 2.71 0.19: exposed tails: x SE = 2.49 0.28; f (22) = 0.63. NS). Discussion Parasitism and host fragmentation In support of the hypothesis of adaptive host response I found that specimens of Pygospio elegans infected by meta- cercariae of Lepocreadium setiferoides hastened their onset of asexual reproduction relative to unexposed controls. By doing so, polychaetes may be expected to achieve greater reproductive success than if they had failed to respond because of increasing costs associated with parasitism over time (Forbes, 1993). However, my observation that early fragmentation also occurred in exposed polychaetes that remained uninfected complicates this interpretation. In a study that separated hosts by exposure and infection status, Minchella and Loverde ( 1981 ) found that freshwater snails of the species Biomphalaria glahrata increased their rates of early egg laying when infected by Schistosoma mansoni, but that the rates for exposed but uninfected individuals and unexposed controls did not differ. These authors argued that only infected snails responded because successful parasit- ism was associated with a high cost to future reproduction (castration). For individuals of P. elegans exposed to, but not infected by, cercariae. early reproduction could still be an adaptive host response if exposure to cercariae in nature is a reliable indicator that costly infections will soon result (Minchella. 1985). Support for this idea comes from the observation that Ilyanassa obsoleta infected by L. setiferoides, although uncommon across mudflats, can remain for several months in small patches where some P. elegans are found (Mc- Curdy et til., 20()0c). As a result, thousands of cercariae are shed in areas where infections are most likely to occur. Additional information on the infection process of L. setif- crnitles is necessary to determine whether polychaetes de- tect cercariae, and whether the exposure-related response resulted from the presence of cercariae or from failed at- tempts at penetration. There is evidence from other parasite- host systems that invertebrates can detect and exhibit anti- parasite behaviors to minimize the likelihood of infection (e.g.. Leonard et al.. 1999). Early fragmentation of P. elegans is unlikely to be a parasite adaptation, because it apparently does not increase transmission rates for cercariae or metacercariae. Specifi- cally, fragmentation was not associated with increased sus- ceptibility to parasitism: most polychaetes fragmented after free-living cercariae would have (>48 h; Stunkard, 1972). For metacercariae. residing in small fragments would not appear to benefit transmission to final hosts, because floun- der select prey at larger sizes relative to conspecifics, and even small differences in prey size preference can pro- foundly influence the energy budgets of predators foraging on mudflats (MacDonald and Green. 1986: Boates and Smith, 1989; Keats. 1990). To assess whether early frag- mentation is actually adaptive for parasites or hosts, the consequences of early fragmentation could be further ex- plored by constructing a model derived from empirical observations of parasites, their intermediate hosts, and the predators that are their final hosts. This approach was used recently to show that the early onset of receptivity to mating observed in females of the amphipod Corophium volntator infected by the trematode G\naecotyla adunca resulted in greater reproductive success for the amphipods than if they had waited to become receptive at the optimal time for uninfected females (McCurdy et al., 2001). I found no evidence that fragmentation of P. elegans served to isolate or remove metacercariae, in that fission produced only two fragments, the smaller of which almost never contained metacercariae. It is unclear whether the greater presence of metacercariae in larger fragments is adaptive for the parasite or its host or whether larger frag- ments merely represent larger targets for parasites. Meta- cercariae might benefit from residing in larger fragments because of the availability of additional resources for para- site development or the possibility of a greater transmission rate to final hosts (as stated above, flounder tend to select larger prey). If residing in larger fragments is parasite- mediated, the observation that metacercariae develop near the site of initial penetration (Stunkard. 1972; pers. obs) indicates that the mechanism does not involve movements by metacercariae through the host coelom and into larger fragments. Fragmentation could also be interpreted as a host response: If larger fragments are better able to tolerate stresses associated with parasitism, the result would be a net reproductive benefit to hosts. In fact, host response need not 50 D. G. McCURDY be exclusive of benefits to parasites, depending on the timing of altered behavior of infected hosts (McCurdy et ai. 1999). Simulated parasites such as Sephadex beads (Suwan- chaichinda and Paskewitz. 1998) could be used to help separate effects mediated by the parasite from those medi- ated by the host. Experiments with simulated parasites would provide cues to the host that it has become infected while removing the possibility of parasite manipulation. Across all experiments, I found no evidence for onset of sexual reproduction, observing neither eggs nor spermato- phores. Seasonal constraints may have precluded sexual reproduction, which usually occurs only during the winter in P. elegans (Rasmussen, 1953; Gudmundsson, 1985; Wil- son, 1985). However, even if the polychaetes had shown evidence of sexual reproduction, this tactic might be ex- pected to increase reproductive success only if mates were available; an unlikely event given the rarity of parasites in natural populations of P. elegans (above). Parasitism and host asexual reproductive success I found that even a low level of exposure to cercariae (on average, 8% of cercariae that a single snail sheds in 30 h) reduced the asexual reproductive success of P. elegans (45%, measured in populations 8 weeks after exposure). In a related finding from another experiment, both head and tail fragments were less likely to survive to complete regen- eration than were unexposed fragments. Direct effects of parasitism are not sufficient to account for these results given that few exposed polychaetes actually became in- fected in either experiment. One possibility is to explain the reduced reproductive success of exposed but uninfected hosts as the result of a trade-off between host reproductive effort and costly activities associated with defenses against parasites. Recent work has shown that hosts exposed to parasites may trade off energy used in reproduction for behaviors or immune responses to resist parasites (Sheldon and Verhulst, 1996; Leonard et ai, 1999). Regardless of the underlying causes, the dramatic reduc- tion in reproductive success of P. elegans after exposure to cercariae has implications for natural populations of this species and for soft-bottom intertidal communities. Pygos- pio elegans often dominates such communities, and thus can directly affect the distribution and abundance of other infauna (Wilson, 1983: Brey, 1991; Kube and Powilleit, 1997). In addition, it is possible that parasitism of P. elegans may influence the structure of intertidal communities by altering or creating engineering functions in hosts. Engi- neering functions are those that produce new habitat as a result of changes in behaviors or life history associated with parasitism (Thomas et ai, 1999). 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We explored the effects of temporal variation in sperm availability on fertilization and subsequent larval development in the colonial ascidian Botryllus schlosseri. a brooding hermaphrodite that has a sexual cycle linked to an asexual zooid replacement cycle. We developed a method to quantify the timing of events early in this cycle, and then isolated colonies before the start of the cycle and insemi- nated them at various times. Colony-wide fertilization lev- els (assayed by early cleavage) increased from zero to 100% during the period when the siphons of a new generation of zooids were first opening, and remained high for 24 h before slowly declining over the next 48 h. Because embryos are brooded until just before the zooids degenerate at the end of a cycle, delayed fertilization might also affect whether em- bryos can complete development within the cycle. Conse- quently, we also determined the effect of delayed insemi- nation on successful embryo development through larval release and metamorphosis. When fertilization was delayed beyond the completion of siphon opening, there was an exponential decline in the percentage of eggs that ultimately produced a metamorphosed larva at the end of the cycle. Thus, even though the majority of oocytes can be fertilized when insemination is delayed for up to 48 h, the resulting embryos cannot complete development before the brooding zooids degenerate. Introduction Field experiments have contributed greatly to current understanding of fertilization processes in free-spawning marine invertebrates (reviewed by Levitan and Petersen, 1995; Yund, 2000). In response to the evidence of potential Received 20 October 2000; accepted 8 March 2001. * To whom correspondence should he addressed. E-mail: jssavage@ uno.edu sperm limitation reported in some field studies, many lab- oratory studies have started to explore diverse related as- pects of invertebrate reproductive biology such as gamete viscosity (Thomas, 1994a,b). egg size and sperm swimming speed (Levitan, 1998), egg longevity (Meidel and Yund, 2001 ), sperm morphology (Eckelbarger et at., 1989a,b), and the kinetics of fertilization (Young. 1994; Levitan. 1998; Powell et a/., 2001 ). However, results from laboratory stud- ies have in turn led some authors to question the extent to which simple field fertilization experiments adequately mimic the details of fertilization processes in nature (e.g., Thomas, 1994a,b; Meidel and Yund, 2001). Field experi- ments may often circumvent aspects of reproductive strat- egies that have evolved to mitigate sperm limitation (Yund, 2000). Hence laboratory experiments still play a vital role in understanding reproductive strategies, and field fertilization studies should endeavor to incorporate the details of the fertilization process gleaned from laboratory work. Performing realistic field experiments with marine inver- tebrates that brood embryos presents challenges that are very different from those faced when dealing with broadcast spawners. The biggest challenge with field fertilization studies of broadcasters is interpreting results obtained by artificially holding eggs in a concentrated group (e.g., Levi- tan and Young. 1995; Wahle and Peckham, 1999) or by removing them from the water column after only a brief interval (Levitan, 1991; Coma and Lasker, 1997). This issue is moot with brooders, who by definition retain eggs and have internal fertilization. However, a different set of prob- lems merits further consideration. The precise timing of egg viability, sperm release, and fertilization itself is often less well understood than in broadcasters. Sperm function may be regulated by the female through sperm chemotaxis (Miller, 1985), activation (Bolton and Havenhand, 1996), or storage (Bishop and Ryland, 1991). In the latter case, the 52 EFFECTS OF DELAYED INSEMINATION 53 temporal pattern of fertilization within a female may be uncoupled from the pattern of sperm release by males. In hermaphrodites, the potential for self-fertilization is a con- cern, and genetic analyses of paternity may be required to conclusively exclude selting in some taxa (Yund and Mc- Cartney, 1994). For some brooders, the actual path of sperm access to eggs is poorly understood. Information on all of these topics is critical both to the design of more realistic field fertilization studies and to the interpretation of existing studies. The colonial ascidian Botryllus schlosseri is a useful model for field fertilization studies (Grosberg, 1991; Yund and McCartney, 1994; Yund, 1995, 1998). Fertilization is internal, and embryos are brooded until released as tadpole larvae (Milkman, 1967). When colonies are grown on glass surfaces, egg production can be quantified non-destructively (Yund et cil.. 1997), thus permitting estimation of fertiliza- tion levels by comparing egg and embryo counts (Yund, 1995, 1998). Although the general time of fertilization within the life cycle (i.e., temporal resolution on the order of a day) has long been known (Milkman, 1967), the finer- scale timing (temporal resolution on the order of hours) has not been explored. Many authors have assumed that the apparent temporal separation of fertilization and sperm re- lease prevents self-fertilization (e.g., Milkman, 1967; Gros- berg, 1987; Yund and McCartney, 1994), but we have recently shown (Stewart-Savage and Yund, 1997) that sperm release commences several days earlier than previ- ously thought. Although sperm storage has been demon- strated in another colonial ascidian (Bishop and Ryland. 1991; Bishop and Sommerfeldt. 1996), past workers have implicitly assumed that storage is unlikely in B. schlosseri (Milkman, 1967; Grosberg. 1991; Yund, 1995, 1998). To the best of our knowledge, this assumption has never been explicitly tested. To address this interrelated set of issues, this paper explores the effect of variation in the timing of fertilization on fertilization levels and subsequent larval development in B. schlosseri. and compares those results with published information on the timing of sperm release. Materials and Methods Study organism Colonies of Botryllus schlosseri are composed of asexu- ally produced zooids arranged in clusters, or systems, with all zooids in a system sharing a common exhalant siphon. Throughout the life of a colony, all zooids periodically undergo a synchronous asexual zooid replacement cycle in which a new generation of zooids, termed buds, forms between the existing zooids (Berrill, 1941; Izzard. 1973). At the end of the life span of adult zooids (about 8 days at 16C; cycle length is temperature dependent), the buds expand, take over the function of the previous generation of zooids (which are quickly resorbed). and then commence their sexual reproductive cycle. The sexual cycle includes the internal fertilization of the mature eggs soon after the inhalant siphons open (Milkman, 1967); the continuous release of sperm starting 16 h later (Stewart-Savage and Yund. 1997); and the brooding of developing embryos, which are released just before the zooids degenerate at the end of the cycle (Milkman, 1967). Standard methods The colonies of B. schlosseri that were employed in this study were collected from the Damariscotta River. Maine. Animals were grown on glass microscope slides in the flowing seawater system at the University of Maine's Dar- ling Marine Center. Field-collected colonies that had been established in laboratory culture were divided to provide clonal replicates (ramets) of genotypes. Colonies employed in all experiments were monitored for the approach of takeover (the transition between zooid generations). When colonies were about to commence takeover (late stage 5 through early stage 6 by the criteria of Milkman, 1967), they were isolated in 50 ml of sperm-free (aged >24 h) seawater. Isolated colonies were housed in an incubator at 16C (range: 14-18 C) and fed phytoplankton (Duniella sp.) at densities of approximately 10 5 cells/ml. Water and food were changed twice daily. Colonies were monitored for siphon opening and then isolated in individual 250-ml con- tainers with algae (water and food were changed daily) until exposed to sperm. Sperm exposure was accomplished by placing colonies in a flowing seawater tank in proximity to numerous male-phase colonies (>24 h after siphon open- ing; Stewart-Savage and Yund, 1997) for 1 h. After insem- ination, colonies were rinsed with aged seawater and re- turned to isolation. Experimental protocols To standardize insemination times, we first had to accu- rately quantify the start of the reproductive cycle (i.e., the functional opening of siphons). Inhalant siphons are formed early in the takeover process, but the common exhalant siphon of a system generally does not form until near the end. However, it is difficult to ascertain functional siphon opening on morphological criteria alone. In the course of other work, we observed that the consumption of green algae immediately turned the digestive systems of actively feeding zooids (i.e., those that must have open siphons) green. Consequently, we used algal uptake as an assay for siphon opening. To establish the temporal pattern of siphon opening, we isolated 14 colonies and briefly exposed them to algae three to four times during the process of takeover. At each sample interval we recorded the percentage of siphons that were open (% of zooids with green digestive systems). From these data we calculated an average rate of siphon opening. This approach subsequently allowed us to 54 J. STEWART-SAVAGE ET AL. make single observations of the percentage of siphons that were open and back-calculate the time of the first siphon opening. Both of our other experiments use this approach to estimate the time of initial siphon opening, and the timing of insemination is expressed relative to this event. To examine the effect of the timing of fertilization on fertilization levels, we exposed colonies to sperm through a range of different times after siphon opening (0.5 to 96 h; n =- 79). Colonies with about 20 eggs (mean of 20.0 standard error of 11.6) were utilized throughout, and all eggs and embryos in a colony were surgically removed 10-18 h after insemination and scored for successful de- velopment. Initial studies indicated that embryos should be in the 8-cell to the 32-cell stages during this time range. Uncleaved eggs were scored as unfertilized, as were em- bryos with an abnormal cleavage pattern (arrested cleavage, abnormal cell number or shape). A few embryos at ad- vanced developmental stages (e.g., gastrula) were excluded from the data set since fertilization was by either contami- nating or self sperm. To examine the effect of timing of fertilization on sub- sequent development and metamorphosis, colonies were initially fertilized in sets of multiple ramets per genotype. For each genotype, one ramet was left unfertilized (to assess the level of sperm contamination or self-fertilization), one ramet was fertilized about 22 (2) h after the beginning of siphon opening (when results from the previous experiment indicated that all siphons should be open), and remaining ramets (2-3) were fertilized at various times up to 85 h after initial siphon opening. Because fertilization was consis- tently minimal in unfertilized controls and the availability of genotypes with multiple egg-bearing ramets was often lim- ited, later trials were conducted without the control treat- ment. Before takeover, we counted the number of eggs produced by each colony (minimum egg production was set at 25 eggs). After insemination, colonies were returned to isolation until all ramets of a genotype had been fertilized and at least 24 h had elapsed since the last insemination. Colonies were subsequently housed in a flowing seawater table with an independent seawater supply while embryonic development proceeded: they were re-isolated at stage tour (Milkman. 1967). After each isolated colony had started the next reproductive cycle, all metamorphosed juveniles in the isolation container were counted. Data from colonies that died or became visibly unhealthy during the experiment were discarded. Results Timing of siphon opening Feeding did not begin until after the organization of zooids into new systems and formation of the common exhalant siphon. Although the rate of siphon opening varied among colonies (Fig. 1: range of 3.0%/h 17.8%/h), the 100 n o o N so H 40 - -o u u 20 - 4 Time from Initial Observation (h) Figure 1. Rate of siphon opening in colonies of Botryllus schlosseri as assayed by the presence of algae in the digestive system. Colonies were isolated in 50 ml aged seawater with 2 x 10 5 algae/ml and monitored at intervals of from 1 to 12 h. Zero time is the first observation of algae in the gut. Temporal patterns for 14 individual colonies are shown. Differences in the v-intercept simply reflect how far the takeover process had proceeded when colonies were first observed; slopes indicate the rate of siphon opening. average rate of siphon opening of the colonies was 7.8%/ h 4.5%/h (X SD). We used the average rate of siphon opening to normalize the time of sperm exposure to the start of siphon opening for colonies in the other two experiments. Effect of timing of insemination on fertilization levels To determine the time frame during which eggs can be fertilized within the female, we exposed virgin females to a 1-h pulse of sperm at various times after the beginning of siphon opening and assayed successful fertilization by the percentage of normally cleaved embryos present (Fig. 2). When virgin females were exposed to sperm during the period in which their siphons were opening (first 24 h), the level of fertilization increased with time (Fig. 2B). In col- onies fertilized during siphon opening, there was no spatial relationship between fertilized and unfertilized eggs either within or among systems; it was common to find both in the same zooid. Because the rate of increasing fertilization (5.4%/h) is similar to the rate of siphon opening (7.8%/h 4.5%/h), we conclude that fertilization of the eggs within a zooid occurs shortly after the opening of the siphon. After the completion of siphon opening, fertilization suc- cess remained high (>90%) for 24 h and then declined over the next 48 h with a 7" 500 , of 72 h (Fig. 2B). In a subset of genotypes where multiple ramets were inseminated at dif- ferent times in the same reproductive cycle, thus controlling for potential genotype and cycle effects, the effect of EFFECTS OF DELAYED INSEMINATION 55 72 96 B 24 48 72 96 Insemination Time (h after start siphon opening) Figure 2. Effect of insemination pulse timing on fertilization levels. Colonies were isolated before the start of siphon opening, monitored for the timing of siphon opening, and exposed to sperm for 1 h: the number of cleaving embryos was determined 10-18 h later. (A) Fertilization levels in different ramets of seven genotypes fertilized at different points in the same reproductive cycle. (Bl Overall effect of insemination time on fertilization success in ramets from 25 genotypes. The line represents a polynomial regression of the data (R 2 = 0.580). delayed insemination on fertilization varied by genotype (Fig. 2 A). Of the seven genotypes in which different ramets were inseminated at different times, five genotypes had a decline in fertilization that mirrored the population data. In the other two genotypes, fertilization levels declined rapidly in one. but remained relatively stable over 60 h in the other. Excluding the genotype that exhibited little decline in fer- tilization, the average T 50Vf for the reduction of fertilization was 62 15 h, a value similar to the population-wide regression. Effect of liming of insemination on embr\o development and metamorphosis The maximum duration of gestation is fixed by the length of the asexual zooid replacement cycle. Since eggs could be fertilized well after siphon opening, but the time of embryo release is fixed, we examined the effect of delayed insem- ination on reproductive success. Successful embryo meta- morphosis was selected as an assay of reproductive success because it integrates possible effects on fertilization, devel- opment, larval behavior, and settlement. In five trials that included unfertilized (low control), insemination at 22 h (high control), and ramets inseminated at different times after siphon opening, the percentage of eggs that success- fully developed through metamorphosis consistently de- creased with the time of insemination (Fig. 3A). The unfer- tilized controls resulted in either zero or very low (<5%) levels of larval metamorphosis (Fig. 3 A). However, the percent of eggs developing through metamorphosis varied substantially among 22-h insemination controls (Fig. 3A). Because of the low levels of successful metamorphosis in two genotypes fertilized at 22 h. we calculated the T 50% relative to the maximum value for each genotype. The relative T 50C7c for the reduction of metamorphosis success was 41 6 h after the start of siphon opening (about 19 h after the completion of siphon opening). When data from all 12 trials were combined (Fig. 3B), larval metamorphosis exhibited an exponential decline with fertilization time be- yond 22 h. No larval metamorphosis occurred when colo- nies were fertilized more than 78 h after the start of siphon opening. Two outliers (both ramets of the same genotype) had disproportionately high levels of metamorphosis when fer- tilized about 48 h after siphon opening (Fig. 3B. open squares). Independent evidence (i.e.. observations of suc- cessful embryo development in isolated colonies) suggested that this genotype may sometimes be able to self-fertilize. Alternatively, the high fertilization levels in these two col- onies may be the result of sperm contamination. Because these inconsistent values are limited to one genotype, we have excluded these values from the regression in Figure 3B. Inclusion of the two points in the regression has little effect on the equation parameters, but it substantially re- duces the coefficient of determination. Note that many other ramets of this genotype were employed in this experiment (Fig. 3B, open squares) and produced results consistent with those of the other genotypes. Discussion Although more than 50% of Boti-yllus schlosseri eggs can be fertilized 38 to 48 h after the completion of siphon opening (Fig. 2), few viable larvae are produced unless fertilization occurs within the first 19 h (Fig. 3). The de- crease in embryo production after delayed fertilization could be caused by either egg aging or limitations on the duration of brooding. As in most invertebrates, the time required to complete development is a function of temper- ature in B. schlosseri. Since the asexual zooid replacement 56 J. STEWART-SAVAGE ET AL. _c =0 .5 o " p. I o g E Q - 100 r 75 - 50 25 ~^^T! \ . " Tj HI 3 24 48 72 Unfert o - a? . o -- .2 e- o o | Q 2 100 r 75 - 50 25 - B 24 48 72 96 Insemination Time (h after start siphon opening) Figure }. Effect of insemination pulse timing on embryo development and larval metamorphosis. Colonies with quantified egg production were isolated before the start of siphon opening, monitored for the timing of siphon opening, and exposed to sperm for 1 h: the number of settled juveniles was determined 5-7 days later. (A) Developmental success of different ramets from five genets. In three of the genets, one ramet was never exposed to sperm (unfertilized, solid symbols). (B) Overall effect of insemination time on successful development. The open squares are the ramets from the putative self-fertilizing genotype; closed symbols repre- sent the other 1 1 genotypes. The line is an exponential regression of the data except for two outliers at 48 h (R 2 = 0.713). cycle is also a function of temperature (Grosberg, 1982). delayed fertilization could cause the brooding zooids to degenerate before the embryos have become competent to undergo metamorphosis. The deleterious effects of egg ag- ing have been demonstrated in mammals (Juetten and Bavister. 1983; Xu et <(/., 1997), but such effects are usually manifested early in development. Since early development was normal in all but one colony with delayed fertilization (pers. obs.), the decreased gestational duration caused by delayed fertilization is more likely to be responsible. Nev- ertheless, additional work on the mechanism by which de- layed fertilization decreases larval production could more fully resolve this issue. In spite of the narrow temporal window in which both fertilization and development are likely to be successful (Figs. 2 and 3), field experiments indicate that colonies of B. schlosseri are very adept at acquiring sperm. A single male- phase colony can fertilize most eggs of a nearby female- phase colony with very few sperm (Yund, 1998). If several males are present, they compete to fertilize eggs (Yund. 1995. 1998), and closer males can be successful at the expense of more distant males (Yund and McCartney, 1994). Although sperm transfer usually occurs among nearby colonies (Yund. 1995). sperm can also be obtained from very distant locations when insufficient local sperm are available (Yund, 1998). Even eggs of colonies isolated from the nearest natural populations by tens of meters can be fertilized at appreciable levels (Yund and McCartney, 1994). The apparent ease of fertilization under field condi- tions, in spite of a very limited temporal window for suc- cessful fertilization and development, suggests that the pro- cess of sperm capture by colonies must be extremely efficient. Nevertheless, in low-density populations where sperm may be in short supply (Yund. 1998). or in marginal habitats in which sperm production is suppressed (Stewart- Savage et a/.. 2001). our work suggests that reproductive failure may occur in spite of successful fertilization if fer- tilization occurs too late in the reproductive cycle. Recent field sampling has demonstrated this phenomenon in natural populations near the end of the annual reproductive season (Yund and Phillippi, unpubl. data). Unlike the colonial ascidian Diplosoma listerianum, in which fertilization can be temporally disassociated from sperm exposure and colonies can store sperm for up to one month (Bishop and Ryland. 1991 ; Bishop and Sommerfeldt, 1996). B. schlosseri colonies apparently cannot store sperm. The evidence for this conclusion is, first, that colonies isolated in sperm-free seawater were not fertilized until we experimentally supplied a sperm pulse, indicating that sperm are not stored and transferred from one asexual generation of zooids to the next. The apparently complete resorption of all zooid tissue at the end of the cycle further suggests that transmission between cycles is unlikely. Sec- ond, the tight temporal relationship between siphon opening and fertilization (Fig. 2B) suggests that sperm cannot enter until the new generation of zooids opens its siphons and starts to feed. Third, the narrow window of time in which fertilization is both possible (Fig. 2) and results in viable offspring (Fig. 3) eliminates any apparent fitness advantage to sperm storage within a single asexual generation. The route of sperm access to eggs in B. schlosseri is unknown, but there are at least two possible points of entry (Ryland and Bishop. 1993): sperm enter through the EFFECTS OF DELAYED INSEMINATION 57 inhalant siphon and cross the pharyngeal basket to reach the eggs, or sperm enter through the exhalant siphon and then swim to the eggs. During takeover in B. schlosseri, the exhalant siphon of each system is formed before the inhal- ant siphons of all of the component zooids open, and the precise timing of exhalant siphon formation varies among systems (pers. obs.). If sperm enter via the exhalant siphon, fertilization levels in the early time intervals of our fertili- zation timing experiment should have varied among sys- tems, but should not have varied within a system. However, we routinely found mixtures of fertilized and unfertilized eggs within the same system, suggesting that sperm entry to each zooid required an open inhalant as well as exhalant siphon. Although further work is required to determine the route of sperm entry into Botryllus colonies, we think it is unlikely that sperm enter via the exhalant siphon. Hermaphroditism creates another challenge for success- ful reproduction in B. schlosseri. Inbreeding depression (Sabbadin, 1971) is likely to exert selective pressure to prevent self-fertilization, even though selting would be a possible mechanism to assure fertilization in the narrow time window in which fertilization can produce functional embryos. When the data in this paper are combined with previous data on the timing of sperm release (Stewart- Savage and Yund. 1997). it is apparent that the male and female phases of the reproductive cycle overlap in B. schlosseri (Fig. 4). Sperm release overlaps for about 48 h with the window for successful fertilization, but there is substantially less overlap with the narrower window in which fertilization results in viable embryos (Fig. 4). Con- sequently, B. schlosseri is not a true sequential hermaphro- dite (Milkman, 1967), but the male and female phases are functionally separated in time. This functional segregation of the reproductive phases probably plays some role in 24 48 7: 96 120 144 168 192 216 240 Time From Completion of Siphon Opening (h) Figure 4. Relationship between male and female reproductive phases in Botryllus schlosseri. Data collected at different temperatures have been normalized to a 10-day cycle length. The zero time point is the completion, rather than the initiation (as in Figs. 2 and 3), of siphon opening. The sperm release curve is redrawn from Stewart-Savage and Yund (1997) with permission. ensuring that few metamorphosing embryos result from self-fertilization. However, the very success of our experi- mental protocols indicates that one or more additional mechanisms to prevent self-fertilization must exist. Eggs of colonies isolated in small volumes of water until points in the reproductive cycle at which substantial self-sperm should have been present (Fig. 4) nevertheless remained unfertilized until we introduced a pulse of sperm (with the possible exception of the two outliers in Fig. 3B). Conse- quently, some form of self-incompatibility, as described in other colonial and solitary ascidians (Rosati and De Sands. 1978: Bishop, 1996), appears likely in B. schlosseri (see also Scofield et /., 1982). Acknowledgments Financial support was provided by the National Science Foundation (OCE-97-30354). This is contribution number 366 from the Darling Marine Center. Literature Cited Berrill, N. J. 1941. The development of the bud in Bolryllus. Biol. Bull. 80: 169-1X4. Bishop, J. D. D. 1996. 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(August 20(11) Morula Cells as the Major Immunomodulatory Hemocytes in Ascidians: Evidences From the Colonial Species Botryllus schlosseri LORIANO BALLARIN 1 -*, ANTONELLA FRANCHINI 2 , ENZO OTTAVIANI 2 , AND ARMANDO SABBADIN 1 1 Department of Biologv, University of Padova, via U. Bassi 58/B, 35100 Padova. Italy: and ^Department of Biologv, University of Modena and Reggio Emilia, via Campi 213/D, 41100 Modena, Italy Abstract. Immunocytochemical methods were used to study the presence and distribution of IL-1 -a- and TNF-a- like molecules in the hemocytes of the colonial ascidian Botryllus schlosseri. Only a few unstimulated hemocytes were positive to both the antibodies used. When the hemo- cytes were stimulated with either mannan or phorbol 12- mono-myristate. the phagocytes were not significantly changed in their number, staining intensity, or cell morphol- ogy. In contrast, stimulated morula cells were intensely labeled, indicating that these cells play an important immu- nomodulatory role. Introduction Phagocytes and morula cells are two types of circulating hemocytes that play a key role in ascidian immunobiology. Phagocytes can easily recognize and ingest non-self cells and particles (Smith, 1970; Anderson. 1971; Fuke and Fu- kumoto. 1993: Ballarin et ai. 1994: Ohtake et ai, 1994; Dan-Sohkawa et ai, 1995; Cima et ai, 1996) and are able to synthesize and release opsonic agglutinins (Coombe et ai, 1984; Kelly et ai, 1992; Ballarin et ai. 1999). Morula cells, a ubiquitous hemocyte type among ascidians. take part in a variety of biological functions of irnmunological rele- vance, such as hemolymph clotting, tunic synthesis, and Received 18 July 2000; accepted 10 May 2001. * To whom correspondence should be addressed. E-mail: ballarin@civ.bio.unipd.it Abbreviations: FSW. filtered seawater; HA. hyaline amoebocytes: IL. imerleukin; MLC. macrophage-like cells: PMM. phorbol 12-mono-myris- tate; TNF. tumor necrosis factor. encapsulation of foreign bodies (Endean, 1955b; Smith. 1970; Anderson, 1971; Chaga, 1980; Wright. 1981: Za- niolo. 1981). They are by far the most frequent circulating ascidian cell-type (Endean. 1955a: Andrew. 1961; Smith. 1970; Kustin et ai, 1976; Ballarin et ai, 1995). and their abundance suggests direct involvement in other important defense reactions. Although most of their roles in ascidian immune responses still remain unclear, morula cells can induce cytotoxicity after recognition of foreign molecules or cells (Parrinello. 1996; Cammarata et ai. 1997: Ballarin et til.. 1998), and they are also required for phagocytosis (Smith and Peddie. 1992). Cytokines are soluble molecules that mediate communi- cation among various immunocyte types in vertebrate im- mune systems. In the last decade, much evidence has accu- mulated indicating that cytokine-like molecules are also involved in invertebrate immune responses, and their pres- ence has been demonstrated in hemocytes of molluscs, annelids, arthropods, echinoderms, and tunicates (Beck and Habicht. 1991; Ottaviani et ai. 1995a.b. 1996: Franchini et iii. 1996). Cytokine-like molecules stimulate cell prolifer- ation, increase hemocyte motility and phagocytic activity, and induce nitric oxide synthase (Raftos et ai, 1991: Otta- viani ft ai, 1995b). As regards ascidians, the activities of interleukin-l (IL-1 )- and IL-2- but not tumor necrosis factor (TNF)-like molecules have been revealed in various spe- cies, either solitary or colonial (Beck et ai. 1989). Tunicate IL-1 -like molecules modulate immune responses and are secreted by hemocytes in response to exogenous stimuli (Raftos et ai. 1991. 1992. 1998: Beck et ai, 1993; Kelly et ai, 1993). 59 60 L. BALLARIN ET AL. We have studied in hemocytes of the colonial ascid- ian Botryllus schlosseri the presence and distribution of molecules that are immunoreactive to antibodies raised to human IL-l-a and TNF-a. The results indicate that these immunoreactive molecules are mainly detectable in stim- ulated morula cells, suggesting that these cells have a role in immunomodulation. Moreover, previous results in other ascidian species are supported (Smith and Peddie, 1992). Materials and Methods Animals Wild colonies of Botryllus schlosseri from the lagoon of Venice, Italy, were used. They were kept in aerated aquaria, attached to glass slides, and fed with Liquifry Marine (Liquifry Co., England) and algae. Hemocyte monolayers Colonies were rinsed in filtered seawater (FSW), pH 7.5, containing 10 mM L-cysteine as anticoagulant. The tunic marginal vessels were then punctured with a fine tungsten needle, and hemolymph was collected with a glass micropi- pette. Hemolymph was centrifuged at 780 X g for 10 min, and pellets were resuspended in FSW to a final hemocyte concentration of 8-10 X 10 6 cells/ml. Samples of the he- mocyte suspension (50-100 /xl) were cytocentrifuged onto slides with a Shandon Instrument Cytospin II running at 500 rpm for 2 min. Hemocytes were then stained with May Griinwald-Giemsa for morphological examination with a Leitz Dialux 22 light microscope. Hemocyte stimulation Cell suspensions were placed in 1-ml tubes on a revolv- ing mixer, and hemocytes were stimulated by incubation for 5, 15, 30, and 60 min with mannan at 5 mg/ml or phorbol 12-mono-myristate (PMM) at 20 nM in FSW containing 10 mM L-cysteine to prevent cell clotting. Mannan. a quite common microbial polysaccharide, is easily recognized by mannose receptors, the presence of which has been indi- rectly interred on the surface of Botryllus phagocytes (Bal- larin et al., 1994). PMM is a well-known activator of protein kinase C that mimics the action of diacylglycerol (Wolfe, 1993). The above-reported concentrations of the two com- pounds were previously demonstrated as the most effective in stimulating Botryllus phagocytes and the related respira- tory burst (Ballarin et al.. 1994; Cima et al.. 1996). FSW was used for controls. The viability of hemocytes, after the incubation, was assessed by the trypan blue exclusion assay (Gorman et al., 1996). Immunocytochemistry The immunocytochemical procedure described by Otta- viani et al. (1990) was performed. The following two pri- mary antibodies were used: polyclonal anti-human IL-l-a (1:250, 1:500, 1:1000) (Santa Cruz Biotech., USA) and monoclonal anti-human TNF-a (1:25, 1:50, 1:100) (Neo- Markers, USA). Cells were incubated with primary antibod- ies overnight at 4C, and reactivity was revealed by immu- noperoxidase staining using avidin-biotin-peroxidase complex (Hsu et al.. 1981). The best results were obtained with anti-IL-1-a and anti-TNF-a diluted 1:500 and 1:25, respectively. In control preparations, the primary antibodies were either substituted with non-immune sera or absorbed with homologous antigen (i.e., human IL-l-a and TNF-a) before addition to hemocyte monolayers. Moreover, a poly- clonal antibody raised against Botryllus agglutinin (BA) (Ballarin et nl., 2000) was also assayed as a control for specificity. Nuclei were counterstained with hematoxylin. The frequency of positive hemocytes, phagocytes, and morula cells was reported as the percentage of the total hemocyte number, which was determined by counting at least 600 cells in 10 fields under the light microscope. Statistical analysis All experiments were repeated in triplicate, and statistical analysis was performed using the chi-square test (^ 2 ). Results Morphology of cytocentrifuged Botryllus hemocytes The main hemocyte types present in B. schlosseri hemo- lymph were identifiable under the light microscope after cytocentrifugation. Lymphocyte-like cells, representing 2%-4% of circulating hemocytes. contain a large round nucleus surrounded by a thin layer of basophilic cytoplasm. Phagocytes, which include hyaline amoebocytes (HA; ac- tively phagocytosing cells) and macrophage-like cells (MLC) (Ballarin et al.. 1994). have roundish nuclei and neutrophilic cytoplasm which, in the case of MLC, sur- rounds one or more vacuoles containing ingested material (Fig. la, b). Phagocytes constitute 30%-40% of circulating blood cells. Morula cells, the frequency of which is 30%- 50% of total hemocytes, are characterized by the presence of several yellowish-green vacuoles (Fig. 2a, c). Nephro- CYTOKINE-LIKE MOLECULES IN BOTRYLLUS 61 LL . HA MLC * N a Figure 1. Cytocentrifuged Botry/liis schlosseri hemocytes stained with May Griinwald-Giemsa solution, (a) Lymphocyte-like cell (LL) and hyaline amebocyte (HA); (b) macrophage-like cell (MLC; n: nucleus; v: vacuole); (cl nephrocyte (N) with several empty vacuoles (arrowheads). Bar = 10 /xm. cytes and pigment cells (6%-10% of circulating hemocytes) were not well preserved after cytocentrifugation; they ap- peared as giant cells with empty vacuoles (Fig. Ic). Response of unstimulated hemocytes to anti-cytokine antibodies Using anti-IL-1-a and anti-TNF-a, only some phago- cytes and a few morula cells were labeled after immuno- peroxidase staining (Table 1). Thus, most HA, MLC, and morula cells were not immunoreactive with either antibody (Fig. 3). Moreover, no other cell-types stained positively for stimulated cvtes to antibodies raised to human cytokines Antibodies' 1 Cell type Anti-iL-1-a Anti-TNF-a Phagocytes'" Morula cells 0.4 0.3 1.1 0.9 0.9 0.4 4.5 1.2 a Values are percentage of total hemocytes plus or minus the standard deviation. h Phagocytes include hyaline amoebocytes and macrophai -li; 62 L. BALLARIN ET AL. anti-BA l&r d e anti-cytokine Figure 3. Immunocytochemistry on Botryllus schlosseri hemocytcs with anti-BA (a, h), and anti-cytokine (c-e) antibodies, (a) Positive HA; (hi negative morula cells; (c) unlabeled. unstimulated HA; (d) stimulated HA positive for IL-l-a; (e) stimulated MLC positive for TNF-a. Bar = 1? /xm. Discussion In the present work, we demonstrate that molecules rec- ognized by antibodies raised to human IL-l-o and TNF-o are present in immunocytes of the compound ascidian Bot- tyllus schlosseri. After stimulation, only morula cells, among all hemocytes, show a marked and significant in- crease in immunoreactivity. The increase in the number of immunoreactive cells depends on the length of the time of hemocyte incubation with the stimulating agents. In con- trast, among unstimulated hemocytes, only some morula cells and a few phagocytes are immunoreactive. Therefore, although the ligands recognized by the antibodies used are unknown and notwithstanding that serological cross-reac- tivity is not sufficient proof of evolutionary homology be- tween those ligands and vertebrate cytokines. still our data indicate that the morula cells have an important immuno- modulatory role in ascidian blood. We hypothesize that morula cells are the main source of cytokine-like molecules in Botryllus hemolymph, which can better explain their abundance in the circulation. Indeed, these cells are able to encapsulate foreign bodies (Anderson, 1971; Wright, 1981; De Leo el al, 1996) and are involved in clotting after blood vessel damage (Vallee, reported by Wright, 19X1). In many ascidian species, they can also induce cytotoxicity after recognition of foreign molecules or cells (Parrinello, 1996; Cammarata el al.. 1997: Ballarin ct uL. 1998). All these events can be modulated by cytokine- like molecules produced by activated cells. In agreement with this view, TNF-a-like molecules are involved in insect encapsulation (Franchini et ui, 1996), and IL-1-like mole- cules have been shown to stimulate echinoderm coelomo- cyte aggregation, which occurs in encapsulation (Beck and Habicht, 1991 ). Moreover, in vertebrates, both TNF-a and IL-l-n stimulate immune and inflammatory responses, and TNF-a is required for blood coagulation (Abbas et al.. 1991). The induction of cytokine-like molecules in hemocytes after stimulation has already been reported in bivalve mol- luscs and insects: in all these cases, phagocytes are the immunoreactive cells (Hughes et al.. 1990; Franchini et al., 1996). Analogously, in vertebrates, mononuclear phago- cytes are the main source of both IL-l-a and TNF-a (Abbas ct al.. 1991 ). Nevertheless, the situation in Botryllus appears peculiar in that positivity to anti-cytokine antibodies is absent from the majority of phagocytes without significant differences in its distribution between unstimulated and stimulated cells. Although morula cells have no phagocytic activity, they are reported to promote phagocytosis by ascidian phago- cytes (Smith and Peddie, 1992). Thus, the stimulatory effect on phagocytes and the enhancement of phagocytosis by morula cell lysates (Smith and Peddie, 1992) may easily be explained by the immunomodulatory role of the cytokines they produce. This idea is strongly supported by the obser- CYTOK1NE-L1K.E MOLECULES IN BOTRYLLUS 63 35- 25- f 15 60 15 30 time (min) 60 Figure 4. Morula cells positive to anti-IL-1-a and anti-TNF-a, ex- pressed as percentage of total hemocytes. after stimulation with either mannan at 5 mg/ml (circles) or PMM at 20 nM (triangles) for 5, 15, 30, and 60 min. *P < 0.001 vs. control (unstimulated hemocytes, t = 0). vation that the time-dependent increase of immunoreactive morula cells closely resembles the time-dependent increase in the frequency of phagocytizing hemocytes in in vitro assays (Ballarin et al., 1997). The opsonic role of tunicate IL-1-like molecules reported by Kelly et at. (1993) is in agreement with this view. Acknowledgments The authors wish to thank Mr. M. Del Favero, Mr. R. Mazzaro, and Mr. C. Friso for their technical assistance. This work was supported by a grant from the University of Padova to one of us (L.B.). Literature Cited Abbas, A. K., A. H. Lichtman, and J. S. 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Histology of the ascidian Botryllus schlosseri tunic: in particular, the test cells. Boll. Zoo/. 48: 169-178. Reference: Bio/. Bull. 201: 65-75. (August 2001) Molecular Evidence that Sclerolinum brattstromi Is Closely Related to Vestimentiferans, not to Frenulate Pogonophorans (Siboglinidae, Annelida) KENNETH M. HALANYCH 1 *. ROBERT A. FELDMAN 2 , AND ROBERT C. VRIJENHOEK 1 1 Biology Department MS 33, Woods Hole Oceanographic Institution. Woods Hole, Massachusetts 02543; : Molecular Dynamics, Inc.. part of Amersham Pharmacia Biotech, 928 East Arques Ave., Sunnyvale, California 94086-4250; and 3 Monterey Bay Aquarium Research Institute. 7700 Sandholdt Road. Moss Landing, California 95039 Abstract. Siboglinids. previously referred to as pogono- phorans, have typically been divided into two groups, frenu- lates and vestimentiferans. Adults of these marine proto- stome worms lack a functional gut and harbor endosymbiotic bacteria. Frenulates usually live in deep, sedimented reducing environments, and vestimentiferans inhabit hydrothermal vents and sulfide-rich hydrocarbon seeps. Taxonomic literature has often treated frenulates and vestimentiferans as sister taxa. Sclerolinum has traditionally been thought to be a basal siboglinid that was originally regarded as a frenulate and later as a third lineage of siboglinids. Monilifera. Evidence from the 18S nuclear rDNA gene and the 16S mitochondria! rDNA gene pre- sented here shows that Sclerolinum is the sister clade to vestimentiferans although it lacks the characteristic mor- phology (i.e.. a vestimentum). The rDNA data confirm the contention that Sclerolinum is different from frenulates, and further supports the idea that siboglinid evolution has been driven by a trend toward increased habitat specialization. The evidence now available indicates that vestimentiferans lack the molecular diversity expected of a group that has been argued to have Silurian or possibly Cambrian origins. Introduction Siboglinids were formerly called pogonophorans and in- clude two groups of marine protostomes, frenulates and vestimentiferans, that are commonly referred to as beard- Received 22 November 2000: accepted 1 1 April 2001. * To whom correspondence should be addressed. E-mail: khalanvch@ whoi.edu worms and tubeworms, respectively. Both groups lack a functional gut as adults and rely on endosymbiotic bacteria for nutrition. They have a closed circulatory system and possess a metamerized tail region called the opisthosoma. Vestimentiferans are distinguished from frenulates by the presence of a vestimentum, a winged region near the ante- rior of the organism. Both taxa occur in reducing environ- ments and typically are found at depths below several hundred meters. Due to the limited availability of samples and the difficulty of retrieving live specimens, several as- pects of their biology (e.g.. reproduction, physiology) are still poorly understood. Vestimentiferans, in general, have been better studied than frenulates because they are key- stone species in eastern Pacific hydrothermal vent habitats and in Pacific and Caribbean seeps. The taxonomic literature concerning frenulate and vesti- mentiferan siboglinids has a colorful and confusing history. One taxonomic scheme recognizes frenulates (aka pogono- phorans sensu stricto) and vestimentiferans as distinct phyla (Jones, 1985). Alternatively, vestimentiferans have also been recognized as a class within the phylum Pogonophora (Jones, 1981; Ivanov, 1994). Others place frenulates and vestimentiferans within the phylum Annelida (Land and N0rrevang, 1977; Kojima et al.. 1993; Bartolomaeus. 1995; McHugh, 1997; Rouse and Fauchald. 1997; also see South- ward, 1988). The latter hypothesis has been supported by recent morphological (Rouse and Fauchald. 1995. 1997). embryological (Young et al., 1996; Southward, 1999), and molecular analyses (Kojima et al., 1993: McHugh, 1997; Blacker*-//., 1997; Kojima, 1998; Halanych et al.. 1998). To further complicate matters, a ranked classification scheme has produced different names for the same clade of organ- 65 66 K. M. HALANYCH. R. A. FELDMAN. AND R. C. VRUENHOEK isms. Vestimentiferans have been called Vestimentifera (Jones, 1981). Obturata (Jones, 1981; Southward, 1988; Southward and Galkin, 1997). and Afrenulata (Webb. 1969). Frenulates have been called Pogonophora (Jones. 1985), Frenulata (Webb, 1969), Perviata (Southward, 1988), and originally Siboglinidae (Caullery, 1914). Hereafter we apply the following nomenclature: (1) Ves- timentifera are equated with Obturata and Afrenulata; (2) Frenulata are equated with Perviata and Pogonophora (sensu Jones, 1985); (3) Monilifera is a third monogeneric clade that includes Sclerolinum; and (4) Siboglinidae refers to the clade that includes Vestimentifera, Frenulata, and Monilifera. We recognize that the term "Pogonophora" is more commonly used and that rules of priority for nomen- clature do not apply to higher taxa. However, we have opted to use the term "Siboglinidae" throughout this manuscript to emphasize that this group of organisms represents derived annelids (McHugh, 1997; Rouse and Fauchald. 1997). We restrict the term "pogonophoran" to common usage. Even among siboglinids, there has been one group. Sclerolinum, that has been particularly problematic in terms of phylogenetic position. Unlike most frenulates that live in the mud, Sclerolinum species can live on decaying organic material like wood or rope made from natural fibers (Webb, 1964a; Southward, 1972). This taxon was originally con- sidered a member of the frenulate family Polybrachiidue (Southward. 1961 ). but Webb ( 1964b), mainly citing differ- ences in the postannular region, argued that Sclerolinum could not be ascribed to either of the two orders (Theca- nephria and Athecanephria) of siboglinids recognized at the time (vestimentiferans had not been discovered yet). He erected a new family, Sclerolinidae, that he states should "have order rank." Ivanov ( 1991 ) more formally recognized the unique nature of Sclerolinum, and in 1 994 he proposed that Frenulata (= Perviata), Monilifera (= Sclerolinidae), and the Vestimentifera be regarded as three taxa with equal rank (i.e.. classes within the phylum Pogonophora). Addi- tionally, Ivanov ( 1994) further suggested that Monilifera are allied to the Vestimentifera on the basis of the common absence of several characters (e.g.. spermatophores, teloso- mal diaphragm, metasoma preannular and postannular re- gions) relative to the Frenulata. Southward (1999) sug- gested that Monilifera might be similar to the ancestral siboglinid form, thus predicting that it should occupy a basal position in siboglinid phylogeny. Distinguishing between these hypotheses on the placement of Sclerolinum will allow us to test the notion of Black el al. (1997) that habitat preference or specificity may be an important factor in siboglinid evolution. If Black et al. are correct, Sclerolinum is expected to occupy a position between frenulates and vestimentiferans (which may be consistent with Ivanov's ideas), and not a position basal to the frenulate-vestimen- tiferan clade. To date, molecular studies that include siboglinids have either focused on vestimentiferans (Williams et al.. 1993; Black et al.. 1997; Kojima et al., 1997; Halanych et al., 1998) or have addressed siboglinid origins (Winnepen- ninckx et al., 1995a; Kojima et al., 1993; Kojima, 1998; McHugh, 1997). Most studies have included only one frenu- late representative. Although Black et al. (1997) included two "frenulate" siboglinids, one of these, the Loihi worm, was undescribed. Additionally, several 18S sequences were reported in a symposium contribution (Halanych et al., 1998) for which page limitations did not permit detailed analyses or explanation. Herein we extend these previous analyses by increasing the sampling of frenulates. including Sclerolinum, and using novel 18S rDNA and 16S rDNA data. The present findings support the notion that habitat requirements have been important in siboglinid evolution. Additionally, frenulates are sister to a Sclerolinum-vesti- mentiferan clade, the latter of which showed limited diver- sity suggestive of a recent radiation within the clade. Materials and Methods Taxa employed Table 1 lists the species analyzed and GenBank accession numbers for the rDNA sequences used in this study. The frenulate and vestimentiferan operational taxonomic units (OTUs) included in this study represent all of the currently recognized genera available to the authors. The addition of closely related species within a genus would have increased OTUs without increasing the phylogenetic signal for the issues under examination and were therefore excluded. For example, there are no nucleotide differences observed in the 18S rDNA of Escarpia spicata (Guaymas Basin) and E. laminata (Florida Escarpment). Limiting the number of OTUs also reduced computation time, allowing for more thorough analyses. Unless otherwise noted, collection local- ities correspond to those given in Black et al. (1997). Siboglinum ekmani, S. fiordicum. and Sclerolinum brattstromi were collected near Bergen, Norway, and iden- tified by Eve Southward. Marine Biological Association of the United Kingdom. Identification of the frenulates Spiro- brachia and Polybrachia were made by Eve Southward on the basis of animal and tube morphology. Both specimens were collected by TVGrab from the Aleutian Trench (5727.394'N, 14800.013'W) at a depth of 4890 m on the German research vessel Sonne. The non-siboglinid annelid OTUs for the 18S data were chosen to represent a diversity of lineages for which se- quences were available. The arthropod (Anemia) sequence was designated as the most distant outgroup for rooting purposes. Based on both morphology (e.g., Eernisse et al., 1992) and molecular studies (e.g.. Halanych et al.. 1995; Winnepenninckx et al., 1995a; Aguinaldo et al., 1997; Eernisse, 1997), arthropods are clearly outside of the proto- Timi used in rDNA anal\ses SIBOGLINID EVOLUTIONARY HISTORY TABLE 1 67 Organism GenBank Accession' 1 GenBank Accession 11 18S rDNA I6S rDNA Organism 18S rDNA 16S rDNA Pogonophora Frenulata Galalheiiliiuiin brachiosum AF168738 Polybrachia sp. AF 168739 Siboglinum fiordicinn GB X79876 h Siboglinum fiordicwn AF3 15060 Siboglinum ekmani AF3 15062 Spirobriit-liiti sp. AF 168740 Vestimentifera Escarpia spicata AF 168 741 Escarpiid n. sp. Lumellihriichia barhami AF168742 Oiisisia alvinae AF168743 Ridgeia piscesae AF 168 744 Ridgeia piscesae GB X79877 h Chaetopterida Chaetopterus variopedatus U67324 C AF3 15040 Hirudinea AF3 15037 Haemopis sanguisuga X91401 J Hinulu nit'dk-iniilix AF3 15058 AF3 15039 Oligochaete AF3 15038 Enchytraeus sp. Z83750 d AF315036 Phyllodocida Glycera americiina U19519 e AF3 15041 Polynoidea AF3 1 5053 Lepidonotopodium fimbriutum AF3 1 5056 AF3 15043 Branchipolynoe symmytilida AF3 15055 AF3 15044 Sabellida AF315045 Sabella piminniti U67144' AF3 15047 Tubificidae AF3 15052 Tubifex sp. AF3 15057 AF3 15048 Echiura AF3 15051 Ochetostoma erythrogrammon X79875 h AF3 15054 Urechis sp. AF3 15059 Sipuncula Riftia pachyptila AF 168745 AF3 15049 Phascolosoma gnnuilaiiini X79874 b AF3 15050 Nemertea Tevnia jerichonana AF168746 AF3 15042 Linens sp. X79878" Monilifera Mollusc Sclerolinum brattstromi AF3 15061 AF3 15046 Scutopux ventrolineatus X91977' Annelida Priapulida Alvinellidae Priiipuliix caudatus X80234 1 Puralvinella pabniformis AF 168747 Arthropod Anemia salina X01723 h a Unless otherwise noted, sequences were obtained in this study. b Sequence from Winnepenninckx et al. ( 1995a). c Sequence from Nadot and Grant (unpublished). J Sequence from Kim el al. ( 1996). Sequence from Halanych et al. ( 1995). ' Sequence from Winnepenninckx et al. ( 1996). g Sequence from Winnepenninckx et al. ( 1995b). h Sequence from Nelles et al. ( 1984). stome worm radiation. Because siboglinids are not closely related to molluscs and because of rate heterogeneity prob- lems within the Mollusca, only a single representative (the aplacophoran Scutopus) was used. Due to alignment limi- tations, outgroups employed in the 16S analyses a leech, an oligochaete, two polynoid polychaetes, and an echiu- rid were more limited (see Table 1). Because different investigators collected the data at different times, there was not a 1:1 correspondence in OTUs between data sets. We felt it more important to present all the relevant data rather than trim taxa from the data sets. The aligned data sets are available at the journal's Supplement's page (http:// www.mbl.edu/BiologicalBulletin/VIDEO/BB.video.html) and at TREEBASE (http://phylogeny.harvard.edu/treebase). Data collection Total genomic DNA was extracted using a modified hexadecyl-trimethyl-ammonium bromide (CTAB) protocol (Doyle and Dickson. 1987). The entire 18S nuclear rDNA gene was amplified via PCR (polymerase chain reaction), using the universal metazoan oligonucleotide primers 18e and 18P (Halanych et al.. 1998). A region of the 16S mitochondria! rDNA was amplified using 16Sar-5' and 16Sbr-3' primers (Palumbi, 1996). Each 50 /xl reaction consisted of about 50 ng of template DNA, 0.5 /u,A/ of each primer, 2.5 mM MgCl 2 , 200 pM dNTPs, 5 ju.1 of manufac- turer's 10X reaction buffer, and 1.5 U Tag polymerase (Promega Inc.. Wisconsin). Cycling profiles were as fol- 68 K. M. HALANYCH. R. A. FELDMAN, AND R. C. VRIJENHOEK lows: 18S initial denaturation at 95 C for 3 min, 35 cycles of amplification (denaturation at 95 C for 1 min, annealing at 50 C for 2 min, extension at 72 C for 2 min 30 s), and a final extension at 72 C for 5 min: 16S initial denaturation at 94 C for 2 min, 40 cycles of amplification (denaturation at 94 C for 30 s, annealing at 46 C for 30 s, extension at 72 C for 1 min), and a final extension at 72 C for 7 min. PCR products were purified using the QIAEX II gel extraction kit (Qiagen Inc., California). Approximately 60 ng of purified PCR product was used in sequencing reactions according to the manufacturer's instructions (FS Dye Termination Mix or Big Dye, Applied Biosystems Inc., California). The reaction profile was 25 repetitions of de- naturation at 94 C for 30 s, annealing at 50 C for 15 s, and extension at 64 C for 4 min. Dye-labeled fragments were separated by electrophoresis on a Perkin Elmer ABI 373A or 377 DNA sequencer. Both strands of the PCR product were sequenced. In addition to the PCR primers, the oligo- nucleotide primers used for sequencing are given in Halanych el al. (1998) or Hillis and Dixon (1991). The sequences were assembled and verified using the AutoAs- sembler and Sequence Navigator programs (Applied Bio- systems Inc., California). The terminal primer regions were not included in the sequences submitted to GenBank or in the phylogenetic analyses. Phylogenelic analyses Sequence alignment was produced with a Clustal W program (Thompson el al., 1994) and subsequently cor- rected by hand using the protostome secondary structure models available through the Ribosomal Database project (http://rdp.cme.msu.edu/html/). Regions that could not be unambiguously aligned (e.g., divergent loop domains) were excluded from analyses. Tree reconstructions were imple- mented with the PAUP* 4.0b4b2 program (Swofford, 2000), and MacClade 3.06 (Maddison and Maddison, 1992) was used for character and tree analyses. Neighbor-joining (NJ), parsimony, and maximum likelihood (ML) analyses were performed and yielded similar results. In the interest of brevity, results and discussion will focus on ML analyses. NJ trees were reconstructed under Jukes-Cantor, Kimura- 2-parameter, Tamura-Nei. general-time-reversible, and log/ det models. All except log/det were examined under equal rates of among-site rate variation using the empirically derived gamma shape parameter, a, of 0.3 (see Swofford el al.. 1996, for summary of different assumptions used in these models). A Kishino-Hasegawa ( 1989) likelihood eval- uation of the resulting topologies revealed no significant differences between models for either the 16S or the 18S data. Kishino-Hasegawa evaluations estimated a six-substi- tution-type rate matrix for which nucleotide base frequen- cies were set to empirical values and a was estimated. NJ bootstraps consisted of a log/det correction (model was arbitrarily chosen) with 1000 iterations. Parsimony analyses consisted of heuristic searches with 100 random sequence additions and tree-bisection-reconnection (TBR) branch swapping. Transitions (Ti) and transversions (Tv) were given equal weighting. ML evaluation of parsimony topol- ogies was the same as for NJ topologies. One thousand iterations were used for parsimony bootstrap analyses. When using likelihood to search for the "best" tree (as opposed to evaluating given trees), computation time was limiting. Therefore, we used a nucleotide model with two substitution types where the Ti/Tv ratio was set to the value estimated for the best parsimony tree (empirical base fre- quencies were used). ML searches were heuristic with 10 random sequence-addition replicates. ML bootstraps em- ployed the "Faststep" option with 100 iterations. Results The 18S rDNA data set consisted of 26 OTUs and 1935 nucleotide positions. Of the 1614 nucleotide positions that could be unambiguously aligned. 34.6% (559 positions) were variable and 18.7% (303 positions) were parsimony informative. Figure 1 shows the single best likelihood tree (Ln likelihood = -8260.55148) recovered. All search methods in all analyses found a monophyletic siboglinid clade (bootstrap support was >98% for all methods). Res- olution within the vestimentiferan clade, as well as between annelid groups, was poor, however. The moniliferan Sclerolinum brallslromi falls out with the vestimentiferan taxa in all analyses (bootstrap S 98%). The remaining trenulates form a distinct sister-clade to the Sclerolinwn- vestimentiferan clade with >99%> bootstrap support. Resolution among annelid taxa and within the vestimen- tiferans was poor due to the lack of phylogenetic signal. Because this paper does not focus on the annelid radiation, we did not try to enhance resolution among all annelid taxa. However, we did attempt to boost the signal within the vestimentiferan clade by employing a less inclusive taxo- nomic alignment. For metazoan 18S sequences, inclusion of broader taxonomic diversity can often create larger regions of ambiguous alignment that should not be included in analyses, due to poor assumptions about positional homol- ogy. Thus by reducing the taxonomic breadth examined, the phylogenetic signal can potentially be increased by a "bet- ter" alignment (Halanych, 1998). Unfortunately, even when just the siboglinids were aligned, little genetic diversity was observed, and the vestimentiferan taxa were still poorly resolved (not shown). The exception was Lamellibrachia harhami, which was consistently placed as the most basal vestimentiferan. Table 2 shows the logdet/paralinear dis- tances (below diagonal) and absolute distances (above di- agonal) for this less-inclusive, siboglinid-only alignment (in which most divergent domains could be unambiguously aligned). Even though the distance values for the siboglinid- SIBOGLINID EVOLUTIONARY HISTORY 69 e; 99 100 100 1 100 ipirooracnia I Polybrachia _l g1 Galathealmum ^ Siboglinumekmani 100 r Siboghnum fiordicum GB Siboglinum fiordicum 86 58 (D 96 59 Escarpia Ridgeia RidgeiaGB Oasisia Riftia Tevnia Lamellibrachia Sderolinum ^ Enchytraeus -Oligochaete ^^^^^^ ^^ Haemopis -Leech Moniliferan Sabella - Polychaete Paralvinella - Polychaete Phascolosoma -Sipunculid Ochetostoma - Echiund Chaetopterus - Polychaete Glycera Polychaete ^^ Lineus Nemertean Scutopus Mollusk Artemia - Arthropod Priapulus- Priapulid 0-01 substitutions/site Figure 1. Results of 18S rDNA phylogenetic analyses. The single best likelihood tree (Ln likelihood = 8260.551481 found. Analysis details are given in the text. Maximum likelihood bootstrap values of >50% are given in bold. Parsimony (italicl and neighbor joining (underlined) values are also given for the major nodes of interest (values for other nodes were omitted in the interest of space). Branch lengths are drawn proportional to the inferred amount of change along the branch (scale shown). only alignment are only slightly greater than the full align- ment values, the greatest distance within vestimentiferans was only 0.02 (with a maximum of 25 nucleotide differ- ences), revealing that there was very little 18S genetic diversity within this group. The 16S rDNA data set consisted of 24 OTUs, each with 497 nucleotide positions. Of the 465 nucleotide positions that could be unambiguously aligned, 60.4% (281 positions) were variable and 47.7% (222 positions) were parsimony informative. The reconstructed topology (Ln likelihood = -3967.21062). Figure 2, was qualitatively similar to the 18S topology. Siboglinids are divided into two major lin- eages: vestimentiferans plus the moniliferan Sderolinum brattstromi (bootstrap support 83% for ML and 100% for NJ and parsimony) and a frenulate sister-clade (bootstrap support >94% in all analyses). Again. 5 1 . brattstromi was basal to the vestimentiferans. In a departure from the 18S analyses, Riftia pachyptila, not Lamellibrachia barhami. often fell out as the most basal vestimentiferan. However, this was never supported by >54% bootstrap support; ML analyses that excluded the non-siboglinid outgroups re- vealed that the base of the Vestimentifera was poorly re- solved with 16S data. A comparison of genetic divergence values (Table 3) indicates that there was limited genetic 70 K. M. HALANYCH. R. A. FELDMAN, AND R. C. VRIJENHOEK TABLE 2 Paim'ise distances for the siboglinid-only IKS rDNA data set: absolute distances above diagonal and log/det distances below diagonal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 Spirobrachia _ 109 113 87 132 131 124 122 125 124 131 125 121 120 2 Polybrachia 0.07 9 104 138 137 139 140 140 140 147 142 140 136 3 Galathealinum 0.07 0.01 106 142 141 142 143 143 143 150 145 143 139 4 Siboglinum ekniani 0.05 0.06 0.06 116 117 113 1 III 113 110 121 112 107 112 5 Siboglinum fiordicum 0.08 0.09 0.09 0.07 5 14(1 136 136 140 143 138 134 139 6 Siboglinum fiordicum GB 0.08 0.08 0.09 0.07 0.00 143 139 139 143 146 141 137 140 7 Escarpia 0.08 0.09 0.09 0.07 0.09 0.09 7 14 10 19 6 13 31 8 Ridgeia 0.08 0.09 0.09 0.07 0.09 0.09 0.00 8 7 17 4 12 32 9 Ridgeia GB 0.08 0.09 0.09 0.07 0.09 0.09 0.01 0.00 14 21 11 19 38 10 Oasisia 0.08 0.09 0.09 0.07 0.09 0.09 0.01 0.00 0.01 20 7 14 32 1 1 Riftia 0.08 0.09 0.09 0.07 0.09 0.09 0.01 0.01 0.01 0.01 16 25 39 12 Tevnia 0.08 0.09 0.09 0.07 0.09 0.09 0.00 0.00 0.01 0.00 0.01 1 1 30 1 3 Lamellibrachia 0.07 0.09 0.09 0.07 0.08 0.09 0.01 0.01 0.01 0.01 0.01 0.01 28 14 Sclerolintiin 0.08 0.09 0.09 0.07 0.09 0.09 0.02 0.02 0.02 0.02 0.02 0.02 0.02 variation within vestimentiferans (<0. 1 1 log/del distance; a maximum of 47 nucleotide differences). As for the frenulate clade, neither 18S or 16S supported a monophyletic Siboglinum: but because only two Siboglinuin species were examined, additional taxa are needed to verify the status of this frenulate taxon. Additionally, we performed Kishino-Hasegawa ( 1989) likelihood evaluation for both genes to test the monophyly of the frenulate and vestimentiferan- Sclerolinum clades. To this end, we used the constraints option in PAUP* 4.0b4b2 to conduct parsimony heuristic searches (specifics same as above) to find the best trees that were consistent and inconsistent with the monophyly of these clades. Both the 16S and the 18S data significantly support the monophyly of both groups ( 1 8S frenulates average ML score supporting monophyly = -8244.69, non-monophyly score = -8278.135. P value < 0.01; 16S frenulates monophyly = -3894.889. non-monophyly = -3927.49, P value < 0.005; 18S vestimentiferan-Sc/ero/znMW! monophyly = 8244.69, non-monophyly = -8271.922, P value < 0.05; 16S vestimen- tiferan-Sclerolinum monophyly = 3894.889, non-mono- phyly = -391 1.802, P value < 0.05). Discussion The monophyly of siboglinids (aka, Pogonophora sensu hit 11) is supported by morphological (Southward, 1988, 1993; Rouse and Fauchald, 1995; Rouse. 2001). embryo- logical (Southward, 1999), and molecular (Winnepenninckx et ui. 1995a; Black el ai. 1997; McHugh. 1997; Halanych c/ ai. 1998. this study) evidence. Thus, in agreement with others (Southward, 1988, 1999; Ivanov, 1994; McHugh, 1997), we see no support for the recognition of vestimen- tiferuns and frenulates as having fundamentally different body plans (i.e.. "phyla" sensu Jones. 1985). The assertion made by Webb !l964b) and later by Ivanov (1991. 1994) that Sclerolinitm was notably different from frenulates is validated by the present data. Moreover, we found that Sclerolinitm brtittstroini is closely allied to the vestimenti- ferans. and does not occupy a position basal to a frenulate- vestimentiferan clade, confirming Ivanov's (1991; 1994; Ivanov and Selivanova, 1992) ideas that moniliferans oc- cupy a position intermediate between vestimentiferans and frenulates. Southward (1993) also suggested a possible evolutionary link between Sclerolinum and vestimentiferans. This con- tention is confirmed by the present analysis, as well as a recent morphological cladistic analysis (Rouse, 2001). Us- ing 44 morphological characters coded for all recognized siboglinid genera. Rouse found support for the monophyly of Frenulata. Vestimentifera, and the Sclerolinum-vestimen- tiferan clade. However, our use of nomenclature differs from Rouse with regard to the term Monilifera, which he applies to the Sr/e>w/i'w-vestimentiferan clade. Because this term was originally (Ivanov and Selivanova, 1992) applied to only Sclerolinitm, and because of the morpho- logical differences from vestimentiferans. Rouse's use of the term will inject confusion into the literature. Although we acknowledge that Monilifera, as defined here, is redun- dant with the generic name Sclerolinum, several aspects of siboglinid evolution and taxonomy are in need of additional study. Thus, we have chosen not to name this clade until more is understood about siboglinid evolution. The placement of Sclerolinum was especially interesting in the context of the evolution of habitat preference. Previ- ous studies of vestimentiferans (Black et ui. 1997). clams (Peek et til.. 1997), mussels (Craddock et ai. 1995). and shrimp (Shank et ai, 1999) reveal that vent-endemic organ- isms are related to. and possibly derived from, species associated with hydrocarbon seeps that occur near subduc- tion zones and continental margins. Furthermore, recent observations (Feldman et ai. 1998; Baco et ai. 1999; Distel SIBOGLINID EVOLUTIONARY HISTORY 71 94 e: i Spirobrachia ! 100 r Polybrachia cum * Galathealmum 700 100 I 79 1 Escarpia 1- Escarpiid n. sp. 1- - Tevnia Ridgeia 1 \ 75 ' - Ridgeia 2 53 Ridgeia 3 / 80 , ' 93 56 Oasisia i 80 r Lamellibrachia 1 1 *~ Lamellibrachia 2 5 r 67 Lamellibrachia 3 (0 no Lamellibrachia 4 83 1 99 I- Riftia 1 ; iXY 1 " Riftia2 1 'II Monilife*' 01 " 1 100 1 Lepidonotopodium - Polychaete 61 ifex - Oligochaete (D I (0 ^^ 0.05 substitutions/site Figure 2. Results of 16S rDNA phylogenetic analyses. The best likelihood tree (Ln likelihood = -3967.21062) found. Another tree with a Ln likelihood score of -3967.25739 was found in the same search. The trees differed in relationships within the Ridgeia clade. Analysis details are given in the text. Maximum likelihood (ML) bootstrap values of >50% are given in bold. Parsimony (italic) and neighbor joining (under- lines) values are also given for the major nodes of interest (values for other nodes were omitted in the interest of space). In the ML bootstrap analysis, Lamellibrachia and Sclerolinum formed a clade in 55% of the iterations. That is not shown above because it is incompatible with the "best" ML tree. Branch lengths are drawn proportional to the inferred amount of change along the branch (scale shown). c/ ill.. 2000) reveal that several symbiont-bearing clams, vestimentiferan tubeworms, and mussels can survive on rotting organic material, such as wood or a whale carcass. The moniliferan S. brattstromi and related species (e.g., S. javanicum, S. minor, and 5. major) are typically found growing on decaying organic material such as wood or rope (Webb, 1964a, b; Southward, 1972; Ivanov and Selivanova, 1992). Other members of the genus, (e.g., S. sibogae and S. magdalenae) lived buried in mud (Southward, 1972). These habitat preferences suggest that affinity for a mud or silt habitat was ancestral in siboglinids, allowing us to speculate that a pattern of evolution from low-oxygen, sedimented habitats to decaying organic material to hydrocarbon seeps to hydrothermal vents has occurred within the Sclerolinum- vestimentiferan clade. Although neither the 18S nor the 16S data clearly resolve relationships within the Vestimentifera, the cytochrome c oxidase subunit I (COD data of Black et al. (1997) show 72 K. M. HALANYCH, R. A. FELDMAN, AND R. C. VRIJENHOEK -t -t r- I- so CN m -^ . -f -t rr . ~ P*-, pr, P*-, -t pr, P*-, - 3 OC O OO ON - t- -t >r, -f -f i/ r*~i ON sO C ?S | r-i - pr, \G P - r-i ON m OsC-fsI 3 OC OC vC ^C - ON -^- OC C N 1 d ri -t r -t r-i c: C 3 I/-, _ ON in r* NO VO r*~i t d d '/-, - HI I = ,___ ~ sC oc \o r-j p Oi - CS | C 3 IO rl 1 r*-i ^f 3 d O ~l ON ^ ri r ; -t oc so si 3 Tj- r-j rr, p- -r p*-. r - p- O oc ri C NO | d C 3 -f rn 3 d d ON ~ r i doc 3 NO f) 3 d d >c ?! c - ri ri ri i/ ; ri ci O P d d d c 1 sC OC 3 d d oc N g -' ^ ^ S 3 d d d c 3 NO 1^ 3 d d -t -r -t r^ > 3 rj ON rj- C 3 ifi v~. l^ O N P- in ON - - 1- ON "-, NO r-l 1 c - r-l -f | O C 3 O O O C 3 O O - - OC 3 j co o in - - *c *c in -t - - : ^ Tj ^ ; gj S S S 3 : - -t doc SSSS 3 d d d c 3 r*-< oo -T o 3 d d 3 vO m I r*"i t ^ - = - r*-, ri p* , co ^ en en rr, ri rr, m ON C 3 rn Cfv T C 3 10 "^ 2 ON - r ON OC r*-j r i c -*< v-- r- s~> r\ n < 5 - C ; -t \c n -^ ; r* t- p- p- rj in O C -, r i i o d d d c 3 o' d d c 3 O O H .g -" in c P; 2C g C N CfN CfN f^l ^ - r~ -f g o O a 3 in ON / -. ., ^ | 2 S = 3 O O O C 3 d d 1 - - 883 S FN OC OC OO ON C 2 O' O O O C - r-- f, ' diagonal and OO Q. I 0. 50 - o Growing Restricted n=9 n=10 O O o 100 200 300 400 500 Total protein per colony (ng) Figure 3. Bivariate scatter plots of the number of polyps in a colony and its total protein content. Linear regression using combined data from both treatments yields the equation v = 0.507.x - 2.44 (^-squared = 0.98). This intercept is not significantly different from zero (T = 0.568. P > 0.58). Regression lines for growing and restricted colonies do not differ in slope (ANCOVA, F = 0.84. df = 1. 15, P > 0.37) or elevation (F = 0.97. df = 1. 16. P > 0.34). D) c E o co g 0) _i D. CXI O (a) P carnea o o o o o Growing Restricted n=12 n=13 (b) H. symbiolongicarpus o o Growing n=6 Restricted n=6 500 1000 1500 2000 2500 3000 Colony size (ug protein) Figure 4. Bivariate scatter plots of oxygen uptake rate of growing and restricted colonies, (a) Data for Podocoryna cornea. The slopes of the regression lines for the growing and restricted treatments do not differ (ANCOVA, F = 1.75. df = 1. 21, P > 0.20), but an elevation difference was found (F = 20.54, df = 1, 22, P < 0.0002). These relationships were strengthened by omission of a single outlying data point from the growing data set (slope: F = 0.10. df = 1, 20, P > 0.76; intercept: F = 41.06, df = I. 21. P < 0.0001). (b) Data for Hydractinia svmbiolongicarpus. The slopes of the regression lines for the two treatments were not significantly different (ANCOVA. F = 0.83. df = 1. 8, P > 0.39), and neither were the intercepts (F = 0.98, df = 1. 9. P > 0.35). Although no significant difference in oxygen consumption rate was found between treatments for H. symbiolongicar- pus (Fig. 4b), a trend may be discerned in the data that would indicate agreement with the result found for P. car- nea. The sample size is too small to render this trend statistically significant, however. Characterization of colonv morpholog\ Growing colonies of both species had a more runner-like morphology than their restricted counterparts (Fig. 5; H. symbiolongicarpus, F = 12.56, df = 1, 20, P < 0.002; P. carnea. F = 6.16, df = 1, 22, P < 0.0212). Growth rate after regression A growth assay was performed 4 to 6 months after the pronounced winter regression. At this time, no significant EFFECT OF CLONING RATE IN HYDROIDS 81 30 (Runner-like) o o (Sheet-like) n=12 T H symb/olongicarpus P camea Figure 5. Comparison of growing and restricted colonies after 18 months of experimental treatment in terms of colony morphology as given by the shape metric (colony perimeter)/\ (colony area). Means and stan- dard errors are represented. difference was detected between treatments in either species for growth as measured by total colony polyp counts (Fig. 6a; H. symbiolongicarpus, F = 0.06, df = 1, 16, P > 0.806; P. carnea, F = 0.44, df = 1, 18, P > 0.516. data for both analyses log-transformed) or by total colony protein (Fig. 6b; H. symbiolongicarpus, F = 0.17. df = 1. 16. P > 0.689; P. carnea, F = 1.04. df = 1, 18, P > 0.321; data for both analyses log-transformed). Discussion Two experimental treatments were used in this study of hydroid colonies. One group of replicates was allowed to completely overgrow and remain undisturbed on 12-mm coverslips ("restricted" colonies); a second group was re- peatedly cloned as vegetative growth continued, without being allowed to enter into a gamete-producing sexual phase ("growing" colonies). A clear difference in growth rate was found between treatments in both species studied. with restricted colonies exceeding growing colonies in growth rate during controlled assays. Since only one clone was used per species, this result is not replicated at the level of the species. Nevertheless, at a higher level (i.e., species within family), the two clones provide replication of this primary result. Assays of the oxygen uptake rate between treatments revealed that the growing colonies of Podocoryna carnea exceeded the restricted ones in oxygen consumption. Al- though no significant statistical difference was found for Hydractiniu symbiolongicarpus, the sample size was small, and a trend seems to be discernible in the data that would suggest agreement with the result for P. ciirneu. Such a result may seem counterintuitive: the colony that uses more oxygen might also be expected to grow faster. On the other hand, higher oxygen uptake may be correlated with lower growth rate if the former indicates greater metabolic expen- diture on, for instance, somatic maintenance. Such a hy- pothesis is not entirely implausible. These hydroid colonies are ecologically space-limited, typically inhabiting small hermit crab shells. It is likely that selection favors rapid sequestration of available space to prevent the settlement of competitors; colonies may maximally allocate energy re- sources to growth until the available space is covered. Under such conditions of intense metabolic demand, cellu- lar metabolism may generate high levels of reactive oxygen species (Allen, 1996; Chiueh, 2000). These reactive species can cause various defects in macromolecules. so continu- ously growing colonies might experience defects in the mechanisms of oxidative phosphorylation or allocate greater resources to production of anti-oxidant enzymes (e.g., Blackstone, 2001). Thus the data are consistent with the hypothesis that growing colonies expend more energy on functions other than somatic growth, although further study of this issue is needed. Our interpretation of these results is that the restricted colonies are metabolically more efficient and so can allocate more energy to growth (Lowell and Spiegelman, 2000). g- 40-, ! | 30- to & (a) T H symbiolongicarpus P camea 3 60- o 30 - Q. "ro 5 20 - H 10 (b) H symbiolongicarpus P camea Figure 6. Growth rate comparisons of growing and restricted Hydrac- tinia symbiolongicarpus and Pntlt>ntiti curnea colonies from the ass;iy performed after 32-35 months of experimental treatment. Means and standard errors are represented, (a) Number of polyps per colony (M Total colony protein content. 82 L. M. PONCZEK AND N. W. BLACKSTONE The widespread tissue regression that occurred appar- ently reset to zero the growth rate difference that had been entrained by the experimental treatments. By this view, the physiological basis of the difference prior to regression was transmitted to the clonal fragments of the growing colonies, becoming enhanced over time as shown by the decreasing colony growth rate. This may suggest an epigenetic basis for the phenomenon, wherein a particular state of gene activity underlies the increased rate of oxygen consumption coupled with the reduced growth rate. During the regression event. all colonies lost most of their living tissue, effecting a cell population bottleneck. The elimination of the growth rate difference could perhaps be due to sampling error in the cells that escaped death during the regression, or to some dedifferentiation process involving a return to a metabolic ground state. In any case, cells of similar condition and gene activity seem to have survived the regression. Periodic regressions of this kind have been observed in some clonal taxa and are possibly related to senescence (Bayer and Todd, 1997; Gardner and Mangel. 1997). The life span of the modules (polyps) that make up a colony may be ex- tended through cycles of degeneration and regeneration (Hughes. 1989). Comparing absolute growth rates of colonies undergo- ing both treatments early in the experiment (Fig. 1) with those measured some two years later (Fig. 3) reveals a consistent decline. Furthermore, the growth rate equal- ization after regression occurred not by the growing colonies recovering a rapid growth rate but by the faster growing restricted ones assuming a similarly diminished rate. This reduction in growth rate over time may be considered to be a manifestation of colony senescence (Bell, 1988). By this criterion, growing colonies senesced more rapidly than restricted ones prior to the tissue regression event, suggesting that a high cloning rate accelerates colony senescence relative to uncloned colo- nies. After regression, the degree of clonal senescence (measured by growth rate) became equalized. Hydractiniid hydroid colonies fragment to produce po- tentially viable clonal modules, thus enlarging and dis- persing the genet asexually (Cerrano et al.. 1998). The colony fragmentation rate (equivalent to the cloning rate considered in this study) presumably could vary with the physical environment in which the hydroids are found. In aquaria. Cerrano et al. ( 1998) found that clonal colonies arising from fragments of Podocoryna exigna colonies can grow on a sandy-bottom substratum and that hermit crabs with naked shells placed into this environment were colonized within a few days. If such a process occurs naturally in P. exigna and other hydractiniid hydroids. such as the species used in this study, a genet might extend itself naturally by fragmentation. Clonal lineages may vary in fragmentation rate and growth rate of colo- nial ramets. This study shows that cloning rate could possibly affect the growth rate of a ramet within a lineage through negative feedback, since variation in growth rate may be passed on through some epigenetic mechanism such as cytosine methylation (but see Tweedie and Bird, 2000; and Amedeo et al., 2000). Nevertheless, histocom- patibility data (Grosberg et al.. 1996; Mokady and Buss, 1996) suggest that in at least some populations of H. symbiolongicarpus the rate of fragmentation is low rela- tive to the rate of sexual recruitment. The alteration in morphology with variation in cloning rate might have a bearing on the ecological functioning of a hydroid colony (McFadden et al., 1984; Yund, 1991; Brazeau and Lasker, 1992). Intraspecific competition is common between Hydractinia colonies (Buss and Black- stone, 1991). The present study has shown that a high cloning rate can produce a more runner-like colony mor- phology, thus tending towards a form associated with a "guerrilla" ecological strategy (Jackson et al., 1985). Such a clone might have more limited direct competitive ability, but might also be dispersed to more locations due to its greater rate of fragmentation. Asexual reproduction is an essential part of the life his- tory of all clonal organisms and is thus an important factor in their evolution and ecology. In some taxa, fragmentation rate depends on morphological characters, which are at least in part genetic and thus subject to selection. The fragmen- tation rate of clones of branching coral reef demosponges was found to depend on branch thickness (Wulff, 1985). A coral of the genus Plexaura has evidently evolved morpho- logical characters that make fragmentation more common in this species than in its congeners and produce some popu- lations in which more than 90% of the individuals are clonemates (Lasker, 1990). A possible difference in growth rate dependent on cloning rate would have to be taken into account when considering the demographic impact of frag- mentation. The effects of the two experimental treatments on the clonal replicates of both hydroid species indicate that fre- quently fragmenting colonies exhibit reduced colony growth rates, hence diminished reproductive potential and compromised competitive ability in the space-limited hab- itats in which they are typically found. Moreover, a within- species difference in colony morphology was found be- tween unfragmented colonies and those maintained in a constant state of vegetative growth by repeated cloning (fragmenting); this difference could affect the ecological functioning of the colonies in nature. However, these dis- crepancies may disappear if a large-scale regression of colony tissue occurs. Regardless of the specific physiolog- ical mechanisms producing these differential effects, frag- mentation rate can be important to various aspects of the biology of clonal organisms. EFFECT OF CLONING RATE IN HYDROIDS 83 Acknowledgments Comments were provided by K. Gasser, B. Johnson- Wint, and P. Meserve. The National Science Foundation (IBN-94-07049 and IBN-00-90580) provided support. Literature Cited Allen, J. F. 19%. Separate sexes and the mitochoncirial theory of aging. ./. Thcor. Binl. 180: 135-140. Amedeo, P.. V. Habu. K. Afsar, O. Mittelstein Scheid. and J. Pasz- ko ski. 2000. 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Variation in clone structure of fragmenting coral reef sponges. Binl. J. Linn. Sac. 27: 311-330. Yund, P. O. 1991. Natural selection on hydroid colony morphology by intraspeciric competition. Evolution 45: 1564-1573. Zamer, W. E., J. M. Shick, and D. W. Tapley. 1989. Protein measure- ment and energetic considerations: comparisons of biochemical and stoichiometric methods using bovine serum albumin and protein iso- lated from sea anemones. Limnol. Oceanogr. 34: 256-263. Reference: Biol. Bui!. 201: 84-94. (August 2001) Egg Longevity and Time-Integrated Fertilization in a Temperate Sea Urchin (Strongylocentrotm droebachiensis) SUSANNE K. MEIDEL* AND PHILIP O. YUND School of Marine Sciences, Darling Marine Center, University of Maine, Walpole. Maine 04573 Abstract. Recent tield experiments have suggested that fertilization levels in sea urchins (and other broadcast spawners that release their gametes into the water column I may often be far below 100%. However, past experiments have not considered the potentially positive combined ef- fects of an extended period of egg longevity and the release of gametes in viscous fluids (which reduces dilution rates). In a laboratory experiment, we found that eggs of the sea urchin Strongylocentrotus droebachiensis had high viability for 2 to 3 d. Fertilization levels of eggs held in sperm- permeable egg baskets in the field and exposed to sperm slowly diffusing off a spawning male increased significantly with exposure from 15 min to 3 h. In a tield survey of time-integrated fertilizations (over 24, 48, and 72 h) during natural sperm release events, eggs held in baskets accrued fertilizations over as much as 48 h and attained fairly high fertilization levels. Our results suggest that an extended period of egg longevity and the release of gametes in viscous fluids may result in higher natural fertilization lev- els than currently expected from short-term field experi- ments. Introduction Recent work has started to explore the fertilization dy- namics of free-spawning marine organisms that release one or both gametes into the water column (e.g., algae: Pearson and Brawley, 1996; corals: Lasker et al., 1996; starfish: Babcock et al.. 1994; sea urchins: Levitan et al.. 1992; ascidians: Yund, 1998; fish: Petersen etui, 1992). Although the details of scientific approaches vary, studies can be Received 22 September 2000; accepted 26 April 2001. * To whom correspondence should be addressed. E-mail: meidel@maine.edu broadly grouped into experiments in which a limited num- ber of manipulated organisms are induced to spawn, and surveys of natural spawning events (Levitan. 1995; Yund, 2000). Experimental studies that control spawning syn- chrony and spatial relationships to test specific mechanistic hypotheses generally suggest that fertilization levels may be limited by sperm availability unless males and females spawn simultaneously, at close range, or under nearly ideal flow conditions (see Levitan and Petersen, 1995; and Yund, 2000, for reviews). In contrast, many surveys of natural spawns report fairly high fertilization levels, at least at the times and places in which most members of a population spawn (Yund, 2000). However, comparisons between exist- ing experiments and surveys are complicated by two major factors. First, results from experimental studies can success- fully predict fertilization levels in natural spawns only if experimental conditions (both biotic and abiotic) accurately mimic natural spawning conditions; however, experiments often circumvent reproductive strategies that may have evolved to enhance fertilization (Yund, 2000). Second, ex- periments and surveys are rarely conducted with the same species, so it is virtually impossible to distinguish between taxonomic and methodological effects in existing studies. Echinoderms have proven to be a particularly valuable model system for short-term field experiments, and experi- mental fertilization data from echinoderms generally sup- port the paradigm of severe sperm limitation under a wide range of flow and population conditions (e.g., Pennington, 1985; Levitan, 1991; Levitan et al., 1992; Wahle and Peck- ham, 1999; but see Babcock et a!., 1994). However, there are no published surveys of fertilization levels in natural spawns of echinoderms. The absence of survey data is probably due in part to a lack of information on temporal spawning patterns and the proximate environmental cues that initiate spawning (though multiple cues have been 84 SEA URCHIN FERTILIZATION DYNAMICS 85 proposed and investigated: Hirnmelman, 1975; Starr et al., 1990. 1992. 1993). Two interrelated adaptations that have been largely by- passed in previous experimental studies may have consid- erable effects on fertilization levels in natural spawns of temperate echinoderms. The first is an extended period of egg viability, which potentially allows fertilizations to ac- crue over time. Short-term experiments make one or both of the following assumptions: that most eggs are fertilized within the first few seconds of release (Denny and Shibata. 1989; Levitan et al., 1991) and that gametes are quickly diluted to concentrations below which fertilization can oc- cur. Consequently, extended egg viability has implicitly been presumed to have little influence on fertilization levels in the field. Meanwhile, recent estimates of egg longevity have steadily extended what was presumed to be a relatively short period of viability. Pennington ( 1985) reported a min- imum viability period of 24 h for eggs of the temperate sea urchin Strongylocentrotiis droebachiensis (Muller), and eggs of a West coast sea urchin are now known to be viable for up to 2 wk when stored under axenic conditions (Epel et ai. 1998). If eggs can be fertilized for a long period of time, extended or repeated exposure of eggs to sperm during long-duration spawning events (or events in which multiple males spawn successively) could result in high time-inte- grated levels of fertilization, even if sperm are limiting in the short term. A second adaptation that may interact with extended egg longevity to increase fertilization levels is the release of gametes in viscous fluids, which reduces gamete dilution rates and potentially increases the duration of egg exposure to sperm. Thomas ( 1994) has shown that three species of sea urchins (Tripneustes gratilla, Echinometra miitliaei, and Colobocentrotus at rants) release gametes in such viscous fluids that eggs and sperm remain on the test and spines at current speeds less than 0.13 m s~'. When the current speed increases, gametes are transported away from this reservoir in long (3-4 cm) strings or clumps, which led Thomas (1994) to hypothesize that sea urchins may achieve high fertilization levels if gametes encounter each other in these structures. Sperm concentrated in clumps presumably also have greater longevity because of a reduction in the respiratory dilution effect (Chia and Bickell, 1983). In con- trast to natural sperm release, fertilization experiments often mimic "males" with syringes from which diluted gametes are extruded at a fixed (and fast) rate, thus circumventing the potentially beneficial effect of "sticky" sperm that cling to the test and spines and slowly diffuse away. In this study, we investigate the effects of these two aspects of sea urchin reproductive biology on fertilization levels in Strongylocentrotiis droebachiensis. We initially determine the duration of egg viability at two points during the reproductive season. We then explore whether extended (3 h) exposure of eggs to sperm diffusing off a male sea urchin enhances fertilization levels relative to short-term ( 15 min) contact at various downstream distances. Finally, we use the full period of egg viability to assay time-inte- grated fertilization levels during natural sperm release events in small populations and use the distribution of developmental stages in these field samples to evaluate the temporal distribution of fertilization events. Materials and Methods General procedures To obtain fresh eggs and sperm for use in experiments and field sampling, sea urchins (Strongylocentrotus droe- bachiensis) were injected through the peristomial mem- brane with 0.2-2.0 ml of 0.5 M KC1. Females spawned into 50-ml glass beakers containing chilled seawater that had been aged (~ 15-20 h; hereafter referred to as aged seawa- ter) to eliminate ambient sperm. Female spawn was checked to confirm the absence of immature oocytes (as indicated by the presence of a large nucleus and nucleolus) and then washed three times with aged seawater. Dry sperm was pipetted directly from the aboral surface of spawning males and kept refrigerated until use (maximum 2 h). To assay fertilization levels in the field, unfertilized eggs were deployed in sperm-permeable containers. These egg baskets consisted of a 0. 1-m-long frame of PVC pipe (in- ternal diameter 0.05 m) with the sides (90% of circum- ference) cut away, covered with 35-jim Nitex mesh (after Wahle and Peckham, 1999, as modified from Levitan et ai. 1992). and two Styrofoam floats attached for positive buoy- ancy. Baskets were suspended from the surface or deployed on the bottom in different spatial arrangements as described in the following sections. Egg longevity To determine the viability period of eggs of Strongylo- centrotus droebachiensis, we performed laboratory experi- ments at the beginning (experiment 1 : February 28 to March 2. 2000) and in the middle (experiment 2: March 28 to April 1, 2000) of the spawning season along the coast of Maine (March to May, Cocanour and Allen, 1967). In each exper- iment. 120 JJL\ of freshly spawned eggs (mean SE of 1651 69 eggs) from each of four females were added to 10 ml aged seawater (aerated for 1 h prior to use) in 20-ml glass scintillation vials. At the start of each experiment (0 h) and after 24. 48. 72. and 96 h (experiment 2 only), eggs in each of four replicate vials per female (only one replicate per female at h in experiment 1 ) were fertilized with 20 /xl of a 10-fold sperm dilution ( 10 jul fresh dry sperm from 3 males. 90 /u,l aged seawater). Vials were gently agitated three times during a 15-min period, following which the fertilization process was stopped with the addition of 2.5 ml 37% formaldehyde. At each time point, one additional via! 86 S. K. MEIDEL AND P. O. YUND per female was fixed without fertilization, as a control for false fertilization envelopes (from causes such as egg dam- age or low egg quality). Vials were kept at ambient seawater temperature ( 1-3C) during both experiments. Fertilization levels were calculated as the percentage of a random sub- sample of 300 eggs with a fertilization envelope. Two-way analyses of variance (ANOVA) with the fixed factors Female (four levels) and Time (three levels in ex- periment 1; five in experiment 2) were used to analyze variation in fertilization levels (% fertilization). To achieve homogeneity of variances, percent fertilization values were arcsine transformed for experiment 1 (O'Brien's test, F = 1.20. P > 0.32) but not transformed for experiment 2 (O'Brien's test, F 1.35, P > 0.19). The Student- Newman-Keuls (SNK) test was used for post-hoc compar- isons of levels within main effects in the absence of a significant interaction effect. Cumulative fertilization in the field: 15 min vs 3 h In this experiment, we determined whether extended (3 h) exposure of eggs in baskets to sperm from a spawning male enhanced fertilization levels relative to short-term (15 min) exposure. We constructed a fertilization platform that was mounted on a concrete block (L X W X H: 0.36 m X 0.33 m X 0.14 m) deployed by a rope. The platform consisted of a pine board (1.59 m X 0.24 m X 0.02 m) bolted to the concrete block so that it extended 0.31 m upstream of the block and 0.92 m downstream. The board housed one male and two female stations. The male station was simply a surface-mounted PVC plate (0.08 m X 0. 12 m X 0.003 m), located 0.30 cm from the upstream end of the board, to which a spawning male could be fastened. Female stations consisted of eyebolts anchoring ropes that extended to the surface and were located 0.3 and 1.0 m downstream of the male station. Experiments were performed on a sandy substratum be- low the dock of the University of Maine's Darling Marine Center in the Damariscotta River estuary (ME. 4350'N, 6933'W) at a depth of 4.30 m at mean low water (MLW). For each trial (n = 8), four egg baskets (two side by side 0.05 m above the platform at each of two female stations) containing 500 ju.1 freshly spawned eggs (mean SE: 7613 455 eggs) from one female were attached to the eyebolts. A male was induced to spawn by injection of 2.5-4.5 ml 0.5 M KC1 and then attached to the male station with rubber bands. The fertilization platform was then im- mediately deployed. In addition to the platform, two mobile female stations (baskets on weighted lines with the lower basket 0.35 m above the substratum) were deployed 2 m upstream (control for ambient sperm: one basket) and 2.60 m downstream (two baskets spaced 0.1 m apart vertically, omitted from trial 1 ) from the male station. After 15 min, one egg basket from each of the three downstream female stations was retrieved without disturbing the remain- der of the array, by pulling it to the surface on its own line. The remaining baskets were retrieved after 3 h, and the presence or absence of sperm on the aboral surface of the male was recorded. Eggs were immediately collected and fixed with formaldehyde. To determine fertilization levels, 300 eggs per vial (200-300 in five cases, 154 in one case) were randomly sampled and scored for the presence or absence of a fertilization envelope. Where sufficient num- bers of eggs were retrieved (82% of baskets), small sub- samples were taken before fixation and scored after about 15-20 h for the presence or absence of later developmental stages. During trials 2 through 8, current velocity was recorded with a 3D- ACM acoustic-doppler current meter (Falmouth Scientific). Each trial took place around mid-tide (i.e., com- menced 1.5 h after high [or low] water and ended 1.5 h before low [or high] water) to minimize variation in the flow regime. Three laboratory controls (held at 3C), consisting of 200 ju.1 freshly spawned eggs in 10 ml aged seawater, were assayed for ( 1 ) fertilization at the start of each trial; (2) fertilization at the end of each trial; and (3) the presence of false fertilization envelopes, scored twice (after retrieval of 15 min and 3 h samples). Laboratory controls were scored in the same manner as field samples. A two-way ANOVA with the fixed factors Time (two levels) and Distance (three levels) was used to determine differences in fertilization levels (%) in field samples. Per- cent fertilization values were arcsine transformed prior to analysis to achieve homogeneity of variances (O'Brien's test. F = 0.94, P > 0.47). Sperm availability in nature We measured cumulative (over 24, 48, or 72 h) fertiliza- tion levels of eggs retained in baskets during natural spawn- ing events of Strongylocentrotus droebachiensis. This sam- pling design is a hybrid between an experiment and a true survey of natural spawns, because any sperm present were naturally released, but egg locations were under experimen- tal control. Sampling started in mid-February and ended in early April in 1999 and 2000 but varied in intensity (both spatial and temporal) during the two years. In 1999, samples were collected at a single station at Christmas Cove (ChC, mouth of the Damariscotta River estuary); in 2000, samples were collected from three stations at ChC and four stations at Clarks Cove (C1C. 1 km seaward of the Darling Marine Center and -9 km from the ChC site). Both sites were relatively sheltered with a sandy substratum, and surveys of the immediate surroundings indicated the absence of sea urchin populations other than those sampled (pers. obs.). A small population of 5. droebachiensis ( 150 animals in 1999, -60 in 2000) occurred naturally at ChC. At C1C, we SEA URCHIN FERTILIZATION DYNAMICS 87 released about 350 sea urchins on a rock ledge around the lower low water line on January 29, 2000, but this popula- tion appeared to have declined to about 30 animals by April 7, 2000. At each site, multiple stations were positioned to provide samples at different nominal distances from the sea urchins. At ChC, station 1 was within 1 m of a rock wall that was inhabited by sea urchins during the autumn months; station 2 was on the shoreward end of a floating dock, 5 m straight offshore of the wall: and station 3 was on the seaward end of the same dock, about 13 m from the wall. The shallow depth of station I ( 1 .4 m at MLW) allowed sampling at only one depth (0.15 to 0.35 m above the substratum). At stations 2 and 3, we sampled the surface waters during each interval (1.4 to 6.2 m above the substratum, depending on the tidally variable water depth): at times of anticipated sperm pres- ence (based on 1999 results) we also sampled the bottom water 0.15 to 0.35 m above the substratum. During 1999. only station 3 was sampled, and egg baskets were deployed only near the surface. Because the sea urchins were free to move, the positions of our stations relative to spawning males could not be known precisely. However, likely loca- tions can be inferred from sea urchin movement patterns. In 1999, sea urchins mainly remained on the rock wall or wandered between stations 1 and 2. whereas in 2000 many animals spent the spawning season on a piling adjacent to station 2. We employed a similar sampling scheme at C1C. with minor modifications to accommodate local dock structures. Station 1 was within 1 m of the rock ledge to which sea urchins were transplanted: station 2 was 1 m straight off- shore of station 1 (along a fixed wooden dock): and stations 3 and 4 were on floating docks about 12 m from station 1, at 45 angles to either side of the transect from stations 1 to 2. Because of minimal water depth ( 1 .0 to 1 .4 m at MLW). all stations were sampled at only a single depth (stations 1 and 2: 0.15 to 0.35 m above the substratum; stations 3 and 4: 0.4 to 3.5 m above the substratum, depending on the tidally variable water depth). Stations 3 and 4 were sampled only when sperm were expected to be present. At each site, sets of three replicate egg baskets (spaced 0.1 m apart vertically) were deployed at each station and depth and retrieved 24 h (1999 only), 48 h. or (on only three occasions) 72 h later. In 1999. baskets contained 500 fil of eggs ( 7600 eggs) from one female, and in 2000 they contained 800 /j.1 of eggs (mean number SE: 11216 787 eggs) pooled from two to three females. Laboratory controls (200 /xl of eggs in 10 ml aged seawater) were used to determine the incidence of fertilization membranes prior to basket deployment (presumably reflecting sperm contam- ination) and at the time of retrieval (presumably reflecting false membranes). To determine fertilization levels. 300 eggs per basket or vial were randomly subsampled and scored in three categories: unfertilized, presence of a fertil- ization envelope, or development through a later stage (2-64 cells, unhatched/hatched blastula. gastrula). Eggs with fertilization envelopes present were judged to have been fertilized only if the sample also contained later de- velopmental stages. From 41% of baskets (181 out of 441 ). fewer than 300 eggs were retrieved; in these cases, all retrieved eggs were scored. For the calculation of mean fertilization levels, only baskets with more than 50 retrieved eggs were used, resulting in a loss of replicates at some sites and times. We estimated the approximate distribution of fertilization events during a sample interval from the distribution of developmental stages in a sample and the known rate of development to each stage. We used Stephens' (1972) de- velopmental times for S. droebachiensis at 4C from fertil- ization to 32-cell stage (2-cell: 5 h: 4-cell: 8 h: 8-cell: 10.5 h; 16-cell: 14 h; 32-cell: 18 h). From the 64-cell stage to gastrulation, we used our own observations of develop- mental times (64-cell: 21 h; blastula: 24 h; hatching: 40 h; early gastrula: 48 h). We calculated the distribution of fertilizations (%) in time as the percent at each stage (i.e., of a certain age, in h) of all embryos detected (pooled from three replicate baskets). To establish the extent to which spawning had occurred during the 2000 sampling period, we collected sea urchins for analysis of gonad index (wet weight of gonads as a percentage of total wet body weight) from ChC l/i = 10) and C1C (;; = 1 1 ) on April 7 and 11. 2000, respectively. Results Egg longevity Egg viability in aged seawater in the laboratory (as as- sayed by fertilization with fresh sperm) varied significantly among time intervals and females in both experiments (Fig. 1). In experiment 1 (February 28 to March 2, 2000). the effects of both Female (F 3 . 3f) = 5.68, P = 0.003) and Time (F 2 36 = 8.94, P < 0.001 ) were significant, but the interaction between the two main factors was not (F 6 36 = 1.74. P = 0.14). Post-hoc comparisons revealed that fertilization levels were significantly lower for female 2. but similar for females 1. 3. and 4 (SNK-test. P < 0.05: Fig. 1A). Fertilization levels were highest at h, similar at 24 and 48 h (SNK. P > 0.05 ). and significantly lower by 72 h (SNK, P < 0.05). In experiment 2 (March 28 to April 1, 2000), there were again significant Female (F 3 60 = 18.0. P < 0.001 ) and Time (F 4 60 = 273. P < 0.001 ) effects, as well as a significant interaction between the two main factors (F,, 60 = 32.9, P < 0.001). Fertilization of eggs from females 1 and 4 remained relatively high at 72 h. while levels declined markedly for females 2 and 3 (Fig. IB). For females 1 and 2. fertilizations dropped to very low levels by 96 h. while fertilizations for females 3 and 4 were higher at 96 h than at 72 h (Fig. IB). Of a total of 36 control sample- 88 S. K. MEIDEL AND P. O. YUND A) February 28 - March 3. 2000 loo-,. , i, li IL LL ,L ED" 1148 D : B) March 28 - Apnl 1.2000 LL Female Figure 1. Mean ( +SE) fertilization levels (%) over time of eggs from four female sea urchins (A) at the beginning (experiment 1 ) and (B) in (he middle (experiment 2) of the spawning season. Replication is four vials for eac I). me expermen o e spawnng season. epcaon s each female/time combination (except experiment 1 at h: replication = ( 16 and 20 in experiments 1 and 2. respectively), 5 had 0.3% false fertilization envelopes and 1 had 0.7%. In spite of the significant variation among sample times and females in both experiments, egg viability was basically quite high for 48 to 72 h (Fig. 1). With the exception of female 2 in experiment 1. more than 75% of eggs held in aged seawater in the laboratory were viable for 48 h (Fig. 1 ). At 72 h, viability was in the 50%-75% range for eggs from 6 of the 8 females (Fig. 1 ). Cumulative fertilization level (15 min vs 3 li) When eggs in baskets were exposed to a continuous sperm supply from a spawning male, fertilization levels increased from 15 min to 3 h at distances of 0.3 and 1.0 m downstream from the male, but remained similar over time at 2.6 m (Fig. 2). In the 15-min samples, fertilization de- creased with distance from 0.3 to 1.0 m. but remained similar between 1 and 2.6 m (Fig. 2). In the 3-h samples, fertilization decreased monotonically with distance. The two-way ANOVA indicated significant Time (F, , 9 = 31.3, P < 0.001) and Distance (F 2 39 - 40.1, ' P < 0.001 ) effects, as well as a significant interaction between the two main factors (F 2 __, 9 = 4.87, P = 0.013). In 5 out of 8 trials, the male still had sperm on its test at the end of the 3-h deployment, suggesting that fertilization would have continued well beyond the end of our sample interval. Upstream controls for ambient sperm levels (Fig. 2) gen- erally had SO. 3% fertilization except in trials 1, 6, and 7 when fertilization levels reached 5.3%, 9.0%, and 2.0%, respectively. We attribute fertilizations in trial 6 to a large boat wake that probably created oscillatory water motion and transported sperm towards the upstream control sample immediately before retrieval of the 15-min samples, and we attribute fertilizations in trial 7 to false envelopes (see below). Fertilizations in trial 1 could not be attributed to any obvious cause, and the recorded value was subtracted from the fertilization levels recorded in experimental baskets for that trial. The apparent absence of a decline in fertilization between the 1- and 2.6-m samples at 15 min and the lack of an increase in fertilization between the 15-min and 3-h samples at 2.6 m are both attributable to one exceptional sample. During trial 5. we recorded a fertilization level of 48% at 2.6 m at 15 min. while values in other trials ranged only from 0.0%' to 3.3% (mean SE %: 1.4% 0.5%; n = 6) at 15 min and from 3.7%- to 15.3% (6.9% 1.8%; n = 6) at 3 h. If this outlier is excluded, fertilization declines from 1 to 2.6 m at 15 min and increases from 15 min to 3 h at 2.6 m. In laboratory controls, fertilization levels were always very high at the beginning (mean SE: 94.6% 1.7%; /; = 8) and the end (94.8% 1.6%; n = 8) of a trial. Controls for sperm contamination or false fertilization en- velopes mostly indicated 0% envelopes (15 min, 0.3% 0.2%; 3 h, 0.5%. 0.4%; n = 8) except in trial 7 where n n i. Distance from male (m) * Current direction Figure 2. Fertilization as a function of distance and duration of sperm exposure in the field experiment. Mean ( +SE) fertilization levels (%) are reported for each time/distance combination. Spawning male is located at 0.0 m mark. Upstream basket was retrieved after 3 h (hatched bar); downstream baskets after 15 min (stippled bars) or 3 h. Replication is 8 trials, except 7 trials for 2.6 m after 15 min. and 6 trials for 2.6 m after 3 h. SEA URCHIN FERTILIZATION DYNAMICS 89 \.T7c and 3.3% envelopes were found after 15 min and 3 h, respectively. These percentages were subtracted from the fertilization levels recorded in the Held for that trial. Current velocities varied widely during trials 2 through 7 and ranged mainly from 0.08 to 0.20 m s~ ' (Fig. 3). Mean velocities varied 5-fold among trials during the initial 15- min period (from 0.026 to 0.130 m s~') but were quite similar over 3 h (from 0.121 to 0.155 m s~"). Sperm availability in nature In both years of the survey (1999, 2000) and at both sites (ChC, C1C), no fertilizations were recorded during most of the sample intervals. However, in both years several sperm- release events of variable magnitude were detected. In 1999 at ChC (only station 3 surface was sampled), fertilizations occurred on March 5 (mean time-integrated fertilization level 4.7% ). March 23 (57.3%). March 31 (6.6%). and April 1 (24.69r ). In 2000 at ChC, fertilizations occurred on Feb- ruary 19 (station 1 only. 39.5%), March 10 (station 1. 10.3%; station 2, 9.3% surface; no bottom samples were deployed and no fertilization was detected at station 3), March 19 (station 1, 62.3%; station 2. 34.3% surface and 11.3% bottom; station 3. 30.4% surface and 5.3% bottom), and March 29 (station 1, 3.4%-; station 2, 4.6% surface; station 3, 4.5% surface; no bottom samples were deployed). At C1C (sampled only in 2000), fertilizations were detected on March 10 (station 1, 24.1%; no fertilization was detected at station 2; stations 3 and 4 were not sampled), March 17 (station 1, 27.7%: station 2. 10.4%; station 3. 26.2%; station 4, 3.7%), and April 3 (station 1, 6.9%; station 2, 3.3%; stations 3 and 4 were not sampled). In laboratory controls, fertilization levels were always very high at the start of each sample interval (mean SE %: 1999, 96.7% 0.7%, // -- 19; 2000, 93.8% 0.9%, n = 20). Controls for false fertilization envelopes (stored in the laboratory and fixed upon retrieval of the corresponding field sample) had very low levels of false envelopes (1999, 0.8% 0.5%, n = 16; 2000, 0.2% 0.1%; n = 20). Based on the distribution of developmental stages (two- cell to early gastrula) at the time of collection, we estimated that the temporal fertilization pattern varied markedly among the major sperm release events that we detected (Figs. 4-6). Because the discrete developmental stages that we scored are separated by longer time intervals later in development, the 24-h sample interval utilized in 1999 at ChC produced far better resolution of the time of fertiliza- tion (~3 h) than did the 48- to 72-h intervals employed in 2000 ( 3-h resolution for the 24 h immediately preceding sample collection, but 10 h for the portion of the interval >24 h prior to collection). In 1999. fertilizations occurred in fairly continuous trickles over about 48 h (March 3-5; Fig. 4A) or 24 h (March 22-24; Fig. 4B) or in two distinct pulses of similar magnitude about 24 h apart (March 30-April 1; 8 ^ C 1 . Iceland (64N. 22W) 1 Voucher specimens are being maintained in the marine invertebrate collections of C. W. Cunningham at Duke University. the two Atlantic species is entirely due to long-term isola- tion. Thus, subsequent physiological adaptations to warmer water in A. forbesi (Franz et ai, 1981 ) are independent of the speciation event. Essentially, the distinction between these species reflects either primary divergence due to se- lection or secondary contact following vicariance (Endler. 1977). In this study, mitochondrial and nuclear sequence data were collected from populations of A.forbesi and A mbens throughout North America and Europe, as well as from populations of the Pacific sister taxon A. tinnirensis (Clark and Downey. 1992). Phylogenetic and population genetic assays were used to test the hypotheses described above. It appears that Worley and Franz (1983) were remarkably accurate in suggesting a Pliocene speciation followed by a recent invasion of A. mbens from Europe, even in their prediction of details of timing, mechanisms, and effects. Although selection may have driven some of the diver- gence, it now seems clear that the initial separation of A. mbens and A. forbesi is due to late Pliocene changes in climate and ocean current flow, whereas North American populations of A. mbens are very recent arrivals. Materials and Methods Asterias specimens were collected from intertidal sites listed in Table 1 . Tube feet were immediately placed in 95% ethanol or DMSO buffer (0.25 M EDTA pH 8.0. 20% DMSO, saturated NaCl; Seutin et cil., 1991). Species were identified on the basis of key morphological characters described in Clark and Downey (1992) and Hay ward and Ryland (1995). DNA extraction and amplification DNA was phenol-extracted from each specimen follow- ing the protocol in Hillis et ul. (1996). These extractions were stored at 80C. PCR amplification of an approxi- mately 700-bp portion of the mitochondrial cytochrome c oxidase I (COI) protein-encoding gene was performed using the primers LCO1490 and HCO 2198 from Folmer et al. (1994). Amplification was performed in 50-;u,l reactions containing 10-100 ng DNA, 0.02 mM each primer, 5 jul Promega 10X polymerase buffer, 0.8 mM dNTPs (Pharma- cia Biotech), and 1 unit Taq polymerase (Promega). Reac- tions took place in a Perkin-Elmer 480 thermal cycler with a cycling profile of 94 : (60 s) -40 (90 s) -72 ( 150 s) for 40 cycles. The internal transcribed spacer (ITS) region was amplified under similar conditions, with an annealing tem- perature of 50C and with primers ITS4 and ITS5 (White et ul.. 1990). For each individual, sequences were obtained for three to four clones, and the consensus sequence was ob- tained to eliminate Taq error. PCR products were prepared for sequencing and were cycle-sequenced as in Wares (2001) using both PCR prim- ers. COI sequences representing each individual in this study have been deposited with GenBank (AF240022- 240081 ); ITS sequences were only obtained for 10 individ- uals, representing each species and region, and are also accessible in GenBank (AF346608-AF346617). Sequences were aligned and edited for ambiguities using complemen- tary fragments in Sequencher 3.0 (Genecodes Corp., Cam- bridge, MA). No gaps or poorly aligned regions occurred in the COI alignment, but missing characters were trimmed from the ends of the alignment to produce equal sequence lengths for all individuals. In the ITS alignment, all missing or ambiguous characters, including gaps, were removed. Consensus sequences were exported as a NEXUS file for subsequent analysis in PAUP*4.0b4a (Swofford. 1998). Phylogenetic analysis A heuristic search for the set of most-parsimonious trees based on the COI data was performed using PAUP*4.0b4a (Swofford, 1998). Trees were rooted using Leptasterias polaris (Asteriinae) and individuals of A. tinnirensis. Start- ing trees were obtained via stepwise addition, with simple addition sequence. Tree-bisection-reconnection was used for branch swapping, and branches were collapsed if the maximum branch length was zero. Maximum-likelihood (ML) phylogenies were also gener- ated in PAUP*. The best-fit model for all likelihood anal- yses (HKY with F-distributed rate variation; Hasegawa et ai, 1985; Yang, 1994) was determined by adding parame- ters until the likelihood description of the neighbor-joining tree did not significantly improve (Goldman. 1993; Cun- ningham et 0.10), though the substitution rate is significantly different (P < 0.05). Bootstrap replicates of the ITS data also indicate strong support for differentiation among these species. The ITS data do not reject a molecular clock model. Divergence among these species is indicated in Table 2. HKY -I- F distances in the COI fragment indicate that A. amurensis, A. forbesi, and A. nibens have been isolated from each other for a similar amount of time; assuming trans-Arctic isolation around 3.5 Ma. A. nibens and A. forbesi have been separated for at least 3.0 Ma. Although the estimated divergence date is higher when all codon positions are included (Table 2), and these data do not reject a molecular clock, neutrality tests (see below) suggest that some second-position substitutions may be under selection. Therefore, third-position sites may be more appropriate for the divergence estimate. The estimated divergence time is also higher when the ITS data are used; however, there is no reason to believe that speciation predated the appearance of Asterias in the North Atlantic, and the long branch leading to A. forbesi is not easily explained since it appears in both phylogenies (one using a protein-coding gene, one using untranslated spacer region data). This longer branch appears to influence the age estimates of the COI (all positions) and ITS data sets strongly. A McDonald-Kreitman test (McDonald and Kreitman, 1991) rejects a pattern of neutral substitution between A. nibens and A. forbesi (P < 0.01 , Table 3). Despite branch lengths that do not reject the molecular clock model, there is an excess of amino acid replacement substitutions be- tween the Atlantic species. The replacement substitutions between A. nibens and A. forbesi do not include any first- position substitutions. Half (8/16) of the amino acid substi- tutions do not involve a change in charge or polarity, whereas almost half (7/16) of the changes substitute a basic residue for an uncharged or nonpolar residue. However, there does not seem to be an obvious pattern to these changes between A. nibens and A. forbesi. Other species comparisons do not reject the neutral model of substitution (Table 3). Within each species, Tajima's (1989) test is nonsignificant (A. amurensis, D = 0.837, P > 0.10; A. forbesi, D = -0.705, P > 0.10; A. nibens, D = -1.482, P > 0.10), indicating that there is no reason to suspect non-neutral evolution in the intraspecific comparisons. Additionally, Bayesian analysis (Templeton el at., 1992; Clement ct ai, 2000) of the COI data within A. rubens indicates greater than 95% confidence that the intraspecific gene tree is parsimonious. The ML root haplotype is found on both coasts of the Atlantic (Fig. 1A, Haplotype B), and this haplotype is at least an order of magnitude more likely to be the ancestral haplotype than any other haplotype of A. rubens (likelihood index = 0.857). All North American haplotypes are also found in Europe; the unique haplotypes found in Europe contribute to a significantly higher allelic diversity (P < 0.0 1 . Table 4). The ITS data are consistent with the COI data in that there is no allelic diversity among North American and European individuals of A. nibens (n = 6). Discussion Understanding the mechanisms that are responsible for the divergence of Asterias nibens and A. forbesi first re- quires that the timing of their divergence be estimated. Estimates based on the molecular calibrations reported here suggest that these species last shared a common ancestor at least 3.0 Ma (Table 2), not long after the genus first arrived in the North Atlantic (around 3.5 Ma; Worley and Franz, 1983; Vermeij, 1991 ). Note, however, that asterozoan skel- etons are rarely preserved in the fossil record, because they lack rigidly articulated skeletons and rapidly disintegrate (Barker and Zullo, 1980); indeed, fossils of A. forbesi have been reported only twice, each time in Pleistocene intergla- cial sediments. Thus, little direct evidence points to the first appearance of Asterias in the North Atlantic (Durham and MacNeil, 1967; Worley and Franz, 1983), and the biogeo- graphic data used in this paper is therefore based on con- BIOGEOGRAPHY OF ASTERIAS 99 1 A. forbesi 10C> -L 1 H Norway Norway Haplotype A (n=16) 1 Ireland _l rubens 100 Haplotype B (=14) - Ireland 1 Ireland A Haplotype C (n=l) - France - France Norway Haplotype D (=2) e and 100 100 ^ r _jT 100 * 1~ 0.01 substitutions/site B r 100 1 Ireland Iceland i Iceland Newfoundland A. ruutis 100 Maine Maine -1 A. forbesi 99 0.005 substitutions/site Figure 1. Phylogenetic trees for Asierias generated using the best-tit maximum likelihood model in each data set (COI: HKY + T; ITS: F81 ). (A) Cytochrome c oxidase I phylogeny of inter- and intraspecific Asterias relationships. Here all characters (first, second, and third position) are included; an identical topology is found using parsimony or distance methods, or looking at third-position characters alone. Bootstrap support for each species is indicated by the numbers below each branch. These data do not reject a molecular clock model. The divergence across the Arctic (between A. amurensis and the Atlantic speciesl is considered to be 3.5 Ma; this generates an estimate of about 3.0 Ma for the divergence between A. rubens and A. amurensis (see Table 2 and Discussion). Haplotypes A D of A. rubens are found on both the North American and European coasts (A: Maine (n = 8), Nova Scotia (n = 2), Newfoundland (n = 2), Iceland in = 1 I. Norway (H = 2), Ireland (H = I ); B: Maine (n = 2). Nova Scotia (n = 4). Newfoundland (n = 3). Iceland (H = 1 ). Ireland (H = 2). France (n = 2); C: Maine (n = 1 ). Norway (n = 3), Ireland (n = 2). and France (n = 1 ); D: Ireland (n = 1 ), and Maine (n = 1 )). Amphi-Atlantic haplotype B is the maximum likelihood root (index = 0.857). (B) Internal transcribed spacer ( ITS ) phylogeny of inter- and intraspecific Asterias relationships. Likelihood ratio tests do not reject a hypothesis of proportional branch lengths (P > 0. 10) suggesting that, aside from substantial differences in substitution rate, the two phylogenies are equivalent representations of interspecific differentiation. A nearly identical phylogeny is reconstructed when indels are included in the ITS data. sistent fossil evidence from other cold temperate species that participated in the trans-Arctic exchange. Nevertheless, there is reason to believe that Asterias also spread from the Pacific to the Atlantic at about 3.5 Ma (Worley and Franz. 1983). Miocene and early Pliocene temperatures were around 5-6C warmer in the North Atlantic and Arctic, permitting the initial trans-Arctic passage of temperate spe- cies (Berggren and Hollister. 1974; Vermeij, 1991 ), but then two dramatic changes were initiated around 3.0 Ma that appear to play a role in speciation within the North Atlantic. 100 J. P. WARES Table 2 Internal branch lengths (based on best-fit likelihood model} separating Asterias species (lower triangle*, all 3 matrices) All characters A. atnurensis A. rubens A. forbesi A. anmrensis tL = 1.954 x 10~ 8 8.63 x 10" 9 IJL = 2.665 X 10~ 8 9.59 X 10~" A. nibens 0.13678 0.06044 3.59 Ma A. forbesi 0.18658 0.06715 0.16576 0.04595 3rd position only A. anmrensis A. rubens A. forbesi A. amurensis ju, = 6.689 x 10"" 3.36 x 10~ 8 M, = 9.751 x 10~ 8 3.74 x lO"" A. rubens 0.48084 0.2352 2.96 Ma A. forbesi 0.68254 0.26168 0.49270 0.15661 ITS-1 A. anmrensis A. rubens A. forbesi A. amurensis p. = 5.142 x 10~ 9 2.04 x 10~ 9 /M = 7.188 x 10~ 9 2.40 X 10~" A. nibens 0.0361 0.0143 3.84 Ma A. forbesi 0.0500 0.0168 0.0470 0.0163 The calibration date of 3.5 Ma is used to obtain the mutation rate ^. for comparisons between A. amurensis and the Atlantic species. The estimated divergence time between A. rubens and A. forbesi is based on the mean of this calibrated mutation rate (cytochrome c oxidase I [COI] all positions, top; COI 3rd position only, middle; internal transcribed spacer (ITS) 1. bottom). * In each matrix, the lower triangle containing the internal branch lengths is made up of the matrix cells below the diagonal line of empty cells representing comparisons within the same value. The upper triangle contains the estimated mutation rates and estimated divergence data. At that time, warm North Atlantic currents were dis- placed by the formation of the cold-water Labrador Current. This event created a significant thermal gradient in the North Atlantic, and tropical-temperate faunas were abruptly replaced with polar and subpolar faunas on the continental Table 3 McDonald-Krehman tests on each Asterias species pair using cytochrome c oxidase I (COI) translated data Species pair Fixed differences Polymorphisms A. rubens-A. forbesi Synonymous 39 19 Nonsynonymous 16 ; P < 0.001 Synonymous 36 21 Nonsynonymous 12 1 P > 0.05 A. forbesi-A. amurensis Synonymous 44 15 Nonsynonymous 14 1 /' > 0.15 Only the comparison between A. rubens and A. forbesi indicates a significant departure from neutral evolution. A two-tailed Fisher's exact test was used for each set of comparisons. shelf off Nova Scotia and the rest of New England (Berg- gren and Hollister, 1974; Worley and Franz, 1983; Cronin, 1988). As Northern Hemisphere glaciation began, (lie present-day latitudinally controlled faunal provincialization was established as well (Berggren and Hollister, 1974). This dramatic cooling of the northwestern North Atlantic prob- ably initiated the separation of North Atlantic Asterias into European and North American populations with very little genetic contact (Worley and Franz, 1983). Subsequent Pleistocene glaciation would have prevented the long-term Table 4 Comparisons of haplotype diversity (\\, see eqn. 8.4 in Nei 1987. calculated in DNAsp 3.50, ROMS and Rozas 1999) for the cytochrome c oxidase I fragment in each species and population of A. rubens Species/Population Haplotype diversity (H) cr Asterias rubens 0.793 0.00138 North America 0.597 0.00395 Europe 0.893 0.00143 Asterias forbesi 0.964 0.00596 Asterias amurensis 0.999 0.03125 European populations of A. rubens have significantly higher allelic diversity than North American populations (P < 0.01); this finding is supported by nonparametric haplotype sampling in Wares (2000). BIOGEOGRAPHY OF ASTERIAS 101 establishment of populations in New England, as most of the North American coast from Long Island Sound north- ward was covered by a kilometer of ice during glacial maxima (Kelley et til., 1995). Pacific and Atlantic populations of other species appear to have had more recent trans-Arctic genetic contact than the estimates above would suggest for Asterias (Palumbi and Kessing. 1991; van Oppen el al., 1995). Moreover, rapid climatic fluctuations (Cronin, 1988: Roy et al.. 1996) during the Pleistocene could have permitted large-scale changes in the geographic range of cold temperate species. However, both the sea urchin Strongylocentrotus pal/idus (Palumbi and Kessing, 1991) and the red alga Phycodrys nihens (van Oppen et al., 1995) appear to have greater tolerance for Arctic waters than Asterias does. Worley and Franz (1983) report that expansion of Asterias populations into habitats as far north as Greenland only occurs period- ically, and that these populations cannot tolerate colder waters (Franz et al.. 1981 ). However, the indirect morpho- logical and paleontological evidence is bolstered by the molecular evidence, which strongly suggests that A. rubens and A. forbesi diverged shortly after their ancestral lineage separated from the Pacific A. amurensis. The estimates of mutation rate presented here are very similar to other esti- mates for both the COI fragment (Knowlton and Weigt, 1998; Schubart et al.. 1998; Wares, 2001; Wares and Cun- ningham, in press) and the ITS fragment (Schlotterer et al.. 1994; van Oppen et al.. 1995). Thus these data strongly support earlier inferences of a late Pliocene trans-Arctic passage and subsequent speciation within the Atlantic. An analysis of genealogical patterns within A. rubens confirms that the North American populations of this spe- cies are descendants of a recent colonization from Europe that probably followed the most recent glacial maximum (about 20.000 BP, Holder et al.. 1999). The genealogical data presented here fit several important patterns that sug- gest a recent range expansion (Wares. 2000). All North American haplotypes are identical to the most-common European haplotypes (Fig. 1A). Generally, invading haplo- types are the most deeply nested haplotype in the European (putative source) population. This is to be expected, because deeply nested ancestral haplotypes are often the most com- mon (Castelloe and Templeton. 1994), and therefore have a higher probability of participating in long-distance dispersal events. Haplotype B (Fig. 1 A) is a good illustration of this expectation it is closely related to each other haplotype and has a high copy number in both European and American populations. These observations contribute to the high like- lihood (85.7<7r, more than an order of magnitude greater likelihood than any other haplotype) that this is the ancestral allele in A. rubens. Additionally, allelic diversity is significantly lower in North American A. rubens than in Europe (Table 4), a signal of recent range expansion (Hewitt, 1996; Austerlitz et al.. 1997). However, the North American colonization is diffi- cult to date because there are no unique haplotypes in North America; ancestral allelic polymorphism tends to inflate indirect estimates of population size and age (Kuhner el al.. 1998: Edwards and Beerli, 2000). The lack of unique di- versity in North America also prevents the meaningful use of other phylogeographic methods; for instance, statistics of the geographic dispersion of haplotypes (for review see Templeton, 1998) are uninformative (Wares, unpubl. data). This is primarily because even closely related individuals (identical haplotypes) are distributed across the entire geo- graphic range of A. rubens. It is possible that the multiple shared alleles between Europe and North America represent a multiple-invasion history; Asterias larvae are planktotro- phic and disperse in the water column for 6 or more weeks (Clark and Downey, 1992). There is evidence that natural selection has played some role in the overall divergence between these species. A significant number of amino acid replacement substitutions distinguish A. rubens from A. forbesi (Table 3), all of them reflecting second- or third-position nucleotide substitutions. There is no obvious pattern to the amino acid replacements, as most of them involve substitutions among uncharged or nonpolar amino acids. Two of the three species in the genus Asterias are found in cold-temperate waters, while A. forbesi is found in the warmer mid- Atlantic region (Schopf and Murphy, 1973; Franz et al., 1981 ). Many of the phys- iological differences between A. rubens and A. forbesi (Franz et al., 1981 ) reflect this latitudinal distribution. How- ever, the possibility that these amino acid substitutions are related to physiological differences in the warm-temperate A. forbesi has never been tested. The difference in temper- ature between the habitats of A. rubens and A. forbesi is unlikely to contribute to differences in metabolic rate that could accelerate the mutation rate (for review see Rand, 1994). Nevertheless, this hypothesis is worth examination, because A. forbesi is supported by relatively long branches in both the COI and the non-coding ITS region (Table 2, Fig. IB). If natural selection is playing a role in the amino acid divergences of the mitochondrial COI gene between A. rubens and A. forbesi, there is no reason why a noncoding nuclear sequence should reflect the same increase in diver- gence rate. In conclusion, the biogeographic response of Asterias to late Pliocene climatic and oceanographic change fits a pat- tern predicted by Worley and Franz (1983). Following the arrival of Asterias in the North Atlantic around 3.5 Ma (Worley and Franz, 1983; Venneij, 1991). populations were established on both the European and North American coasts during a period when the North Atlantic was as much as 5-6C wanner (Berggren and Hollister. 1974). The for- mation of the Labrador Current 3.0 Ma rapidly changed the faunal composition of the intertidal Canadian Maritimes and New England coast, and Asterias populations in this region J, P. WARES probably went extinct. An American population survived under the conditions of the mid-Atlantic coast and Gulf Stream waters (A.forbesi), and the European population (A. rubens) has recently recolonized the cold-temperate shores of New England and the Canadian Maritimes. Thus, the zone of sympatry between these two species appears to be a zone of secondary contact. Hybridization is considered rare between these species (Schopf and Murphy, 1973; Worley and Franz, 1983), but whether behavioral mechanisms (Franz et ai, 1981) or gametic recognition mechanisms (Hellbergand Vacquier, 1999; Fernet, 1999) are responsible is unclear. The genetic data presented here illustrate a strong con- cordance between paleoceanographic changes and indirect estimates of speciation between the North Atlantic Asterias species. The species boundaries are phylogenetically quite distinct, and the divergence estimates based on these genetic data appear to support a late Pliocene, rather than late Pleistocene or Holocene, separation. 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Bull. 201: 104-119. (August 2001) Systematics and Biogeography of the Jellyfish Aurelia labiata (Cnidaria: Scyphozoa) LISA-ANN GERSHWIN Cabrillo Marine Aquarium, San Pedro, California 90731 and CSU Northrid?>e, California 91330 Abstract. The hypothesis that the common eastern North Pacific Aurelia is A. aurita is falsified with morphological analysis. The name Aurelia lahiata is resurrected, and the species is redescribed, to refer to medusae differing from A. aurita by a suite of characters related to a broad and elon- gated manubrium. Specifically, the oral arms are short, separated by and arising from the base of the fleshy manu- brium. and the planulae are brooded upon the manubrium itself, rather than on the oral arms. Aurelia aurita possesses no corresponding enlarged structure. Furthermore, the num- ber of radial canals is typically much greater in A. lahiata, and thus the canals often appear more anastomosed than in A. aurita. Finally, most A. labiaia medusae possess a 16- scalloped bell margin, whereas the margin is 8-scalloped in most A. aurita. Separation of the two forms has previously been noted on the basis of allozyme and isozyme analyses and on the histology of the neuromuscular system. Partial 18S rDNA sequencing corroborates these findings. Three distinct moiphotypes of A. lahiata, corresponding to sepa- rate marine bioprovinces, have been identified among 17 populations from San Diego. California, to Prince William Sound. Alaska. The long-undisputed species A. limhata may be simply a color morph of A. labiata, or a species within a yet-unelaborated A. lahiata species complex. The first known introduction of Aurelia cf. aurita into southern Cal- ifornia waters is documented. Although traditional jellyfish taxonomy tends to recognize many species as cosmopolitan or nearly so, these results indicate that coastal species, such as A. labiata, may experience rapid divergence among iso- lated populations, and that the taxonomy of such species should therefore be scrutinized with special care. Received 16 December 1998; accepted 5 April 2001. Current address: Dept. of Imegrative Biology, University of California. Berkeley. CA 94720. E-mail: gershwin@socrates.berkeley.edu Introduction Perhaps had Darwin not been afflicted with seasickness, he might have noticed the bewildering array of geographi- cally varying jellyfish morphologies. Some of his contem- poraries documented species separated by only short dis- tances but differing greatly in appearance (Eschscholtz. 1829; Brandt. 1835, 1838; Agassiz, 1862; Haeckel, 1879, 1880). Morphological distinctions have since been reported for populations of Cassiopea from separate islands of the Caribbean (Hummelinck, 1968), Mastigias in different lakes of Palau (Hamner and Hauri, 1981), and Aurelia scyphistomae from various parts of the Thames estuary (Lambert, 1935). In his studies of the genus Cyanea, Brewer ( 1 99 1 ) reported distinct morphotypes that could be corre- lated with isolated locations in Long Island Sound, USA; these observations resurrected a long-standing argument about species distribution and recognition criteria of North Atlantic Cyanea. Nineteenth-century taxonomists recog- nized different species, corresponding to a latitudinal gra- dation, on both sides of the Atlantic. Cyanea arctica Peron and Lesueur, 1810, was known as the boreal species from Europe to North America. In the western Atlantic. C. fulva L. Agassiz. 1 862, was found along the mid-Atlantic states, while the form south of the Carolinas was recognized as C. versicolor L. Agassiz. 1862. In the eastern Atlantic, C. capillata (Linnaeus. 1746) was established as the northern European species, while C. lamarckii Haeckel, 1880. was identified in warmer southern European waters. This pattern of biodiversity was largely overlooked by twentieth-century taxonomists. who often lumped the forms and recognized only C. capillata (Mayer. 1910; Bigelow. 1914; Stiasny and van der Maaden. 1943; Kramp, 1961; Calder, 1971; Larson, 1976). The scarcity of biogeographic studies of jellyfishes may be, in part, attributable to the unclear systematics of these 104 SYSTEMATICS AND BIOGEOGRAPHY OF AURELIA LAB/ATA 105 Figure 1. Original illustrations of Aurelia labiata, showing greatly enlarged munubrium: (A) lateral view of medusa; (B) oblique view of subumbrella. (Reprinted from Chamisso and Eysenhardt. 1821). animals. Color differences, patterns of pigmentation, and anatomical variation led to the description of many nominal species during the expeditions of the nineteenth century (see Mayer, 1910; Kramp, 1961). The range of variation in jellyfishes is not well understood, and species definitions are often vague, focusing only on the few most obvious char- acters. For example, if one sees a flat, whitish medusa with four horseshoe-shaped gonads. most tend to think it must be Aurelia aiirita. The details of anatomy have not been scru- tinized closely. Therefore, significant morphological differ- ences have not been detected, and inappropriate identifica- tions and erroneous conclusions regarding biogeography have been made. The systematic tangle and biogeographic mistakes are common throughout the medusan taxa, though I focus herein on Aurelia. Mayer (1910) recognized 13 unique forms of Aurellia (the spelling was later formally changed back to Aurelia by Rees, 1957), and sorted these forms into three morpholog- ical groups: 1. A. aiirita (Linnaeus, 1746) sensu Lamarck, 1816, and its seven varieties, described as A. cniciata Haeckel. 1880, A. colpota Brandt, 1835 [sensu Gotte, 1886] (as =A. coerulea von Lendenfeld, 1884), A. flavidula Peron and Lesueur. 1810 [incorrectly listed as 1809) (as =A. habanensis Mayer, 1900). A. hyalina Brandt. 1835. A. dubia Vanhfiffen, 1888. A. vitiana Agassiz and Mayer. 1899. and A. imirginalis L. Agassiz. 1862 2. A. labiata Chamisso and Eysenhardt, 1821 [incor- rectly listed as 1820[. with three varieties, described as A. clausa Lesson, 1829, A. limbata (Brandt. 1835) [incorrectly listed as 1838], and A. inaldivensis Big- elow. 1904 3. A. solida Browne, 1905 Mayer distinguished A. labiata and its varieties from the other two groups based primarily on the degree of scallop- ing of the bell margin, being 16-notched in the former and 8-notched in the latter. He subsequently found a specimen of A. iinritti at Tortugas. Florida, closely resembling A. labiata, leading him to conclude that A. labiata was prob- ably derived as a mutation from A. aiirita (Mayer, 1917). Kramp also wavered on the validity of A. labiata, first recognizing the species in his 1961 synopsis, then later regarding it as doubtful (1965, 1968). Most recently, au- thors such as Russell (1970). Larson (1990), and Arai ( 1997) have recognized two valid species: A. limbata, which is primarily arctic and has a conspicuous brown bell margin, and A. auritci. whose name has been treated as the senior synonym of all others. Russell (1970) followed Kramp (1965, 1968) in regarding all other species as varieties, whereas Larson (1990) and Arai (1997) simply did not mention any other species. The source of this confusion is unclear, as the original description of A. labiatu was quite specific. Translated from Latin, "It differs from A. aiirita by its very long oral lips. Marginal tentacles were not observed, but are without a doubt present. Arms appressed to the bell. Diameter of the bell nearly a foot" (Chamisso and Eysenhardt. 1821). The focus of the description and its accompanying illustrations is the strikingly unique elongated manubrium (Figs. 1,2). 106 L. GERSHWIN Figure 2. Aurelia labiata. adult medusa, from Monterey Bay. Califor- nia. although this character is rarely mentioned in later revisions. Furthermore, the characteristically short oral arms arising from the base of the manubrium were mentioned as being held close to the bell, a trait that is readily apparent in live specimens. Ironically, the commonly accepted character of 16 marginal scallops is not mentioned, although it is subtly illustrated. It is unclear why certain key characters of the original description have been ignored by later workers. Disorder in the nomenclature of Aurelia worldwide has caused confusion about the identity of the species in the eastern North Pacific. Depending on the author, one to three species have been recognized. Most authors have applied the name A. aurita to all forms. Some distinguish ,4. lim- bata, although this appears to have been occasionally con- fused with A. labiata (Zubkoff and Lin, 1975; Greenberg et al., 1996). When A. labiata has been recognized, it has been separated from A. aurita only by the doubling of marginal scallops (Hand. 1975; Kozloff, 1974). Although A. labiata was originally described from California, most reports of the species (apparently incorrectly) are from regions outside the eastern North Pacific. Throughout all the confusion, several studies have re- ported differences between the eastern North Pacific Aurelia and those of other regions, yet failed to elaborate the sys- tematics. Chia et al. ( 1984) found that the muscle system in Puget Sound polyps is distinct from that of polyps from Plymouth. England. Zubkoff and Lin (1975) observed pe- culiar banding in the isozyme patterns of Aurelia scyphis- tomae from Puget Sound, Washington, that caused them to wonder whether this population may belong to a species other than A. aurita. Similarly. Greenberg et al. (1996) could distinguish two groups on their allozyme patterns: one group consisted of two populations of A. "aurita" from Japan (one from Tokyo Bay, and one aquarium-raised) plus a population that was apparently introduced to San Fran- cisco Bay; and the second group consisted of wild medusae from Monterey Bay, California, and Vancouver, British Columbia. They further distinguished the two groups on the basis of morphology, using manubrium length and the highly anastomosed condition of the radial canals. To test the hypothesis that the common eastern North Pacific Aurelia is A. aurita, I compared the morphology of 17 populations of Aurelia from San Diego, California, to Prince William Sound, Alaska, to the morphology of A. aurita from Europe, and A. flavidula from the eastern United States, as described and figured by Agassiz (1862), Mayer (1910), Kramp (1961). Russell (1970), and many of the references therein. The conclusions that I have drawn on morphological characters are consistent with those emerg- ing from the enzyme analyses of Zubkoff and Lin (1975) and Greenberg et al. (1996), the neuromuscular study of Chia et al. (1984), and the DNA sequencing results of J. Lowrie of the Cnidarian Research Institute (pers. comm., June 2000) that is, that the common eastern North Pacific Aurelia is not A. aurita. However, it does match the de- scription of the species previously described as Aurelia labiata Chamisso and Eysenhardt, 1821. Thus, I propose a revalidation of A. labiata, and herein offer a redescription and designate a neotype. In scrutinizing the morphology of A. labiata. I further found that each population possesses unique characters that cluster into three morphotypes cor- responding to well-demarcated biogeographic provinces. The purposes of this paper are to describe the morphological and geographical variation in A. labiata and to stabilize the nomenclature for the species. This is necessary as a basis for further systematic investigation, for ongoing biodiversity studies, and for proper management of species introduc- tions. Materials and Methods Aurelia aurita and other fonns Literature-based comparisons were made using the Euro- pean form, Aurelia nuriia, and are denoted traditionally (e.g., Aurelia aurita). The full breadth of literature used for comparison is too massive to list here, but can be found in the synonymies of Mayer (1910), Kramp (1961), and Rus- sell (1970). Literature-based comparisons were made with A. flav- idula from the eastern United States, primarily following Agassiz (1862) and the references in the synonymy of Mayer (1910). Literature-based comparisons were made to the boreal A. limbata using Brandt (1835. 1838). Vanhoffen (1906), Kishinouye (1910), Bigelow (1913, 1920), Uchida (1934), Bigelow (1938), Kramp (1942), Stiasny and van der Maa- den (1943), Naumov ( 1961 ), Uchida and Nagao (1963), and Faulkner (1974). SYSTEMATICS AND BIOGEOGRAPHY OF AURELIA LABIATA 107 Comparisons were made using live, captive medusae descended from a Japanese population (cultured at Cabrillo Marine Aquarium); although the phylogenetic relationship between the European and Japanese forms is still in ques- tion, they are structurally similar that is. they both lack the enlarged manubrium characteristic of A. labiata. Comparisons were also made on some live, wild medusae from Spinnaker Bay, Long Beach, California, which pos- sessed the A. aurita body form, and on the descriptions of Greenberg et al. (1996) for the introduced San Francisco Bay form. Live representatives of Greenberg's population at Foster City could not be found. References made to forms that possess the A. aurita body type but are of uncertain taxonomic affiliation are denoted non-traditionally (e.g., Aurelia "aurita" or Aurelia cf. aurita). This includes the captive Japanese form, as well as introduced forms. Systematics of Aurelia labiata Attempts were made to locate the holotype at the follow- ing institutions: The California Academy of Sciences (San Francisco) (CAS), Institut Royal des Sciences Naturelles de Belgique (Brussels), Museum fur Naturkunde (Berlin), Mu- seum National D'Histoire Naturelle (Paris), Museum of Comparative Zoology (Harvard). Nationaal Natuurhisto- risch Museum (Leiden), National Museum of Natural His- tory (Washington), Natural History Museum (London), Zoological Institute (St. Petersburg). Zoological Museum (Copenhagen), and the Zoological Museum (Moscow Uni- versity). All would have been reasonable depositories or recipients of a transfer of a holotype of a California species found by European explorers on a Russian expedition of that time. However, none had A. labiata type material nor knew where it might be kept; indeed, it appears doubtful that specimens were originally collected and deposited. Thus, my observations were made on animals from near the type locality and from many other regions along the Pacific Coast of North America. A neotype was designated in order to stabilize the taxon- omy of the species, and is deposited in the California Academy of Sciences in San Francisco. The original type locality could not be identified. Chamisso and Eysenhardt ( 1 82 1 ) recorded the species from "New California," and a map in Schweizer (1973) indicates only somewhere near San Francisco Bay. However, specimens that I collected near San Francisco Bay were in poor shape, so the most intact representative specimen from the available material was selected from Monterey Bay (ca. 100 miles to the south). Morphological differences were not apparent be- tween specimens from San Francisco and Monterey, except- ing those attributable to collection. I preferentially examined live medusae in the wild to avoid artifacts of captivity and preservation; however, cul- tured and captive medusae were observed supplementally. In the wild, mature and immature medusae were collected from July 1995 to March 2000 by hand and by dip nets from nine locations in California (Coronado Island. San Diego: Newport Beach; Spinnaker Bay, Long Beach; Catalina Is- land; Marina del Rey; Santa Barbara; Monterey Bay; Sau- salito, San Francisco Bay; Tomales Bay), and from New- port, Oregon; Poulsbo. Washington; Friday Harbor, San Juan Island, Washington; and Brentwood Bay, Saanich In- let. British Columbia. Cultured and captive medusae were examined at the Birch Aquarium at Scripps. San Diego, California (San Diego A. labiata): Cabrillo Marine Aquar- ium, San Pedro, California (both Japanese Aurelia "aurita" and Long Beach A. labiata): Monterey Bay Aquarium. Monterey, California (Japanese A. "aurita" and Monterey A. labiata): Oregon Coast Aquarium. Newport. Oregon (Japanese A. "aurita" and Newport A. labiata): Point Defi- ance Zoo and Aquarium. Tacoma, Washington (A. labiata from Poulsbo, Washington); and the Seattle Aquarium, Se- attle. Washington (A. labiata from Poulsbo, Washington). In addition to the above observations, characters were as- sessed as much as possible from a videotape taken in July 1996 of medusae from Prince William Sound, Alaska; from photographs of A. labiata from Steamer Bay, Alaska (Barr and Barr, 1983) and A. liiubata from Amchitka Island. Alaska (Faulkner. 1974); and from preserved specimens from the Farallon Islands, California. Measurements were taken on 7-20 live medusae from each of the following locations: Coronado Island. Newport Beach. Spinnaker Bay. Marina del Rey. Monterey Bay, Tomales Bay. Newport (OR). Poulsbo. and Brentwood Bay. Each medusa was individually dipped out of the water with a bucket and measured immediately with a vernier caliper or ruler to the nearest millimeter. Bell diameter (BD) was typically measured with the specimen lying flat on its ex- umbrellar surface. Manubrium length (ML) was usually measured with the animal in the water with the manubrium projecting upward, but captive medusae from Newport (OR) were measured with the manubrium hanging down- ward in the water. Since the manubrium is stiff and carti- laginous, its position did not appear to bias the measure- ments. To account for the difference in size at maturity of medusae from different populations, manubrium lengths were normalized as a percentage of bell diameter. In addition to the measurements described above, about 200 medusae from each population were cursorily examined for the following characters, then released: manubrium shape, number of marginal scallops, oral arm length, num- ber of radial canals emanating from each gastro-genital sinus, bell shape and color, and if female, the location and pattern of larval brood. German papers were translated with Power Translator 6.02 for Windows (Globalink). 108 L. GERSHWIN B FIG. 3. Comparative diagram of three morphotypes ofAurelia labiata with A. aurila, subumbrellar and lateral views. (A) Aurelia aitrila. (B) Southern morph, from Southern California Bight. (C) Central niorph. from Santa Barbara, California, to Oregon. (D) Northern morph, from Puget Sound, Washington, to Alaska. In A. aurita, manubrium is inconspicuous, oral arms meet in the middle, the radial canals are few. and the margin has 8 scallops. In A. labiata. the manubrium protrudes below the bell margin, which has 16 scallops, there are many radial canals, and the oral arms do not meet. Darkened areas along oral arms (A. aurita) and manubrium (A. labiata) indicate position of larval brood. Results Comparison with European Aurelia aurita (Fig. 3) Medusae from every population that I studied in the eastern North Pacific differed from published descriptions of the European A. aurita but closely matched the original description of A. labiata. Specifically, the A. labiata body form is characterized by an enlarged, fleshy manubrium; oral arms arising from the base of the manubrium; planulae brooding upon the manubrium; up to 15 radial canals arising from each gastro-genital sinus, and typically anastomosing in older individuals; and secondary scalloping of the bell margin between rhopalia (Fig. 3B-D). In contrast, the A. aurita body type possesses no such enlarged manubrium structure; the oral arms meet in the middle of the animal; planulae are brooded upon the oral arms; typically only 3-5, sometimes 7. radial canals arise from each gastro-genital sinus; and secondary scalloping is rarely observed (Fig. 3 A). Comparison with western Atlantic Aurelia "flavidula" The nominal species Aurelia flavidula is another taxo- nomic tangle that was somewhat resolved by Kramp ( 1942). Kramp concluded that the yellow Greenlandic form seen by Fabricius (1780) and named by Peron and Lesueur (1810) was identical to A. limbata, later named by Brandt (1835), and that calling the northern Atlantic American form A. flavidula was a mistake by Agassiz (1862). Agassiz had differentiated the western Atlantic A. "flavidula " from the European A. aurita on the former having a marginal net- work of anastomoses, the gonadal pouches closer together and occupying fully 1/3 of the bell diameter, and differences in the mouth fringes. Kramp further cautioned that using the name A. flavidula would be confusing, so he gave the common American Atlantic form the name A. occidentalis, distinguishing it from A. aurita on the heavier anastomosing of the radial canals; he later lumped it into A. aurita without comment (Kramp, 1961). Proper phylogenetic placement of both the Greenlandic form and the common American Atlantic form must await a revision of the genus Aurelia based on live material. For the Greenlandic form, being yellow and having anastomosed canals seem insufficient for concluding conspecificity with the Alaskan A. limbata. Ideally, conspecificity should be based on numerous characters inherited by common de- scent, not by shared color. The importance of anastomosed canals is discussed below. The American Atlantic form, regardless of its identity, does not possess the enlarged manubrium and related characters of A. labiata: whether it is present along the Pacific coast of North America has not yet been determined. Systematics of Aurelia labiata The common moon jellyfish found in 17 populations from San Diego. California, to Prince William Sound, Alaska, is characterized by the body form described by Chamisso and Eysendardt (1821) for A. labiata. Many of the references to Aurelia of the eastern North Pacific do not contain illustrations or photographs; those that do are most often based on the European morphology. In at least one example, the same photograph is used in both West coast and East coast American field guides (Audubon Society, 1981 ). A large body of literature has thus been responsible for perpetuating the misidentification. The synonymy below contains only the references that have figures or descriptions positively referable to A. labiata sensu Chamisso and SYSTEMATICS AND BIOGEOGRAPHY OF AURELIA HHIATA Eysenhardt, 1 82 1 ; thus, even references to A. labiata are not included below if they do not include the enlarged nianu- brium. The remainder of references to eastern North Pacific Aurelia are dealt with below in appropriate sections. Aurelia labiata Chamisso and Eysenhardt, 1821 (Figs. 2; 3B-D) Aurcllia labiata Chamisso and Eysenhardt, 1821: 358. pi. 28. fig, 1A, B. Mayer. 1910: 622. 628, in part, eastern North Pacific records only. Medusa labiata. Eschscholtz, 1829: 64. Aurelia labiata. de Blainville. 1834: 294. pi. 42. figs. 1. 2 (Cham. & Eysen. illustrations). Lesson, 1843: 377. L. Agassiz, 1862: 160. A. Agassiz. 1865: 43. Haeckel, 1880: 557 (monograph). Fewkes, 1889a: 593 (Point Conception. Monterey; manubrium). Torrey, 1909: 1 1 (coll. by Cham. & Eysen.). Barr and Barr. 1983: 80. text fig. 28 (Field Guide (= FG): AK). Wrobel and Mills, 1998: 55 (FG: Pacific coast). Gershwin. 1999: 993-1000. in part (symmetry variation). Aurelia aurita non Linnaeus 1758. Hauser and Evans, 1978: 21 text photo. 81 (commensal crab). Snively, 1978: 152 text fig., pi. 77 (FG: BC, WA, OR). Gotshall, 1994: 24, fig. 40 (FG). Aurelia sp. Campbell, 1992: 12. 13. Back cover (photographs). Greenberg et ai. 1996: 401-409, in part, text fig 3, 4 (allozymes). Moon jellyfish. Malnig. 1985: 40 (photograph). Stefoff, 1997: 9 (photograph). Holotype. Apparently not extant. Neotype. CASIZ 111024, Monterey Bay. CA, coll. 19 April 1997 by D. Wrobel; gravid female, preserved 25-cm bell diameter (BD), 12-cm manubrium length (ML). Additional preserved material. CAS 20, Farallon Islands. East Landing, coll. 14 Sep 1975 by D.R. Lindberg. CAS 95506, same data as CAS 20. CAS 95507, same data as CAS 20. CAS 81306, Monterey Bay, Pacific Grove, coll. 13 Nov 1990 by N. Greenberg, ca. 15-cm BD, manubr. 6.5 cm. CAS 81307, Monterey Bay, Pacific Grove, coll. 13 Nov 1990 by N. Greenberg, BD ca. 15 cm, ML ca. 6 cm. CAS 86767, 2 specimens, Vancouver Island, Sooke Basin, Roche Cove. coll. 11 Sep. 1990 by N. Greenberg. 14.5-cm BD, 6 cm ML. CAS 81304, Monterey Bay, Pacific Grove, coll. 13 Nov 1990 by N. Greenberg, ca. 13-cm BD, ca. 4-cm ML. CAS 81306, Monterey Bay, Pacific Grove, coll. 13 Nov 1990 by N. Greenberg. CAS 107800, 2 specimens, Monterey (CA), coll. 30 July 1966 by Rofen. CAS 111016 and 1 1 1020, Brentwood Bay, Saanich Inlet, coll. 24 June 1996 by LG. CAS 111017. Point Defiance, Puget Sound, coll. 5 April 1996 by LG. CAS 1 1 1021-1 1 1022, numerous specs, Santa Barbara, coll. 30 Nov 1996 by S. Anderson. CAS 1 1 1023, numerous specs, Marshall dock, Tomales Bay (CA), coll. 30 June 1996 by LG. CAS 111227, Spinnaker Bay, Long Beach (CA). coll. Sep 1995-Jan 1997 by L. Gershwin. In addition, preserved, unregistered specimens were examined from collections at Bodega Marine Labora- tory, Cabrillo Marine Aquarium, Friday Harbor Laboratory, and Santa Barbara Museum of Natural History. Diagnosis. Aurelia with manubrium elongated, wide, pro- truding below the bell margin when viewed laterally. Oral arms shorter than bell radius, attached to base of manu- brium. extending outward to bell margin or bent at 90 angle typically counterclockwise. Bell margin 16-scalloped. with a primary indentation at each of 8 rhopalia and a secondary indentation midway between rhopalia. Older in- dividuals typically with many radial canals arising from each gastro-genital sinus; in some, the outer branches are greatly anastomosed. Embryos and larvae brooded on the manubrium or on stiff, shelf-like manubrial extensions, rarely on the oral arms. Redescription. Medusa. (Based on mature tetramerous individuals.) Bell typically quite flat at rest, in some subhemispherical; older individuals may have raised hump over gonadal region. Diameter at maturity ranging from 100 mm to 450 mm, depending on population. Manubrium fleshy, rigid; rectan- gular, pyramidal, or rounded in side view; variably ruffled at 4 corners; width approximately 1/3 of bell diameter; with stiffened, whorled, perradial mesogleal extensions. Index of manubrium length to bell diameter varying geographically, longest in Oregon (.v = 37.2% 3.6%; n = 10. Newport), shortest in southern California (x = 16.7% 2.6%; n = 7, Spinnaker Bay, Long Beach). Oral arms 4, perradial, straight or curved at 90 angles typically counterclockwise (but occasionally variable), arising from base of manu- brium; length short, reaching approximately to bell margin (thus only 1/3 bell diameter); extending laterally outward against subumbrellar surface of bell. In older cultured indi- viduals, oral arms may hang downward. Size of subgenital ostia varying, encircled by raised mesoglea in some indi- viduals. Interradial and adradial canals typically un- branched; perradial canals branched once, or in large indi- viduals the gastro-genital sinus may overgrow the trifurcation causing the perradial canal to appear un- branched. Eradial canals branched. 4-12 arising from each gastro-genital sinus. Some large specimens have conspicu- ous anastomoses of canals on outer third of bell. Gastro- genital sinuses interradial, 4. but varying from 1 to 8 (per- haps more), in rounded to flattened horseshoe-shaped or heart-shaped rings, with adaxially-pointing free ends. Bell with 16 marginal scallops produced by 8 primary indenta- tions at rhopalia located along the perradial and interradial axes, with secondary indentations between adjacent rhopa- lia. Bell transparent and colorless in juveniles and young adults, becoming milky white, or tinted pinkish, purple, peach, or bluish in older medusae. Color of gonad pale pinkish or brownish in mature females, dark purple in mature males, but often appearing white in males ready to spawn. Plumtlu. Elliptical to elongated; ciliated. Color most of- ten white, but other colors found in certain populations: lavender (Monterey), peach (Saanich Inlet), or yellow-ochre (Spinnaker Bay). Planktonic or benthic locomotion by cili- ary movement. Brooded on manubrium or its whorls. Scvphistoma. Polyps 2-3 mm in height, with oral disk 1 -2 110 L. GERSHWIN mm diameter. Manubrium short, cruciform. Septal funnels conspicuous. Typically with 16 tentacles, alternating shorter and longer: number of tentacles highly varied, often corre- sponding to symmetry of parent medusae, parent polyp, or offspring ephyrae. At Friday Harbor, Washington, and Santa Cruz Island. California, scyphistomae typically with 20 tentacles. Color whitish to pale pinkish-orange. Habit benthic. usually hanging downwards from underside of docks, mussel shells, or rocks. Asexual proliferation by side budding, stolon budding, or podocyst formation. See Chia et al. (1984) for a histological study of the neuromuscular system. Strobila. Ranging from monodisk to polydisk with more than 20 developing ephyrae. Color varying with locality: cinnamon in southern California, buff in Monterey. Polyp remaining flesh-colored or whitish. Strobilation time about 7 days; easily induced with periods of chilling. Ephyra. Diameter 2-3 mm at release. With 8 marginal arms, each with a terminal rhopalium flanked by 2 lappets. Nematocysts scattered over the exumbrellar surface. Num- ber of arms and rhopalia highly varied, not always in correspondence with each other or within a clone. Color same as the strobila: cinnamon or pale butt. Type locality. Monterey Bay, California. Distribution. I have collected A. luhinui from Saanich Inlet, British Columbia, to San Diego, California. To the north, I was able to confirm its presence in Prince William Sound, Alaska, from a videotape; the species has also been photo- graphed at Steamer Bay, in southeast Alaska (Barr and Barr, 1983). Its range may extend southward into the waters off Baja California. Mexico. The species generally occurs in bays and harbors where it is easily collected from jetties and boat slips, but medusae have been observed drifting in open waters off Santa Barbara, California (S. Anderson, Univ. California Santa Barbara, pers. comm., Nov. 1996), near Monterey Bay. California (D. Wrobel. Monterey Bay Aquarium, pers. comm.. Oct. 1996; D. Powell, Monterey Bay Aquarium, pers. comm.. May 1997). off Newport. Oregon (D. Compton, Oregon Coast Aquarium, pers. comm., June 1996). and in Puget Sound (LG. pers. obs., June 1996). The polyps generally strobilate in early spring, and the medusae quickly mature, spawn, and die by mid- summer or early fall. In some years and in some localities, the population of medusae is present throughout the year (Spinnaker Bay, LG. pers. obs.; Monterey. D. Wrobel. pers. comm.). Biogeography Observations of 1 7 populations from San Diego. Califor- nia to Prince William Sound. Alaska have shown that the species can be reliably subdivided into three easily distin- 1 g- 0.35 ! 03 s j! 025 | 1 0.2 o E 015 c 01 c 5 005 n Populations Figure 4. Average manubrium lengths of Japanese Aurelia cf. uuriia and nine populations of A. labiata. Japanese = Aurelia cf. auritu. cultured at Cabrillo Marine Aquarium. Northern morph: Saanich = Saanich Inlet. British Columbia; Pt. Def. = Poulsbo, Washington (cultured at Pt. Defi- ance Aquarium); Seattle = Poulsbo. Washington (cultured at Seattle Aquarium). Central morph: Newport = Newport. Oregon (captive at Oregon Coast Aquarium); Tomales : Tomales Bay, California; Monterey = Monterey. California. Southern morph: Marina = Marina del Rey. California; Spinnaker = Spinnaker Bay. California; Coronado = Coronado Island, California. Between morphotype comparison. ANOVA: F = 42.595. df = 3.5, P = 0.001. guishable geographical morphotypes. Though bell diameter is highly variable with environmental conditions, even among nearby populations (Lucas and Lawes, 1998), ma- nubrium length, expressed as a percentage of bell diameter, differs significantly among the three forms (Fig. 4, ANOVA; F == 42.595, df = 3,5. P = 0.001). These three forms are easily distinguished as follows (summary in Table 1 ). Following the synopsis of each form is a list of literature that pertains to Aurelia from the region, but contains insuf- ficient information for positive determination. Southernmost form (Fig. 3B). Manubrium a wide, rounded frilly mound, not distinctly pyramidal. Radial ca- nals few to many, possibly dependent on age; adradials particularly wide in San Diego medusae. Oral arms typically straight, not curved. Planulae ranging in color from white to ochre to bright orange, brooded in a reticulating pattern on frills of manubrium. Bell colorless to milky whitish; some individuals with dark purple tentacles. Male gonads dark purple, female gonads pale pink. Typical maximum size, 35 cm. Marina del Rey medusae with pronounced rhopalial hoods set up off the margin. Known range. California, from San Diego to Marina del Rey. possibly extending north to Ventura and south into Baja California. Populations are apparently isolated and discontinuous; not observed at Oceanside. Dana Point. Los Angeles Harbor, or Malibu. Reported at Catalina Island. Local residents at Ventura Harbor and Channel Islands Harbor tell of seeing an occasional medusa or two; it is currently unclear if they are this form. Typically occurring until late spring, occasionally into autumn. SYSTEMATICS AND BIOGEOGRAPHY OF AURELIA LABIATA Table I Comparison ofmorphotype characters, Aurelia labiata Character Northern morph Central morph Southern morph Manuhrium length (x) (as % hell diam) 22.98% 37.15% 16.73% SD 0.04% 0.04% 0.03% /j 26 10 7 site Poulsbo/Saanich Newport Spinnaker Bay Manuhrium shape pyramidal long and tapered rounded Oral arm length 1/3 bell diam 1/3+ hell diam 1/3 hell diam Oral arm shape straight counterclockwise straight # canals per sinus 7-9 7-15 5-7 Anastomosing heavy very heavy moderate Bell size to 12 cm to 45 cm to 25 cm Bell color whitish or peach purple, pink, or white whitish Planula color white or peach white or purple white, ochre, or orange Literature. Aurelia aurila, MacGinitie and MacGinitie, 1949: 131, text fig. 32 (growth, strobilation, Newport Bay). MacGinitie and MacGinitie, 1968: 131. text fig. 32 (growth, strobilation, Newport Bay). Reish, 1972: 25. text fig. 26 (FG: Southern CA). Allen, 1976: 22. 75 (FG: Southern CA). Reish. 1995: 38, fig. 31 (FG: Southern CA). Central fonn (Fig. 3C>. Manubrium extremely elongated, rectangular and tapering. Canals numerous, typically heavily anastomosed in largest individuals. Oral arms straight or bent counterclockwise. Planulae distinctly laven- der, brooded in teardrop-shaped clumps on the base of manubrium or on shelves. Scyphistomae pale buff colored. Medusae from Monterey, California tending to be distinctly purple; Santa Barbara, California, medusae often pale pink. Gonads dark purple in males, pale brown in females. Di- ameter of captive medusae from Newport, Oregon, recorded to 45 cm, with longest manubrium being 17 cm! Known range. Santa Barbara (including Channel Islands), California to Newport, Oregon. Likely occurring, but un- confirmed, along the outer coast of southern Washington state. Abundant in late summer. Literature. Aurelia labiata. Fewkes, 1889b: 122 (Santa Barbara Channel; pink). Boyd, 1972 (fouling organism; Bodega Harbor, CA). Pearcy, 1972: 354 (Oregon). Hand, 1975: 95 (FG: Central CA). Aurelia aitrita. ?Galigher, 1925: 94 (scyphistomae; Monterey. CA). Hamner and Jenssen, 1974:833-848. text fig. 1 (growth and degrowth. Tomales Bay, CA). Shenker. 1984: 619-630 (abundance; OR). Abbott. 1987: 28 (morphology; Monterey). Keen and Gong, 1989: 735-744 (scy- phistoma clonal growth; Tomales Bay, CA). Niesen. 1997: 43 (FG: Northern CA). Rigsby. 1997: 207 (Monterey Bay). Aurellia labiata. Light ct ai. 1954: 41 (FG: central CA). Aure/lia aurira. Hedgpeth, 1962: 52, text fig. B (FG: Northern CA). Aurelia sp. Gottshall et a/.. 1965: 149 (prey of blue rockfish; Bodega. Monterey, Morro Bay). Pereyra and Alton. 1972: 448 (near Columbia River. OR). Northernmost form (Fig. 3D). Manubrium low. pyrami- dal. Many parallel radial canals in mature individuals, giv- ing a lacy appearance to the bell. Oral arms more or less straight, but may be variable in the same individual in Departure Bay specimens (M. Arai, Pacific Biological Sta- tion. Nanaimo, BC. pers. comm. 2000). Planulae variably colored; brooded at the base of the manubrium and on manubrial shelves. Overall coloration peach or whitish, with gonads dark purple in males, pale brown in females. At Poulsbo, Washington, maximum diameter approximately 12 cm; brooded planulae white, appearing as a wash or haze rather than in discrete bundles. At Saanich Inlet. British Columbia, medusae larger, to approximately 15-cm diame- ter during my study, but reported to range from 14-29-cm (Hamner ct ///- chella in Indonesia, and suggested the presence of a sort of "marine Wallace's line." Even though the stomatopod lar- vae are planktonic, and thus have the means to disperse over great distances, it appears that they do not. Whether the same explanation can be applied to Anrelia remains to be shown. Because so much of the coastline is hospitable to A. labiata, it is helpful to ask whether other similar species may be present as well. Currently there is no evidence of endemic species other than A. labiata, excepting the unre- solved nomenclatural questions relating to A. limbata. How- ever, it is easy to imagine that other forms may have been overlooked in a similar way as A. labiata. or that within the species I herein recognize as A. labiata. numerous cryptic species exist. The recent scientific literature abounds with discoveries of cryptic species, such as one recent startling example, wherein the fungal Gibberella fiijiknroi species complex was found to comprise 45 phylogenetic species (O'Donnell et al.. 1998)! Given that many of the popula- tions of A. labiata along the eastern North Pacific coast are uniquely diagnosable. and that these diagnosable forms partition into the three latitudinal morphotypes, the possi- bility of cryptic species seems high. Indeed, Greenberg et al. (1996) hypothesized restricted gene flow between eastern Pacific populations, based on significant allele frequency differences. Thus, the biogeographic pattern in A. labiata may represent cladogenesis in action, or possibly even a splitting event of the recent past. I hesitate at this time to recognize the three forms as distinct species, or for that matter to assign the eastern North Pacific forms to a new genus, although it is clear that the three forms are quite different from one another and from A. anrita. Although scyphozoan population genetics have not yet been studied in depth, some cnidarians have surprisingly low rates of ge- netic divergence (see Knowlton. 2000), so species conclu- sions should be made cautiously. Thus, until the clade currently known as A. anrita is resolved, it is difficult to comment with confidence on the internal and external rela- tionships of the morphotypes of A. labiata. However, this does beg the questions of species concept and species rec- ognition criteria. Taxonomic characters Throughout most of the twentieth century, it was custom- ary to recognize medusan taxa based on certain key char- acters, reeardless of distribution and discrete forms of vari- ation; that is, all populations possessing a small number of aiven characters were thought to be one species. For exam- ple, in the Pelagiidae. the character of tentacle number has been so highly regarded that a large and conspicuous spe- 114 L. GERSHWIN cies was incorrectly classified, favoring a tentacle number over all other characters combined (Gershwin and Collins, 2001 ). The same reasoning seems to have applied to Aiire- lia, favoring the "essence" of A. aurita over all other char- acters. This appears to have resulted in excessive lumping for many taxa. In contrast, I have employed a phylogenetic perspective, bringing together data from morphology, ge- ography, and genetics to evaluate a lineage's history. How- ever, some characters are still worthy of further comment. as they have led to confusion in the past. Perhaps the most ignored character is the best key in separating A. labiata from A. aurita. Greenberg et al. ( 1996) used manubrium length in distinguishing the American form from the Japanese form, but failed to notice the asso- ciated changes in the relationship of the oral arms to each other and the altered brooding habits (Figs. 1, 2. 3B-D). To summarize, in A. labiata the oral arms are relatively short, about one-third the bell diameter, and project outward from the base of the fleshy manubrium. In addition, the larvae are brooded on the manubrium or on the rigid manubrial shelves. In contrast. A. aurita lacks the fleshy manubrium; consequently, the oral arms meet at the mouth and are about one-half the bell diameter. Furthermore, the brood pouches for the larvae line the upper portions of the oral arms. Thus, the large manubrium of A. labiata relates to a suite of morphological and functional differences from A. aurita. Kramp (1913) considered the anastomosed canals to be a distinctive character in separating the Greenlandic form of A. (inritu (as A. flavidula) from the typical form, and most descriptions of A. limbata include this character. However, the canals of some captive medusae of both A. labiata and A. "aurita" eventually become heavily anastomosed (F. Sommer, Monterey Bay Aquarium, pers. comm., and my own unpublished observations), possibly attributable to the phenomenon of growth and degrowth (Hamner and Jenssen, 1974). This was not taken into consideration by Greenberg et al. ( 1996). in claiming that the anastomoses could be used as a reliable character for distinguishing eastern Pacific Aurelia from western Pacific Aurelia. Indeed, their North American medusae were held captive nearly a year, whereas their Asian medusae were held only for 2 months. Although this character does seem more conspicuous in large speci- mens of A. lahiata than in A. "aurita, " this may be due to the increased number of canals in A. labiata: that is, many canals anastomosing may give the appearance of a finer mesh than one would expect in an individual with fewer canals. This too (extra canals) was not taken into account by Greenberg et al. (1996). A closer study of anastomosis of canals might be helpful in future taxonomic studies. Some authors have reported that the number of canals arising from the gastro-gonadal sinuses is taxonomically unreliable because it is associated with size and rate of growth (Stiasny, 1922; Bigelow, 1938; Kramp, 1942, 1965; Russell. 1970). Indeed. I have observed that older, larger individuals do tend to have more canals than smaller, younger individuals. However, old. large A. aurita typically have 1 or 2 eradial canals arising in each space between interradial and adradial canals (for a total of 5-7 canals arising from each gonad). whereas old, large A. labiata typically have 3-6 eradials per side (for a total of 9-15 total per gonad). However, in the closely related A. limbata, Stiasny (1922) and Bigelow (1938) argued that the number of radial canals and the degree of branching are both useful characters. Curiously, medusae of the northern and central forms tend to possess greater numbers of radial canals than do medusae of the southern form. The taxonomic significance of the 16-scalloped bell mar- gin is currently unclear. Medusae from all endemic eastern North Pacific populations that I have observed possess this scalloping, in some cases quite conspicuously so. However, use of this character to distinguish species has been criti- cized by Kramp (1965). citing that in A. limbata the sec- ondary scalloping is lost in preservation, and agreeing with Bigelow (1913) that the degree of scalloping is merely due to contraction of the bell. Because of its occasional occur- rence in A. aurita. the secondary scalloping should not be used as the distinguishing taxonomic character of A. labiata as has been done in the past. However, it remains one of several useful field characters for A. labiata and may prove useful in similarly distinguishing other species worldwide. Confusion has arisen regarding certain specimens from Nanaimo. British Columbia. Stiasny (1922) and van der Maaden (1939) assigned them to A. limbata: whereas Kramp ( 1942) identified them as a variety of A. aurita based on the width of their radial canals. I have not yet examined these specimens. However, Stiasny's (1922) description is consistent with A. labiata, namely, the 16-scalloped margin and the 5-9 radial canals issuing from each gastrovascular sinus. At present. A. labiata appears to be a temperate endemic restricted to the eastern North Pacific. However, this leaves a series of references to medusae with 16 marginal scallops as A. labiata, although their morphological characteristics and geographic locations suggest that they are not. Avail- able drawings and a photograph all clearly show 16 scallops of the margin, but do not show a protruding manubrium or numerous radial canals (Mayer. 1910. 1917; Uchida, 1928). Since the illustrations of Chamisso and Eysenhardt (1821) indicate a large manubrium. I exclude medusae that lack this character from this classification. However, I have not ex- amined specimens from the following sources for complete diagnostic characteristics. Aiiivlliu /<;/>/<<;. Mayer 1910: 628, fig. 398 (A. limbata as var. of A. Uihiutii; Philippines). Light, 1914a: 294 (harmless); Philippines). Lite, I914b: 200 (Philippines). Mayer. 1915: 160, 1S2 (A. labiata derived from A. aurita). Mayer. 1917: 205. text fig. 11 (Philippines and Tortugas. Flor- ida). Light, 1921: 31 (Philippines). Bigelow. 1938: 167 (synonymous with A. aurita). Aurelia labiata. Stiasny, 1919: 93 (Malay Archipelago). Stiasny. SYSTEMATICS AND BIOGEOGRAPHY OF AURELIA IARIATA 115 1926: 244 (Philippines; ,4. labiata is a variety of A. aiirita). Uchida 1928: 373-376 (pentamerous. Palau). Stiasny. 1931: 140 (-specimen a( British Museum). Stiasny. 1935: 34 (Aroe Islands). Stiasny. 1937: 207 (East Indies). Ranson, 1945: 60. 61 (review of genus). Kramp 1961: 340 (taxonomy). Kramp. 1965: 262-263. plate 1 rig. 1 (A. labiata same as A. auriun. Kramp 1968: 68 (discusses A. labiata). Russell 1970: 140 (discussion of synonymy). Powell, 1975: 6 (New Zealand I. Two reports of A. labiata in Hawaii (Chu and Cutress. 1954: 9; Devaney and Eldredge. 1977: 1 1 1) are worthy of attention. Drawings I made in 1993 from live animals in the Waikiki Aquarium appear to be of A. luhiutii. However, preserved specimens from the same location examined in 1997 lacked the enlarged manubrium. At this time, I pro- visionally include Hawaiian Aurelia with A. labiata. but firm determination must wait until additional live and pre- served material can be examined. The Oahuan form appears to be introduced, as it was not reported until 1954, but the origin of the introduction is not yet known (J. T. Carlton, Mystic Seaport. Mystic. CT, and L. G. Eldredge. B. P. Bishop Museum. Honolulu. HI, pers. cornm.). Thus far, little consensus exists over what characters are taxonomically reliable for jellyfishes over a wide range of populations. To further confound the problem, immature specimens of closely related species often bear a striking resemblance. However, recent rearing of Japanese Aurelia "aiirita" and Monterey A. labiata in the same aquarium yielded distinctive morphs consistent with the two species (M. Schaadt, Cabrillo Marine Aquarium. San Pedro, CA, pers. comm., Oct. 1999). Although I have herein distin- guished only the northern, central, and southern morphs, medusae from each of the 1 1 locations were easily identi- fiable. The ability to distinguish morphological characteris- tics associated with particular populations of Aurelia spp. will not only help to resolve the phylogeny of the group, but may also help in identifying the origins of introductions such as those in Spinnaker Bay, California; San Francisco Bay. California (Greenberg el al, 1996); and Oahu, Hawaii (J.T. Carlton and L.G. Eldredge, pers. comm., 1998). Field key to the eastern North Pacific forms of Aurelia 1. Bell lacking secondary notches between adjacent rho- palia. margin 8-scalloped. Lacking broad and/or elongated manubrium. Currently known only from South San Fran- cisco Bay and Spinnaker Bay cf. A. aitrila 1 '. Bell with secondary notches between adjacent rhopa- lia, appearing 16-scalloped. Possessing conspicuously broad and/or elongated manubrium 2 2. Bell with conspicuous chocolate-brown margin. Pri- marily Arctic A. liiiibuta 2'. Bell lacking brown margin 3 3. Manubrium greatly elongated, tapering rectangular in shape. Generally found Pt. Conception. CA, to northern Oregon. Color variable from white to purple to pink. Often very large, to 45 -cm or more . . . A. labiata. central morph 3'. With manubrium protruding in lateral view, but much less than one-third bell diameter 4 4. Manubrium pyramidal. Generally found in and north of Puget Sound. Color variable from white to peach. Typ- ically small. 12-15 cm A. labiata. northern morph 4'. Manubrium rounded. Generally found south of Pt. Conception. Color typically milky white, occasionally with dark tentacles A. labiata, southern morph Acknowledgments I thank the staff and volunteers of the Cabrillo Marine Aquarium for unwavering encouragement, Susan Gershwin and Norma Kobzina for tracking down obscure references, Richard Harbison for translation of Chamisso and Eysen- hardt (1821). Eric Hochberg for valuable museum and manuscript assistance, Claudia Mills and Allen Collins for stimulating discussions and help in a multitude of ways, Freya Sommer for sharing her knowledge and passion for jellyfishes, Gary Williams for his artwork and taxonomic guidance, Dave Wrobel for the beautiful photograph repro- duced in Figure 2, the countless friends and colleagues who provided valuable suggestions on previous versions of the manuscript. Sincerest thanks to Mary Arai for providing assistance beyond the normal standard for review, and to an anonymous reviewer for additional helpful comments. In addition. I am indebted to the following people and institu- tions for help in obtaining specimens and information (in alphabetical order): Leslee Yasukochi and Eric Johnson at Birch Aquarium at Scripps; Jim Ulcickas at the Bluewater Grill. Newport Beach. California; Cadet Hand and staff at Bodega Marine Lab; Chris Mah at California Academy of Sciences; researchers and students at Friday Harbor Labs; Freya Sommer. Dave Wrobel, Dave Powell, and Ed Seidel at Monterey Bay Aquarium; Dave Compton and Polly Delle at Oregon Coast Aquarium; researchers and staff at Oregon Institute of Marine Biology; John Carlyle at Point Defiance Zoo and Aquarium; Yogi and Kathy Carolsfeld at Saanich Inlet; Erin Johnston and Shaun Larson at Seattle Aquarium; Spinnaker Bay and Spinnaker Cove homeowners; Thomas Shirley and Jennifer Boldt at University of Alaska; Shane Anderson at UC Santa Barbara; Rossi Marx at University of Victoria; and Joyce and Stuart Welch at Tomales Bay. 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Co-Vice Chair John E. Dowling. President of the Corporation John E. Burris, Director and Chief Executive Officer William T. Speck, Interim Director and Chief Executive Officer Mary B. Conrad, Treasurer Robert E. Mainer, Clerk of the Corporation Contents Report of the Director and CEO Rl Report of the Treasurer R6 Financial Statements R7 Report of the Library Director R18 Educational Programs Summer Courses R20 Special Topics Courses R24 Other Programs R32 Summer Research Programs Principal Investigators R35 Other Research Personnel R36 Library Readers R37 Institutions Represented R38 Year-Round Research Programs R43 Honors R57 Board of Trustees and Committees R64 Administrative Support Staff R68 Members of the Corporation Life Members R71 Members R72 Associate Members R83 Certificate of Organization R86 Articles of Amendment R86 Bylaws R86 Publications . . R91 Photo credits: E. Armstrong R3 (bottom), R4 (top), R20, R21, R24, R27, R35, R47, R55 K. Begos R38 D. Buffam R2 (bottom) M. Dornblaser R68 J. Dowling R30 L. Colder R64 Gray Museum of the Marine Biological Laboratory R57 R. Hanlon R43 R. Howard R4 (bottom), R18 A. Kuzirian R6 B. Liles R71 H. Luther R23. R46 J. Montgomery R2 (top) P. Presley Rl A. Rader R86 Report of the Director and Chief Executive Officer It is with great pleasure that I write this report as the Marine Biological Laboratory's newest Director and Chief Executive Officer. My relationship with the MBL has grown and expanded in rewarding and exciting ways during the past twenty-five years. I am now pleased to have the opportunity to serve as Director of this esteemed Laboratory. I first came to the MBL as a student and then returned as an investigator for several summers. My role expanded when I was elected to the Laboratory's Board of Trustees in 1994. and again when I joined the Discovery Campaign Steering Committee. In 1999. I succeeded Mel Cunningham as Chair of the Development Committee. Since being appointed Interim Director upon John Burris's departure in the summer of 2000, I've had a wonderful opportunity to view the inner workings of this remarkable institution. I think it's fair to say that the Marine Biological Laboratory is stronger and healthier both financially and programmatically than it has ever been in its history. In this report, I'll review what has led us to this point, share with you some highlights from the year 2000, and discuss where the Trustees and I see the Laboratory going in the next few years. The Discovery Campaign The Marine Biological Laboratory concluded its first comprehensive fundraising campaign Discovery: The Campaign for Science at the Marine Biological Laboratory in December 2000. Our goal was to raise $25 million for a variety of initiatives at the MBL. When we began planning for the campaign, some felt that this goal was a stretch for the institution. Thanks to the generosity of thousands of Trustees. Corporation Members, Associates, Alumni, Staff Members, Foundations, and Friends of the Laboratory, the MBL far surpassed that goal, raising more than $41 million by the end of the year 2000 in support of research, education, the library and physical plant, and the annual fund. Funds raised through the Discovery Campaign have already had a major impact on the Laboratory's educational and research programs. One of the most obvious achievements of the Campaign is the construction of the C. V. Starr Environmental Sciences Building, which will become the new home of The Ecosystems Center in 2001. Thanks to the Campaign we also established the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution and hired two new assistant scientists there (Michael Cummings and Jennifer Wernegreen); added five new summer courses and the Semester in Environmental Sciences Program for undergraduates to our education roster; created more than a dozen endowed scholarships for students and endowed fellowships for young researchers; established a program in scientific aquaculture in the Marine Resources Center; endowed the director's chair of the Marine Resources Center; and expanded our public outreach efforts through the creation of the Robert W. Pierce Visitors Center. In addition, we raised funds to support endowed lectureships for the summer courses and an annual lecture in Bioethics starting in the summer of 2001, and to help shore up the Laboratory's aging physical plant. Moreover, we received gifts to permanently endow the maintenance of the Waterfront Park and the Pierce Visitors Center. Finally, thanks to gifts to the Discovery Campaign, the Library has been air-conditioned and the Crane House on Millfield Street has been refurbished and added to our year-round housing inventory. Physical Plant We've also been able to tackle some other long- overdue maintenance projects on campus. For example, the crumbling section of seawall near the Lillie Building has been reconstructed. By the summer of 2001, the Brick Dormitory will have been renovated and furnished for year-round use. Cottages at Memorial Circle have been updated and de-leaded, and we have begun renovations at Devils Lane. The research laboratories in the Lillie Building are being renovated to accommodate expanding Rl R2 Annual Report year-round research programs in the Bay Paul Center, BioCurrents Research Center, and Architectural Dynamics in Living Cells Program. We've also added fresh paint and carpeting to the Meigs Room, and have begun painting and replacing lighting and other fixtures throughout the Swope Building. Our plans also include renovating summer research laboratories in the Whitman Building. We expect to begin modestly renovating the Homestead building, which, once vacated by the staff of The Ecosystems Center, will eventually become home to the administrative offices of Financial Services. Education, Human Resources, and The Biological Bulletin. The Biological Bulletin The Marine Biological Laboratory's journal, The Biological Bulletin, celebrated a major milestone in 2000. Edited by Michael J. Greenberg of the University of Florida's Whitney Laboratory, the journal has been publishing peer-reviewed articles of general biological interest for more than 100 years. During the summer of 2001 the journal will launch a new initiative by publishing articles electronically with HighWire Press of Stanford University. Education During the summer of 2000, the MBL's Educational Program offered a record 22 summer and special topics courses. Three hundred and thirty-five course directors and faculty members taught 490 advanced graduate and postdoctoral students in the courses last summer. An additional 315 guest lecturers and instructors participated in the courses as well. From all accounts, the quality of our students improves every year. We offered a symposium on the history of biology and a workshop in microbial diversity designed for middle and high school teachers. Last summer brought quite a few undergraduates to the MBL as well, through a variety of Research Experience for Undergraduate Programs. One program focused on Marine Models, another was coordinated by the Boston University Marine Program. and others were offered by the Marine Resources and Ecosystems Centers. I'm pleased to report that funding has been allocated for two additional research programs for undergraduates beginning in summer 2001. The MBL's own semester-long undergraduate program. The Semester in Environmental Sciences, offered by the staff of The Ecosystems Center, completed its 3 rd year in 2000 with 1 5 students participating. The consortium of colleges whose students come for the fall semester continues to grow, currently numbering more than 40 members. Research The summer research program ran at full capacity during the summer of 2000. One hundred and thirty-two investigators used all of our available lab space. In fact, one applicant had to set up his research in a dark room. The majority of the investigators (60%) were professors/ chief scientists, followed by associate professors (20%) and postdoctoral fellows (10%). The balance was comprised of assistant scientists and graduate students. I'm proud to report that for the second year in a row an MBL Summer Scientist Avram Hershko of the Technion in Israel has won the prestigious Lasker Award (Clay Armstrong won this award in 1999). This award is second only to the Nobel Prize in significance in science. Dr. Hershko will deliver a Friday Evening Lecture during the summer of 2001. I'm also pleased to be able to count two of the year 2000' s Nobel Prize winners as members of the MBL family: Paul Greengard of Rockefeller University, an alumnus of the Embryology Course and a former faculty member of the Neurobiology Course, and Eric Kandel of Columbia, a past MBL Report of the Director and CEO R3 investigator and Corporation Member. These awards validate the tremendous significance and impact the MBL's research and educational programs have on the biology community at large. The MBL's research fellowship program hosted 21 investigators during the summer of 2000. The range of the research being undertaken by these scientists was remarkable, and the caliber of their backgrounds scored high by the Fellowship Committee and our external advisors. The Science Writing Fellowship Program also continued to figure prominently among print and broadcast journalists for the outstanding opportunity it affords them to work alongside scientists to learn about the process of doing science. The Ecosystems Center Research is and will always be a key mission of the MBL. We have seen a continued growth in our resident research programs. The Ecosystems Center, directed by Jerry Melillo and John Hobbie, now numbers more than 60 staff, and its funding base has more than doubled during the past 5 years. It is now is in excess of $7 million. Thirty research projects are underway around the globe, from Siberia to Martha's Vineyard. In 2000 The Ecosystems Center celebrated its 25 th anniversary with a weekend-long celebration. The festivities included an open house, a one-day symposium complete with a visit by Rep. William Delahunt of the Massachusetts 10 lh District, and a reunion clambake at the Swope Center. More than 50 Ecosystems Center alumni from all over the world traveled to Woods Hole to celebrate the success of the Center's first 25 years and to discuss the future of ecosystems science. The Josephine Bay Paul Center The Bay Paul Center for Comparative Molecular Biology and Evolution, under Mitch Sogin's direction. currently has 33 scientists and support staff. The Center's project to sequence the genome of the parasite Giardia is nearly complete. For the first time, the MBL has received a prestigious gift from the Keck Foundation. This $1 million award will establish the W. M. Keck Ecological and Evolutionary Genetics Facility at the Bay Paul Center. Microbial ecologists, molecular evolutionists, and genome scientists from the Bay Paul Center, The Ecosystems Center, and other scientific groups within the Woods Hole community will form a coalition to study how the genes of millions of microbes work together to influence biogeochemical processes within ecosystems. The BioCurrents Research Center The NIH BioCurrents Research Center, directed by Peter Smith, has increased in size and now numbers 1 1 scientists, thanks to the recent addition of Drs. Orian Shirihai and Stefan McDonough to the scientific staff. Among their many research projects. Smith and his colleagues continue to collaborate with Dr. Barbara Corkey of Boston Medical Center on the study of how cells process insulin. They are currently fine tuning instruments that will enable them to monitor the movement and release of glucose, insulin, and calcium within pancreatic beta cells, the goal being to learn more about how diabetes type II works at a cellular level. Another exciting collaboration is underway between the BioCurrents Research Center and the Bay Paul Center to study the evolution, diversity, and physiology of organisms living in extreme environments like the hot vents of the deep oceans and extremely acidic (battery acid-like) ecosystems. The Marine Resources Center Research using DNA fingerprinting to assess paternity and reproductive patterns and population structure in the R4 Annual Report local squid fishery valued at $33 million annually continues in the Marine Resources Center (MRC). under the direction of Roger Hanlon. Work on how polarized vision is used by the squid to help detect prey is also a focal point. During the Campaign, a landmark gift from Honorary Trustee Ellen Grass established the first endowed Directorship at the MBL. This gift, the grant from the Schooner Foundation to establish the Program in Scientific Aquaculture, and a recent anonymous grant of $500,000 ensures future vitality for the MRC. The MRC is also currently in the process of hiring three faculty- level scientists and a scientific aquaculturist. I've only touched on a few of the MBL's resident research initiatives. In addition to these research centers, the MBL is home to a score of investigators' research programs that focus on a range of topics including infertility, microscopy, learning and memory, and the effects of lead poisoning on children. Trustees will start developing a 5- to 10-year strategic plan a map charting the direction that the Laboratory will take in both research and education in the coming years. This plan will further strengthen and position the Laboratory to serve science and society. As we continue to build the year-round research programs, plans have been developed to add a new year- round research program in Global Infectious Diseases and Parasitism. Parasites cause debilitating and often lethal diseases in billions of people around the world. The World Health Organization estimates that one in ten are infected by one or more of the five major parasitic diseases: schistosomiasis, filariasis, malaria, trypanosomiasis, and leishmaniasis. The MBL is already a leader in the field of parasitology and infectious disease, hosting two major international parasitology meetings and offering a world-renowned course in the Biology of Parasitism each summer. This new program will build on the Laboratory's existing strengths in this field and take advantage of the high throughput technologies and scientific expertise available in the Bay Paul Center, creating a one-of-a-kind research environment that fosters interactions between parasitologists and experts in molecular biology, phylogenetics, and environmental microbiology. The Trustees agree that this is a strong and important addition to the MBL's year-round research portfolio. On the education side, Mitch Sogin and Clare Eraser, one of our newest Trustees, are planning to offer an exciting and novel course in genomics. This course will premiere in Fall 2002. We hope to offer more and more cutting-edge courses throughout the year in the future. The Library The MBLAVHOI Library continues to expand both its print and electronic serial collections. More than 2000 full-text electronic journals are now available on our scientists' desktops through the Library's web site. The entire collection has grown to more than 200,000 volumes, occupying all the space the Library has available in Woods Hole. Storage issues are currently being addressed by providing more electronic access to journals and by sending some volumes off campus to the Harvard Depository. Looking Ahead It's an exciting time for the Marine Biological Laboratory. Now more than ever, the Trustees are committed to building and strengthening the MBL's year- round research program. Within the next year, the Trustees The Trustees elected four new Board members and reappointed one Trustee to the Class of 2005 at their November 4, 2000 meeting. Dr. Porter W. Anderson, who completed his first term on the Board this year, was Report of the Director and CEO R5 appointed to a second term. He is joined by Dr. Claire M. Fraser, President and Director of The Institute for Genomic Research in Maryland; Mr. George Logan, Chairman of the Board and Organizer of the Valley Financial Corporation as well as Principal of the Wood Park Capital Corporation in Roanoke, VA; Robert A. Prendergast, Professor of Ophthalmology and Associate Professor of Pathology at The Wilmer Institute at The Johns Hopkins University School of Medicine, Baltimore, MD; and John W. Rowe, M.D., President and CEO of Aetna Inc. Thomas S. Crane, Co- ordinator of Mintz Levin Cohn Ferris Glovsky and Popeo's Health Care Fraud and Abuse and Corporate Compliance practice group serving the firm's Boston and Washington, DC, offices, was elected Clerk of the Corporation. Sheldon Segal, John Dowling, and Mary B. Conrad were reelected to serve as Chairman of the Board, President of the Corporation, and Treasurer, respectively. Trustee Al Zeien was elected Vice Chair of the Board. The Board also thanked retiring members Fred Bay, Marty Cox, Mary Greer, William Steere. and Gerald Weissmann for their tireless efforts on behalf of the Laboratory. In Memoriam As this report was going to press, we were saddened to learn of the tragic deaths of Jim and Alma Ebert, who were killed on May 22, 2001, in a car accident while traveling from Baltimore to Woods Hole for the summer. Jim was President of the MBL Corporation from 1970 to 1978 and again from 1990 to 1998. He was Director of the Laboratory from 1970 to 1978, a Trustee from 1964 to 1968, and was named Director Emeritus in 2000. Alma was active in the MBL Associates, volunteering her time and energy on behalf of the Laboratory, and supporting Jim during his tenure as Director. For five decades the MBL has benefited from Jim's considerable knowledge and experience. He was instrumental in bringing significant funding to the Laboratory, and his guidance and insight were key to the MBL's success. The loss of these dear friends will be deeply felt by the MBL family for many years. William T. Speck : ., Report of the Treasurer The Marine Biological Laboratory had another impressive operating year in 2000 that was partially offset by weak near-term investment portfolio returns. Auspicious growth in Operating Support and the decline in the Equity Markets were the major contributors to the mixed results. Three areas of Operating Support showed double-digit increases. The growth in Government Grants accelerated to 14.7% over 1999 results and represented an all-time high of 45.2% of Total Operating Support. Fees for Conferences and Services grew even faster, up 17.1%. Short-term Investment Income also grew by 13.1% as a result of stronger interest rate returns on a larger portfolio of Cash & Cash Equivalents, Short-Term Investments, and the Assets Held by the Bond Trustee. This had a very favorable impact, particularly on the Change in Unrestricted Net Assets from Operations. It increased from only $138 thousand in 1999 to $1.3 million in 2000. This represented a very strong 9.5% Operating Margin. Reviewing our Non-Operating Activities, we expanded our Investment in Plant to $4.64 million, more than doubling what was done in 1999. Total Contributions, again, exceeded $10 million in the final year of our Discovery Campaign with almost 45% going toward Plant improvements. On the other hand, MBL experienced $2.1 million, or 3.9%, in realized and unrealized investment losses. We also utilized $1.4 million from our standard spending rate draw. This impacted our Long-term Investment portfolio, which fell slightly in value for the first time since 1994. Even with this, MBL reported a $3.2 million Total Change in Net Assets. This represented the sixth year of positive change, but represented only a 4.3% Return on Average Net Assets. MBL's 2000 Balance Sheet experienced some significant changes from 1999. Assets grew by over $1 1 million due to double-digit growth of 16.4% in Net Plant Assets, increased liquidity, and added Assets held by the Bond Trustee, which was a result of the $10.2 million Variable Rate Revenue Bonds issued March 8, 2000. The Bond refinanced $2.3 million of higher cost debt, with the balance of the proceeds being used to make capital improvements to MBL's educational, research, and housing facilities. Even with this increased debt, MBL has a sound Leverage Ratio (Unrestricted and Temporarily Restricted Net Assets-to-Debt) of 5.26X at year-end 2000. Also note our strong operational returns resulted in an improved Debt Coverage Ratio of 11. 6X over previous years. One last positive sign to note is a $3 million increase in the Laboratory's Unrestricted Net Assets. In summary, the Laboratory completed an effective leverage of its financial strength, closed a very successful fundraising campaign, and demonstrated strong operational returns. This more than offset the marginal decline in portfolio performance, and we remain well poised to continue our capital improvement efforts. Mary B. Conrad R6 Financial Statements PricewaterhouseCoopers LLP One International Place Boston MA 021 10 Telephone (f>17) 478 5000 C.\. simile ((,17) 478 3900 REPORT OF INDEPENDENT ACCOUNTANTS To the Board of Trustees of Marine Biological Laboratory: In our opinion, the accompanying balance sheet of Marine Biological Laboratory (the "Laboratory") at December 3 1 , 2000 and the related statements of activities and of cash flows for the year then ended present fairly, in all material respects, the financial position of the Laboratory as of December 31, 2000, and the changes in its net assets and its cash flows for the year then ended in conformity with accounting principles generally accepted in the United States of America. These financial statements are the responsibility of the Laboratory's management; our responsibility is to express an opinion on these financial statements based on our audit. The prior year summarized comparative information has been derived from the Laboratory's 1999 financial statements, and in our report dated April 7, 2000, we expressed an unqualified opinion on those financial statements. We conducted our audit in accordance with auditing standards generally accepted in the United States of America. Those standards require that we plan and perform the audit to obtain reasonable assurance about whether the financial statements are free of material misstatement. An audit includes examining, on a test basis, evidence supporting the amounts and disclosures in the financial statements. An audit also includes assessing the accounting principles used and significant estimates made by management, as well evaluating the overall financial statement presentation. We believe that our audit provides a reasonable basis for our opinion. Our audit was conducted for the purpose of forming an opinion on the basic financial statements taken as a whole. The supplemental schedule of functional expenses as of December 3 1 , 2000 is presented for the purpose of additional analysis and is not a required part of the basic financial statements. Such information has been subjected to the auditing procedures applied in the audit of the basic financial statements and, in our opinion, is fairly stated, in all material respects, in relation to the basic financial statements taken as a whole. April 6. 2001 R7 MARINE BIOLOGICAL LABORATORY BALANCE SHEET As of December 31, 2000 (With Comparative Totals as of December 31. 1999) ASSETS 2000 1999 Cash and cash equivalents $ 3,583,033 $ 1 ,942,285 Short-term investments, at market 3,599,833 3,182,537 Accounts receivable, net of allowance for doubtful accounts of $47,222 in 2000 and $59,978 in 1999 1.109,706 1,158,073 Current portion of pledges receivable 5,026,750 3,974,385 Receivables due for costs incurred on grants and contracts 2,036,734 1,380,766 Other current assets 352.983 306,5 1 8 Total current assets 15.709.039 11,944,564 Assets held by bond trustee 5,423,615 Long-term investments, at market 44,494.649 45,001,493 Pledges receivable, net of current portion 2,433,292 3,498,787 Plantassets.net 23,423,156 20,118,725 Other assets 206.280 Total long-term assets 76,180,922 68,619,005 Total assets $91,690,031 $80,563,569 LIABILITIES AND NET ASSETS Current portion of long-term debt Accounts payable and accrued expenses Deferred income and advances on contracts Total current liabilities Annuities and unitrusts payable Long-term debt, net of current portion Advances on contracts Total long-term liabilities Total liabilities Commitments and contingencies 2.073,375 1.016.060 3.089.435 1,393,735 10.200.000 1.230.743 12.824.478 15,913,913 $ 267,404 1,957,508 656.745 2.881.657 1,460,948 2,056,692 1.574,758 5.092.398 7.974.055 Net assets: Unrestricted Temporarily restricted Permanently restricted Total net assets Total liabilities and net assets 22,903,287 30,752,413 22.120.418 75.776.118 $ 91,690,031 19.887.437 33.349,244 19.352.833 72.589.514 The accompanying notes are an integral part of the financial statements. RS MARINE BIOLOGICAL LABORATORY STATEMENT OF ACTIVITIES For the Year Ended December 3 1 , 2000 (With Comparative Totals for the Year Ended December 31, 1999) Operating support and revenues: Government grants Private contracts Laboratory rental income Tuition, net Fees for conferences and services Contributions Investment income Miscellaneous revenue Present value adjustment to annuities Net assets released from restrictions Total operating support and revenues Expenses: Research Instruction Conferences and services Other programs (Note 2) Total expenses Change in net assets before nonoperating activity Nonoperating revenue: Contribution to Plant: Private Government Release from restriction Invested in Plant Total investment income and gains/losses Less: investment earnings used for operations Reinvested (utilized) investment income and gains/losses Total change in net assets Net assets, beginning of year Net assets, end of year Temporarily Permanently Unrestricted Restricted Restricted 2000 1999 $14,048,464 $ $ $14,048,464 $12,248,442 1.697.062 1.697.062 1,819,240 1.598,373 1,598,373 1,548,168 543,305 543.305 537,835 4.407,311 4,407,3 1 1 3,765,039 1.693.185 2.347.731 1.908,528 5,949.444 8,620.519 1,736.186 594.530 2.330.716 2.060,478 468.482 468.482 466.903 55.176 55,176 (30,533) 4,144.547 (4.249.547) 105,000 30,336.915 (1,252.110) 2,013.528 31,098,333 31,036,091 17.799.627 17,799,627 14,147,645 5.626.223 5,626,223 4,742.287 1.307.458 1,307,458 2,252.842 4,261.327 4,261,327 5,297,773 28,994.635 28,994,635 26.440,547 1.342,280 (1,252.110) 2.013.528 2,103,698 4.595,544 404.018 4.109.597 125.000 4.638,615 1,757.319 198.443 1,615.142 (1.615,142) 2,019,160 2,494.455 125.000 4.638.615 1,955,762 (284,514) (2.484.3811 629.057 (2.139.838) 5,938.476 (61,076) ( 1 .354.795 ) (1.415.871) (1.262.020) (345,590) (3.839,176) 629.057 (3.555,709) 4.676,456 3.015.850 (2,596,831) 2.767,585 3,186,604 11,227.762 19,887,437 33,349,244 19,352,833 72,589.514 61,361.752 $22.903.287 $30,752.413 $22.120.418 $75.776.118 $72,589,514 The accompanying notes are an integral fart of the financial statements. R9 MARINE BIOLOGICAL LABORATORY STATEMENT OF CASH FLOWS For the Year Ended December 3 1 , 2000 (With Comparative Totals for the Year Ended December 31, 1999) Cash flows from operating activities: Change in net assets Adjustments to reconcile change in net assets to net cash provided by (used in) operating activities: Depreciation and amortization Unrealized (appreciation) depreciation on investments Realized gain on investments Present value adjustment to annuities and unitrusts payable Contributions restricted for long-term investment and annuities Provision for bad debt Provision for uncollectible pledges Change in certain balance sheet accounts: Accounts receivable Pledges receivable Grants and contracts receivable Other current assets and other assets Accounts payable and accrued expenses Deferred income Annuities, and unitrusts payable Advances on contracts Net cash provided by operating activities Cash flows from investing activities: Purchase of property and equipment Proceeds from sale of investments Purchase of investments Net cash used in investing activities Cash flows from financing activities: Payments on annuities and unitrusts payable Receipt of permanently restricted gifts Annuity and unitrusts donations received Bond issuance Payments on long-term debt Net cash provided by financing activities Net increase in cash and cash equivalents Cash and cash equivalents at beginning of year Cash and cash equivalents at end of year 2000 $3,186,604 1999 $11,227.762 1.791,975 1,562.487 6.700,396 (3.544.380) (3.886.669) (1.639,795) (55,176) 30,533 (2.033,528) (2,485,624) 36,968 423.982 48.367 47,489 (410,852) (3,010,156) (655,968) 150,317 (252,745) 251,390 1 15,867 (100,233) 359,315 193,872 (73,167) 68,112 (344.015) 302,368 4,914,386 3,091,110 (5.096,406) (2,145.041) 68.837.634 63,101,047 (76,930.252) (65.485.238) (13,189.024) (4,529,232) (96,316) (49,897) 2.033,528 2,438,148 102.270 47,476 10.200.000 (2,324.096) (243,274) 9,915,386 2,192,453 1 ,640,748 754.331 1,942,285 1,187,954 S3.583.033 $1,942.285 The accompanying notes are an integral pan of the financial statements. RIO Financial Statements Rll Marine Biological Laboratory Notes to Financial Statements 1. Background The Marine Biological Laboratory dhe "Laboratory") is a private, independent not-for-profit research and educational institution dedicated to establishing and maintaining a laboratory and station for scientific study and investigation, and a school for instruction in biology and natural history. The Laboratory was founded in 1888 and is located in Woods Hole, Massachusetts. 2. Significant Accounting Policies Basis of Presentation The accompanying financial statements have been prepared on the accrual basis of accounting and in accordance with the principles outlined in the American Institute of Certified Public Accountants" Audit Guide, "Not-For-Profit Organizations." The financial statements include certain prior-year summarized comparative information in total but not by net asset class. Such information does not include sufficient detail to constitute a presentation in conformity with generally accepted accounting principles. Accordingly, such information should be read in conjunction with the Laboratory's financial statements for the year ended December 31, 1999. from which the summarized information was derived. The Laboratory classifies net assets, revenues, and realized and unrealized gains and losses based on the existence or absence of donor-imposed restrictions and legal restrictions imposed under Massachusetts State law. Accordingly, net assets and changes therein are classified as follows: Unrestricted Unrestricted net assets are not subject to donor-imposed restrictions of a more specific nature than the furtherance of the Laboratory's mission. Revenues from sources other than contributions are generally reported as increases in unrestricted net assets. Expenses are reported as decreases in unrestricted net assets. Gains and losses on investments and other assets or liabilities are reported as increases or decreases in unrestricted net assets unless their use is restricted by explicit donor stipulations or law. Expirations of temporary restrictions on net assets, that is, the donor-imposed stipulated purpose has been accomplished and/or the stipulated time period has elapsed, are reported as reclassifications between the applicable classes of net assets and titled "Net assets released from restrictions." Temporarily Restricted Temporarily restricted net assets are subject to legal or donor-imposed stipulations that will be satisfied either by the actions of the Laboratory, the passage of time, or both. These assets include contributions for which the specific, donor-imposed restrictions have not been met and pledges, annuities, and unitrusts for which the ultimate purpose of the proceeds is not permanently restricted. As the restrictions are met. the assets are released to unrestricted net assets. Also, realized/unrealized gains/losses associated with permanently restricted gifts which are not required to be added to principal by the donor are classified as temporarily restricted and maintain the donor requirements for expenditure. Permanently Restricted Permanently restricted net assets are subject to donor-imposed stipulations that they be invested to provide a permanent source of income to the Laboratory. These assets include contributions, pledges and trusts which require that the corpus be invested in perpetuity and only the income be made available for program operations in accordance with donor restrictions. Performance Indicator Nonoperating revenues include realized and unrealized gains on investments during the year as well as investment income on the master pooled investments and revenues that are specifically for the acquisition or construction of plant assets. Investment income from short-term investments and investments held in trust by others are included in operating support and revenues. To the extent that nonoperating investment income and gains are used for operations as determined by the Laboratory's total return utilization policy (see below), they are reclassified from nonoperating as "Investment earnings used for operations" to operating as "Investment income" on the statement of activities. All other activity is classified as operating revenue. Cash and Cash Equivalents Cash equivalents consist of resources invested in overnight repurchase agreements and other highly liquid investments with original maturities of three months or less. Financial instruments which potentially subject the Laboratory to concentrations of risk consist primarily of cash and investments. The Laboratory maintains cash accounts with one banking institution. Investments purchased by the Laboratory are carried at market value. Donated investments are recorded at fair market value at the date of the gift. For closely held non-publicly traded investments, management determines the fair value based upon the most recent information available from the limited partnership. For determi nation of gain or loss upon disposal of investments, cost is determined based on the first-in. first-out method. R12 Annual Report Investments with an original maturity of three months to one year, or those that are available for operations within the next fiscal year, are classified as short-term. All other investments are considered long-term. Investments are maintained primarily with three institutions. In 1924, the Laboratory became the beneficiary of certain investments, included in permanently restricted net assets, which are held in trust by others. The Laboratory has the continuing rights to the income produced by these funds in perpetuity, subject to the contractual restrictions on the use of such funds. Accordingly, the trust has established a process to conduct a review every ten years by an independent committee to ensure the Laboratory continues to perform valuable services in biological research in accordance with the restrictions placed on the funds by the agreement. The committee met in 1994 and determined that the Laboratory has continued to meet the contractual requirements. The market values of such investments are $7,904,545 and $7,275,488 at December 31, 2000 and 1999. respectively. The dividend and interest income on these investments, included in unrestricted support and revenues, totaled $201.407 and $221.882 in 2000 and 1999, respectively. Investment Income and Distribution For the master pooled investments, the Laboratory employs a total return utilization policy that establishes the amount of the investment return made available for spending each year. The Finance Committee of the Board of Trustees has approved a spending policy that the withdrawal will be based on a percentage of the 12 quarter average ending market values of the funds. The market value includes the principal plus reinvested income, realized and unrealized gains and losses. Spending rates in excess of 5%, but not exceeding 7%, can be utilized if approved in advance by the Finance Committee of the Board of Trustees. For fiscal 2000 and 1 999. the Laboratory obtained approval to expend 6% of the latest 1 2 quarter average ending market values of the investments. The net appreciation on permanently and temporarily restricted net assets is reported together with temporarily restricted net assets until such time as all or a portion of the appreciation is distributed tor spending in accordance with the total return utilization policy and applicable state law. Investment income on the pooled investment account is allocated to the participating funds using the market value unit method (Note 4). Held by Bond Trustee Assets held by bond trustee relate to assets held by an outside trustee under the March 1, 2000 loan and trust agreement. Per the prospectus, these funds may be used solely for capital projects as determined by the Laboratory's Board of Directors. At December 31, 2000, these assets were invested in a qualified QIC under a funding agreement with an insurance company. Plant Assets Buildings and equipment are recorded at cost. Donated facility assets are recorded at fair market value at the date of the gift. Depreciation is computed using the straight-line method over the asset's estimated useful life. Estimated useful lives are generally three to five years for equipment and 20 to 40 years for buildings and improvements. Depreciation is not recorded for those assets classified as construction-in-process as they have not yet been placed into service. Depreciation expense for the years ended December 31, 2000 and 1999 amounted to $1.791.975 and $1,562.487. respectively, and has been recorded in the statement of activities in the appropriate functionalized categories. When assets are sold or retired, the cost and accumulated depreciation are removed from the accounts and any resulting gain or loss is included in unrestricted income for the period. Annuities and Unitrusts Payable Amounts due to donors in connection with gilt annuities and unitrusts are determined based on remainder value calculations, with varied assumptions of rates of return and payout terms. Deferred Income and Advances on Contracts Deferred income includes prepayments received on Laboratory publications and advances on contracts to be spent within the next year. Advances on contracts includes funding received for grants and contracts before it is earned. Long-term advances are invested in the master pooled account until they are expended. Revenue Recognition Sources of revenue include grant payments from governmental agencies, contracts from private organizations, and income from the rental of laboratories and classrooms for research and educational programs. The Laboratory recognizes revenue associated with grants and contracts at the time the related direct costs are incurred or expended. Recovery of related indirect costs is recorded at predetermined fixed rates negotiated with the government. Revenue related to conferences and services is recognized at the time the service is provided, while tuition revenue is recognized as classes are offered. The tuition income is net of student financial aid of $579,790 and $527.258 in 2000 and 1999, respectively. Fees for conferences and other services include the following activities: housing, dining, library, scientific journals, aquatic resources and research services. Contributions Contribution revenue includes gifts and pledges. Gifts are recognized as revenue upon receipt. Pledges are recognized as temporarily or permanently restricted revenue in the year pledged and are recorded at the present value of expected future cash flows, net of allowance for unfulfilled pledges. Gifts and pledges, other than cash, are recorded at fair market value at the date of contribution. Expenses Expenses are recognized when incurred and charged to the functions to which they are directly related. Expenses that relate to more than one function are allocated among functions based upon either modified total direct cost or square footage allocations. Financial Statements R13 Other programs expense consists primarily of fundraising, year-round labs and library room rentals, costs associated with aquatic resource sales and scientific journals. Total fundraising expense for 2000 and 1999 is $1,156,656 and $1.008,920, respectively. Use of Estimates The preparation of financial statements in conformity with generally accepted accounting principles requires management to make estimates and assumptions that affect the reported amounts of assets and liabilities and disclosure of contingent assets and liabilities at the date of financial statements and the reported amounts of revenues and expenses during the reporting period. Actual results could differ from those estimates. Tax-Exempt Status The Laboratory is exempt from federal income tax under Section 501(c)(3) of the Internal Revenue Code. Reclassification Certain prior year balances have been reclassified to conform with the current year presentation. 3. Investments The following is a summary of the cost and market value of investments at December 31, 2000 and 1999: Market Certificates of deposit Money market securities U.S. Government securities Corporate fixed income Common stocks Mutual funds Limited partnerships Total investments Cost 2000 1999 2000 1999 $ 40.000 $ 40,000 $ 40,000 $ 40,000 764.969 1,781.128 764,969 1,781,128 2,300.738 69,125 2,165,197 69,951 2,412,548 2,364,068 2,537,913 2.536.808 16,144,089 15,665,205 16,318,538 10,608,588 19.909,549 26,664,204 19,306,250 23,851,004 6,522,589 1,600,300 5.324,442 958.982 $ 48,094,482 $ 48.184,030 $ 46,457,309 $ 39.846,461 Investment portfolios for the years ended December 31, 2000 and 1999 are as follows: Market Short-Term Investments Certificates of deposit Money market Mutual funds Common stocks in transit Total short term Long-Term Investments Pooled investments: Master pooled investments Separately invested: General Chase Trust Library Chase Trust Annuity and unitrusts investments Total long term Total investments Cost 2000 1999 2000 1999 $ 40,000 $ 40,000 $ 40,000 $ 40.000 377,654 233,938 377,654 233.938 3.102,515 2,875,480 3,085.445 2.965.273 79,664 33,119 79,664 33,119 $3.599,833 $34.116.704 6,204.107 1,700,438 2.473.400 44.494,649 $48.094,482 $3,182,537 $35.354.938 5,717,108 1,558,380 2.371.06? 45.001.493 $48.184,030 $3,648,491 $33,153,390 5,654,623 1,543,691 2.522,842 42.874,546 $46.457,309 $3,272,330 $27,514,505 5.335.721 1.448.569 2.275.336 36.574,131 $39.846.461 R14 Annual Report For the years ended December 31, 2000 and 1999. the Laboratory recorded net realized gains of $3.886,669 and $1,639.795; net unrealized losses (gains) of $6,700,396 and $(3,544.380); and dividend and interest income of $1.588,734 and $1,533,579, respectively. 4. Accounting for Pooled Investments Certain net assets are pooled for investment purposes. Investment income from the pooled investment account is allocated on the market value unit basis, and each fund subscribes to or disposes of units on the basis of the market value per unit at the beginning of the calendar quarter within which the transaction takes place. The unit participation of the funds at December 31, 2000 and 1999 is as follows: 2000 7999 Unrestricted Temporarily restricted Permanently restricted Advances on contracts Pooled investment activity on a per-unit basis was as follows: 11,290 40,042 73.724 5,396 1 30.452 8,573 42,351 65,789 5,557 122.270 Unit value at beginning of year Unit value at end of year Total return on pooled investments 2000 $ 283.37 261.53 $ (21.84) 7999 $ 225.51 283.37 $ 57.86 5. Long-Tenn Debt Long-term debt consisted of the following at December 31: 2000 Variable rate (63% at December 31, 1999) Massachusetts Industrial Finance Authority Series 1992 A Bonds payable in annual install- ments through 2012 6.63% Massachusetts Industrial Finance Authority Series 1992B Bonds, payable in annual installments through 2012 5.8% The University Financing Foundation, Inc. payable in monthly installments through 2000 5.8% The University Financing Foundation. Inc. payable in monthly installments through 2002 Variable rate (4.75%) Massachusetts Development Finance Agency Bonds payable in annual installments from 2006 through 2030 10.200.000 $ 10,200.000 7999 $ 890.000 1.175.000 120,929 138,167 $ 2,324.096 In March 2000, the Massachusetts Development Finance Agency issued on behalf of the Laboratory a series of Variable Rate Revenue Bonds (the "Bonds") in the amount of $10.200.000. The initial interest rate on the issue was 3.65% and is reset weekly. At December 31, 2000, the rate was 4.75%. The bonds are scheduled to mature on February 1 , 2030. The Laboratory is required to make interest payments only for the first five years. The first principal payment is due February 1. 2006 with incremental increases through maturity. The proceeds of these bonds were used to finance the capital improvements of the Laboratory's educational, research, and administrative facilities, specifically the construction and equipping of the Environmental Sciences building. A portion of the proceeds was also used to extinguish all of the Laboratory's prior debt obligations. As collateral for the bonds, the Laboratory has entered into a Letter of Credit Reimbursement Agreement which is set to expire on March 15. 2007. The Letter of Credit is in an amount sufficient to pay the aggregate principal amount of the bonds and up to 46 days' interest. The agreements related to these bonds subject the Laboratory to certain covenants and restrictions. Under the most restrictive covenant of this debt, the Laboratory is required to maintain a debt service coverage ratio. In 1992, the Laboratory issued $1,100,000 Massachusetts Industrial Finance Authority (MIFA) Series 1992A Bonds with a variable interest rate and $ 1 .500,000 MIFA Series 1 992B with an interest rate of 6.63%. Interest expense totaled $33.20 1 for the year ended December 3 1 . 2000. The Series 1 992 A and B Bonds were scheduled to mature in December 2012, but were retired on March 8, 2000 with the new bond proceeds. On March 17, 1998, the Laboratory entered into a ten-year interest rate swap contract in connection with the Series 1992A Bonds. This contract was canceled as part of the extinguishment of old debt and new debt issuance on March 8. 2000. Financial Statements R15 In 1 996, the Laboratory borrowed $500.000 with an interest rate of 5.8% per annum from the University Financing Foundation, Inc. The interest expense for the year ended December 31. 2000 was $1.950. The loan was paid off in March 2000 with the new bond proceeds. In 1997, the MBL borrowed $250.000 with an interest rate of 5.8% per annum from the University Financing Foundation. Inc. The interest expense for the year ended December 31. 2000 was $2,140. This loan was scheduled to mature in 2002 but was paid off in connection with the new debt issued in March of 2000. The Laboratory has a line of credit agreement with a commercial bank from which it may draw up to $1.000.000. The line of credit has an interest rate of prime plus 1/2 percent. The line expires May 29, 2001. No amounts were outstanding under this agreement at December 31, 2000 and 1999. 6. Plant Assets Plant assets consist of the following at December 3 1 : Land Buildings Equipment Construction in process 2000 $ 702,908 35,236,087 5,059.022 4,681,629 7999 $ 702.908 33.702.485 4,667.026 1.510.821 Total Less: Accumulated depreciation Plant assets, net 45,679,646 (22.256.490) $23.423.156 40.583,240 (20,464.515) $20,118,725 7. Retirement Plan The Laboratory participates in the defined contribution pension plan of TIAA-CREF (the "Plan"). The Plan is available to permanent employees who have completed two years of service. Under the Plan, the Laboratory contributes 10% of total compensation for each participant. Contributions amounted to $862,850 and $785,509 for the years ended December 31. 2000 and 1999, respectively. 8. Pledges Unconditional promises to give are included in the financial statements as pledges receivable and the related revenue is recorded in the appropriate net asset category. Unconditional promises to give are expected to be realized in the following periods: In one year or less Between one year and five years After five years 2000 $ 5,026,750 3,021,752 7999 $ 3,974,385 3,632,683 202,948 Total 8,048.502 7.810.016 Less: discount of $168,460 in 2000 and $236.844 in 1999 and allowance of $420,000 in 2000 and $100,000 in 1999 (588.460) (336.X44) $ 7,460.042 $ 7,473.172 9. Postretiremen! Benefits The Laboratory accounts for its postretirement benefits under Statement No. 106, "Employers' Accounting for Postretiremen! Benefits Other than Pensions." which requires employers to accrue, during the years that the employee renders the necessary service, the expected cost of benefits to be provided during retirement. As permitted, the Laboratory has elected to amortize the transition obligation over 20 years commencing on January 1, 1994. The Laboratory's policy is that all current retirees and certain eligible employees who retired prior to June 1 , 1 994 will continue to receive postretirement health benefits. The remaining current employees will receive benefits; however, those benefits will be limited as defined by the Plan. Employees hired on or after January I. 1995 will not be eligible to participate in the postretirement medical benefit plan. R16 Annual Report The following tables set forth the Plan's funded status as of December 31: Change in benefit obligation Postretiremen! benefit obligation at beginning of year Service cost Interest cost Actuarial gain Benefits paid Postretiremen! benefit obligation at end of year Change in plan assets Fair value of plan assets at beginning of year Employer contribution Actual return on plan assets Benefits paid Fair value of plan assets at end of year Funded status Unrecognized actuarial gain Unrecognized net obligation at transaction Accrued postretiremen! benefit cost Less estimated amount payable within one year and classified as a current liability Accrued postretiremen! benefit cost, net of current portion Weighted-average assumptions as of December 3 1 Discount rate 2000 2.043,659 23,020 149.574 (87,740) (136.844) 1,991,669 936,149 182.776 56,465 (136.844) 1 .038,546 (953,123) (185,377) 1.128.691 (9,809) $ (9,809) 7.50% 1999 2,171,119 28,231 134,533 (174.966) (115.258) 2,043.659 820,645 192,082 38.680 (115.258) 936.149 (1.107,510) (125,351) 1.215.513 (17,348) $ (17.348) 8.00% For purposes of measuring the benefit obligation, an 8.0% annual rate of increase in the per capita cost of covered health benefits was assumed for 2000. The rate was assumed to decrease gradually to 5% in 2005 and remain at that level thereafter. Components of net periodic benefit cost Service cost Interest cost Expected return on assets Amortization of net obligation at transition Recognized net actuarial loss Net periodic benefit cost Impacl of 1% increase in healih care cost trend: on interest cost plus service cost during past year on accumulated postretiremen! benefit obligation Impact of 1 % decrease in health care cost trend: on interest cost plus service cost during past year on accumulated postretiremen! benefit obligation 2000 $ 23,020 149,574 (69,524) 86,822 (14.655) $ 175,237 14,271 41,263 (11,946) (233,324) 1999 28,231 134,533 (61,425) 86.822 (5,385) $ 182,776 (71,626) (456,863) (10,559) (235,728) Pension plan assets consist of investments in a money market fund. 1C' i 1 i 2 rl rl 2 o ON ~~~" rt- sD <"*"* sO r " ' OC t*~i r] r*- ( S! 1 oo S m o r- r- ON m ^t r- rj ON ^c >n oo n ON s ON' m* ri rl r I be so r, so so fN cs r*i m ri in r- sc oo m, oc sC ^ x: ON -! i 1 oo V~i sD -f n rn r-1 ON {, -C tr, m in so SO sD OO OO m, DO r- a 5 ? = ^ C ON' in rn 1 1 1 r-" r- 1 m 1 t' ri 1 r- 5" ^ ^ n ~~ Q= be So ^o o Tt O oo rj o u r- O ri m oc o -3- < ri oo ~f ON in in o O Q ;.. cs oc o ^' o r-' 1 ON' r-' oc O r- | 3 ri r- ON C/3 g < 00 V~| O w~. cn t*- t- u w (J S; ; S ?. ID in f^ X S ri r-' so' in m m i S ^ -i m, so c 1 V* "*" (3 f-1 2 -t C rt r O O O sC ^ 1' s O fi (N r- ^C r*-, ON sC sC -t DC in, O m, m, S cn u- ^S ^ m" so* sc 1 1 sc DC r- o 1 r-i OC \O DO m r- (N ^c *"' ^ c/: Z Ji ^ & 02 LU c 1 O X UJ r) o K o m r DC o ^ sC C -~ ~ ^ rn ' ^t ""^ O r j m n ON oo 5 _J a "- Si O 1 ~~ '^l r~~ oc m. \O r- ON CN o g Q UJ U u O L? ' = * 1 CO _] (U 'o ac ae ^ m ON - 3 00 m PJ H u ri r- sC 1 sC s r- sC O ri rj r ; m! rr, -t i in oc ri oc z Z . u J sC oc V, <-*-, rj C ri r- ri -rt s : -t oc s 02 ^ S 1 ' 5 ^c -C s J! -S 3 o C .1 it II o '<=, ri ON ON oo m ON O ON ON ON ON oo r~- r- o n rj O rn rn in ON r- O -t -t (-*-, r- 1 ri t a a oc i O ri mi so' ^ o ^ ri r*-, -t ^ 5 ^ 1 g S O sO ri O ri O c " 2 u m ri m, &e Is w 3- -a -T ri r-' oo oc r*- r- O -t oo -t -1- ri I- ri ON r- u~. If = 1 i s -3 , E S SO R17 Report of the Library Director During the past several years a major paradigm shift has clearly taken place in the MBLAVHOI Library. We now have more than 2000 full-text electronic journals available on the network. The library web site is the starting point for content rich information that is being delivered to the much heralded "scientists' desktop." The simple act of checking in a journal and placing it on the shelf or requesting an Interlibrary Loan now requires the use of various pieces of software like Prospero, CLIO, OCLC Microenhancer. OCLC Passport, Ariel, Microsoft Office. EDI, ABLE, URSA, and various modules of Mariner, as well as online delivery service software for statewide courier services: FedEx, UPS. CISTI, NTIS. and ISI. The inauguration of information delivery via our web site also employs the use of SQUID, Geobrowser. LUCID. MySQL, Ultra Edit, Adobe GoLive, Adobe Premiere. Adobe Acrobat, Omni Page, Web Star. Fetch, Quid Pro Quo, Microsoft Office, Portfolio. Graphics Converter. Home Page, SSH, FileMaker. BB Edit, Illustrator. Photoshop, PageMaker, and Apache, and languages such as PHP3, Perl, and SQL. Obviously, "instant" delivery of information requires many hours of staff time implementing major software and hardware infrastructure installations to support this effort. This instant information drive is powerful, but intellectual ownership and archival requirements are elusive in the world of ePublishers and libraries. Print subscriptions still arrive daily, and electronic journals seem to disappear from a web site at whim. We are making choices in an age of disruptive technologies and value -changing economies. Still, much was accomplished in 2000 in the library. The emphasis this year was on expanding the serial collection, both print and electronic. The collection has grown to more than 200,000 volumes occupying all the physical space we have available in Woods Hole. Space anil Renovations Providing space for library resources is a constant concern for library patrons and staff. Some of the storage problems have been addressed by providing more electronic access to journals and sending some volumes off campus to the Harvard Depository. A Feasibility Study performed by Jay Lucker. Library Consultant, and Stephen Hale, Architect, presented several design ideas to the Trustees. Along with the major recommendation for additional space, the study resulted in a redesign of the equipment and furniture in the catalog room, which allowed more computer terminals for patrons, and the installation of a "window" to the reference desk for easy access to "live" reference information. In addition, the WHOI Archives fini hed a compact shelving project that encompasses 2130 square feet and resulted in 1 1 ,200 linear feet; it will allow for more aggressive record management and 15 years' added growth in archival space projections. The major improvement to the current library space in the Lillie building was the installation of a new HVAC system that supplies heat in the winter and cooling in the summer to the stack area, the library office, and the reading rooms. This joint venture, financially supported R1S Library Director's Report R19 by both MBL and WHOI. is preventing the wild temperature swings that can be so damaging to the collections. This is a key improvement and the basis for any conservation and preservation program. Special Collection and Rare Books Dr. Garland Allen and Carol Winn identified nearly 2500 volumes in the open stacks, dating from the early 19th century, that require preservation and increased security to protect their plates and illustrations. Our Rare Books Room is filled to capacity, so we must find additional space for these materials in the coming years. The Mary Sears collection, which included individual pieces of the Challenger and Siboga expeditions, was cataloged and indexed this year. Dr. Arthur Humes' collection was also processed; it included a collection of exotic shells. Also acquired and added to the Florence Gould Collection in the Rare Books was Guillaume Rondelet's Libri de Piscibus Marinis (1554). This volume is now the oldest book in the collection and one of the first books to describe marine organisms and fishes. Electronic Access As access to information becomes more interactive and information retrieval moves at breakneck speed, the importance of web design and accessibility heightens. The library's web page will continue to be in "re-design" mode with the addition of new resources and services. A new staff member, Amy Stout. Digital Systems and Services Coordinator, is in charge of posting and monitoring the use of this integral part of the library's services. Major upgrades to the library's software took place this year, which resulted in a new look and feel to the web interface, allowing more flexibility in customizing displays for patrons. Electronic access to the Oxford English Dictionary and web versions of Zoo Record and PsycINFO were new additions to the library holdings this year. The library joined JSTOR, a project that provides digital archives of classic serials in the general sciences, ecology, and botany. JSTOR gives us access, for example, to the entire run of The Philosophical Transactions of the Royal Society of London from Volume 1, Number 1 in 1665. Cooperating Libraries The Boston Library Consortium (BLC) received grant funding from the Massachusetts Board of Library Commissioners for the implementation of a virtual catalog and interlibrary loan (ILL) direct distance borrowing project (VirCat). This grant has made it possible for a growing number of libraries in the consortium to allow patron initiated borrowing from each other's collections without going through the ILL librarians. A group of BLC libraries, including MBL/ WHOI. purchased ScienceDirect from Elsevier. This increased our full-text electronic coverage of Elsevier titles from 1 1 1 to 850, which represents the combined holdings of Elsevier titles by the BLC members along with an additional 400 Springer- Verlag full-text eJournals. Volunteers and Staff Judy Ashmore, the Assistant Director for MBL Library Operations, Marguerite (Peg) Costa, Cataloger, and Margot Garritt, WHOI Archivist, together representing more than 50 years of experience in the Library, retired this year. Their work has been greatly appreciated by the entire Woods Hole scientific community. Eleanor Uhlinger, former Director of the Pell Marine Science Library, joined the library as Assistant Director in January 2001. Sha Li (Lisa), Director of Information Services Center and Library for the South China Sea Institute of Oceanology, Chinese Academy of Sciences in Guangzhou, China, spent two months in the library on a study visit learning new technology. The volunteers in the Rare Books Room and Archives in the Main library, as well as the volunteers in the Data library, have provided invaluable assistance in helping to organize and make these collections available for future scientists. The oral history project at WHOI has been a great success and will be of inestimable value as the 75th anniversary of that institution approaches. Peg Costa joined the ranks of volunteers and helps Carol Winn with the original cataloging project in the rare books. It is with extreme sorrow that I report that Dr. Robert Huettner died in March 2001. He will be remembered as someone who had a very real element of the spirit of discovery and learning, a teacher who exuded enthusiasm as well as knowledge. Bob and his wife, Millie, have been volunteers in the Rare Books room for more than 10 years. The MBLAVHOI Library hosted the Information Futures Institute at the Jonsson Center in May and welcomed leaders in the field of library science. Participation in these meetings is important not only for the national recognition it affords, but for the leadership these groups exercise in shaping the future of research libraries. The library has embraced the era of informatics. Funded by the Jewett Foundation, extensive research is underway creating an electronic Key system in taxonomy and a taxonomic name server that will serve the academic enterprise over the web. The library committee has finished its strategic plan, which continues to support the library's mission, and looks forward to a future providing a collaborative and collegia! environment, with access to information essential to scientific research, preservation of materials for future generations, and teaching in the Woods Hole scientific institutions. Catherine Norton Educational Programs Summer Courses Biology of Parasitism: Modern Approaches (June 8-August 11, 2000) Directors Pearce. Edward, Cornell University Tschudi. Christian, Yale University School of Medicine Faculty Phillips, Meg, University of Texas Southwestern Medical Center Russell, David, Washington University School of Medicine Scott, Phillip, University of Pennsylvania Selkirk, Murray. Imperial College of Science. Technology & Medicine, United Kingdom Sibley, David, Washington University School of Medicine Ullu, Elisabetta, Yale University School of Medicine Waters, Andrew P., Leiden University Medical Centre Lecturers Allen, Judith. University of Edinburgh Artis. David, University of Pennsylvania Bangs. Jay. University of Wisconsin-Madison Beckers, Cornelis, University of Alabama, Birmingham Beverley, Stephen. Washington University School of Medicine Borst. Piet, Netherlands Cancer Institute Burleigh. Barbara. Harvard School of Public Health Cully, Doris, Merck & Co. Dunne, David, Cambridge University Fidock, David. Albert Einstein College of Medicine Goldberg, Daniel. Washington University School of Medicine Grencis. Richard, University of Manchester, United Kingdom Guiliano. David Gull, Keith. University of Manchester. United Kingdom Hajduk. Steve, University of Alabama, Birmingham Hoffman, Steve Hunter, Christopher, University of Pennsylvania Komuniecki, Richard, University of Toledo Kopf, Manfred, Basel Institute for Immunology, Switzerland Landfear. Scott. Oregon Health Sciences University Langhorne. Jean. Medical Research Council McKerrow, James Mottram, Jeremy, University of Glasgow O'Neill, Scott, Yale University School of Medicine Parsons, Marilyn, Seattle Biomedical Research Institute Preiser. Peter. Medical Research Council Rathod. Pradip. Catholic University of America Sacks. David. National Institutes of Health Scherf, Artur, Institut Pasteur, France Sher. Alan, National Institutes of Health Sinnis. Photini. New York University School of Medicine Tarleton, Rick, University of Georgia Turco. Sam, University of Kentucky Medical Center Wang. Ching Chung, University of California. San Francisco Wirth. Dyann, Harvard School of Public Health Teaching Assistants Beatty, Wandy, Washington University School of Medicine Djikeng, Appolinaire. Yale University School of Medicine Hussein, Ayman, Imperial College of Science. Technology & Medicine. United Kingdom Jackson, Laurie, University of Texas Southwestern Kissinger, Jessica. University of Pennsylvania Lovett. Jennie, Washington University School of Medicine MacDonald, Andrew. Cornell University Morrissette. Naomi. Washington University School of Medicine Reiner. Steven van der Wei, Annemarie, Biomedical Primate Research Centre, The Netherlands Course Assistants Chipperfield, Caitlin Nadine, Cornell University Johnson, Ben, Cornell University Students Andersson, John, Karolinska Institut D'Angelo, Maxinuliano. University of Buenos Aires Dolezal, Pavel, Charles University. Prague Ferreira, Ludmila, Universidade Federal de Minas Gerais Figueiredo, Luisa, Institut Pasteur Gilk. Stacey, University of Vermont Lamb. Tracey. University of Edinburgh Lowell, Joanna, Rockefeller University Martins. Gislaine, University of Sao Paulo Murta, Silvane, Centro de Pesquisas "Rene Rachou," Brazil O'Donnell. Rebecca, University of Melbourne Ralph, Stuart, University of Melbourne Sehgai, Alftca, Tata Institute of Fundamental Research India Tangley, Laura, U.S. News & World Report. Science Writer Triggs. Veronica. University of Wisconsin. Madison Ulbert, Sebastian, Netherlands Cancer Institute Villarino, Alejandro, University of Pennsylvania Embryology: Concepts and Techniques in Modern Developmental Biology (June 18-Jnly 29, 2000) Directors Bronner-Fraser, Marianne, California Institute of Technology Fraser, Scott, California Institute of Technology R20 Educational Programs R21 Faculty Adoutte. Andre, University of Paris-Sud. France Blair, Seth S., University of Wisconsin, Madison Carroll, Sean, University of Wisconsin, Madison Collazo, Andres, House Ear Institute Ettensohn, Charles, Carnegie Mellon University Harland. Richard, University of California. Berkeley Henry, Jonathan, University of Illinois, Urbana Krumlauf. Robb. National Institute for Medical Research Levine, Michael. University of California, Berkeley Martindale, Mark. Kewalo Marine Laboratory Niswander, Lee, Memorial Sloan-Kettering Cancer Center Rothman, Joel, University of California, Santa Barbara Saunders, John, Jr., Marine Biological Laboratory Schupbach. Trudi, Princeton University Shankland, Martin. University of Texas, Austin Soriano, Philippe, Fred Hutchinson Cancer Research Center Wieschaus, Eric, Princeton University Wray. Gregory, Duke University Zeller. Robert, University of California. San Diego Lecturers Davidson, Eric, California Institute of Technology Holland, Linda, University of California, San Diego Hopkins, Nancy, Massachusetts Institute of Technology Joyner. Alexandra, New York University School of Medicine Rosenthal, Nadia. Massachusetts General Hospital, East Smith, William. University of California, Santa Barbara Stern, Claudio, Columbia University Teaching Assistants Allison. Toby, Howard Hughes Medical Institute Atit, Radhika. Memorial Sloan-Kettering Cancer Center Baker, Clare, California Institute of Technology Garcia-Castro, Martin, California Institute of Technology Gendreau, Steve, Exelixis, Inc. Kuhlman. Julie, University of Oregon Lane. Mary Ellen. University of Massachusetts Medical Center Lartillot, Nicolas, University of Paris-Sud. France Liu, Karen, University of California, Berkeley Maduro. Morris, University of California, Santa Barbara Mariani. Francesca, University of California, Berkeley Micchelli, Craig, University of Wisconsin, Madison Ober, Elke, University of California, San Francisco Seaver, Elaine, University of Hawaii Tabin, Clifford, Harvard Medical School Tobey, Allison. Memorial Sloan-Kettering Cancer Center Trainor, Paul. Medical Research Council, United Kingdom Wallingford, John, University of California, Berkeley Walsh, Emily. University of California. San Francisco Williams. Terri A.. Yale University Wilson. Valerie, University of Edinburgh Course Assistants Hurwitz, Mark, Marine Biological Laboratory Stringer. Kristen. Marine Biological Laboratory Wylie. Matthew. Marine Biological Laboratory Students Aspock. Gudrun, University of Basel Ballard. Victoria, University of Surrey. United Kingdom Bates, Damien. Murdoch Childrens Research Institute Beckhelling, Clare. Marine Biology Station, France Bellipanni, Gianfranco. University of Pennsylvania Cheeks, Rebecca. University of North Carolina. Chapel Hill Dichmann, Darwin, Hagedorn Research Institute Dorman, Jennie, University of Washington Ellertsdottir, Elin, University of Freiburg Espinoza, Nora, Louisiana State University Ezin, Max. University of Virginia Field, Holly, University of California. San Francisco Gong, Ying, California Institute of Technology Gross, Jeffrey. Duke University Huber. Jennifer, University of Hawaii Imai. Kazushi. Columbia University Javaherian, Ashkan, Cold Spring Harbor Lab Jiang, Di, National Institutes of Health Khokha, Mustafa, University of California. Berkeley Kyrkjebo, Vibeke, Sars Centre Lee, Vivian, Oregon Health Sciences University Mansfield, Jennifer, Columbia University Marx, Vivien, Freelance Science Journalist Nasevicius, Aidas, University of Minnesota Prud'homme, Benjamin, CNRS Skromne, Isaac, Princeton University Warkman, Andrew, University of Western Ontario Microbial Diversity (June 11 -July 27, 2000) Directors Harwood, Caroline, University of Iowa Spormann, Alfred, Stanford University Faculty Overmann, Jorg, University of Oldenburg Schmidt, Thomas, Michigan State University Lecturers Delong, Edward. Monterey Bay Aquarium Research Institute Gaasterland, Terry. Rockefeller University Greenberg. E. Peter, University of Iowa Groisman. Eduardo A.. Washington University School of Medicine McFall-Ngai. Margaret, University of Hawaii Omston. Nicholas, Yale University Parsek, Matthew, Northwestern University Rainey, Paul. Oxford University Schoolmk, Gary. NIH/NIAID R22 Annual Report Stemmer, Pirn. Maxygen, Inc. Visscher. Pieter, University of Connecticut Walker, Graham. Massachusetts Institute of Technology Weinstock. George, University of Texas. Houston Teaching Assistants Johnson. Hope. Stanford University Leadbetter. Jared. University of Iowa Lepp, Paul, Stanford University Schaefer. Amy. University of Iowa Course Coordinator Hawkins, Andrew, University of Iowa Course Assistant Ament, Nell, Marine Biological Laboratory Students Barak. Yoram, Hebrew University Begos. Kevin, Winston-Salem Journal. Science Writer Blake. Ruth. Yale University Buckley, Daniel. Michigan State University Callaghan. Amy. Rutgers University Goldman. Robert. University of Houston Hansel. Colleen. Stanford University Kadavy, Dana. University of Nebraska. Lincoln Kirisits. Mary Jo, University of Illinois, Urbana-Champaign Lester, Kristin, Stanford University Lin, Li-hung, Princeton University MacRae, Jean. University of Maine McCance, James, Leicester University. England McMullin. Erin, Penn State University Neretin, Lev, Shirshov Institute of Oceanography Powell, Sabrina, University of North Carolina, Chapel Hill Scott, Bari. SoundVision Productions Science Writer Simpson. Joyce. University of Illinois, Urbana Singh, Brajesh. Imperial College Stevenson, Bradley. Michigan State University Ward. Dawn. University of Delaware Zaar. Annette. Universitat Freiburg Neural Systems and Behavior (June ll-August 4, 2000) Directors Carr, Catherine, University of Maryland Levine. Richard. University of Arizona, Tucson Faculty Brodtuehrer, Peter. Bryn Mawr College Dudchenko, Paul. University of Stirling Ferrari, Michael. University of Arkansas French. Kathleen. University of California, San Diego Glanzman. David. University of California. Los Angeles Kelley, Darcy. Columbia University Knierim. James. University of Texas Medical School Kristan, William, University of California, San Diego Nadim. Farzan. Rutgers University Nusbaum, Michael, University of Pennsylvania School of Medicine Prusky, Glen. The University of Lethbridge. Canada Roberts. William, University of Oregon Szczupak. Lidia, Universidad de Buenos Aires Weeks. Janis, University of Oregon Wood, Emma, University of Edinburgh Zakon, Harold, University of Texas, Austin Lecturers Augustine. George. Duke University Korn, Henri, Pasteur Institut Maler. Leonard, University of Ottawa Pflueger, Hans-Joachim, Freie Universitat Berlin Ribera, Angela, University of Colorado Health Science Center Schwartz, Andrew, The Neuroscience Institute Walters, Edgar T., University of Texas Medical School Teaching Assistants Armstrong, Cecilia, University of Washington Beenhakker. Mark. University of Pennsylvania Blitz. Dawn Marie. University of Chicago Bower, Mark, University of Arizona, Tucson Chen, Shanping, House Ear Institute Chitwood, Raymond. Baylor College of Medicine Coleman. Melissa. St. Joseph's Hospital Duch. Carsten, University of Arizona, Tucson Gamkrelidze. Georgi, Lucent Technology Gerrard, Jason, University of Arizona. Tucson Goodman. Miriam B., Columbia University Hill, Andrew, Emory University Masino, Mark. Emory University McAnelly, Lynne, University of Texas, Austin Otis, Thomas. University of California. Los Angeles Parameshwaran, Suchitra, University of Maryland Philpot, Benjamin, Brown University Scares, Daphne, University of Maryhnd Stell, Brandon. University of California, Los Angeles Villareal. Greg. University of California, Los Angeles Yong. Rocio. University of California. Los Angeles Zee. M. Jade. University of Oregon Course Assistants Aimers. Lucy. Marine Biological Laboratory Psujek. Sean. Marine Biological Laboratory Students Akay, Turgay, University of Cologne Archie. Kevin, University of Southern California Billimoria, Cyrus, Brandeis University Black, Michael, Arizona State University Boyden, Edward, Stanford University Bradford, Yvonne. University of Oregon Cardin, Jessica. University of Pennsylvania Dasika, Vasant. Boston University Ding, Long. University of Pennsylvania Froemke, Robert. University of California, Berkeley Grammer. Michael, University of Southern California Hubbard, Aida, University of Texas, San Antonio Hunt. Barbekka. University of Colorado, Boulder Karmarkar, Uma. University of California, Los Angeles Konur, Sila, Columbia University Oestreich, Joerg. University of Texas, Austin Rela. Lorena, University of Buenos Aires Sinha. Shiva. University of Maryland Siuda. Edward, Michigan State University Tobin, Anne-Elise. Emory University Educational Programs R23 Neurobiology (June ll-Aitgmt 12, 2000) Directors Faher. Donald. Albert Einstein College of Medicine Lichtman. Jeff W.. Washington University School of Medicine Section Director Greenberg, Michael. Children's Hospital Faculty Denk, Winfried, Max-Planck Institute for Medical Research Can. Wenbiao, New York University School of Medicine Griffith. Leslie. Brandeis University Harris. Kristen, Boston University Hart. Anne. Massachusetts General Hospital Heuser, John E., Washington University School of Medicine Howell. Brian. National Institutes of Health Khodakhah. Kamran. University of Colorado School of Medicine Lambert. Nevin. Medical College of Georgia Lin, Jen-Wei. Boston University Nedivi. Elly. Massachusetts Institute of Technology Nowak. Linda. Cornell University Reese. Thomas. National Institutes of Health Sanes. Joshua, Washington University Medical School Schweizer. Felix. University of California. Los Angeles Shaman. Steven. Children's Hospital Smith, Carolyn. National Institutes of Health Terasaki. Mark, University of Connecticut Health Center Thompson. Wesley J., University of Texas Van Vactor. David, Harvard Medical School Wong. Rachel, Washington University School of Medicine Lecturers Barres. Ben A.. Stanford University School of Medicine Bean, Bruce. Harvard University Conchello. Jose-Angel, Washington University Ghosh, Anirvan, Johns Hopkins University School of Medicine Linden. David, Johns Hopkins University McCleskey. Edwin, Oregon Health Sciences University McMahan, Uel, Stanford University School of Medicine Miller, Chris. Brandeis University Sigworth, Fred. Yale University Smith, Stephen, Stanford University School of Medicine Tsien, Roger, University of California, San Diego Turrigiano, Gina Teaching Assistants Pereda. Alberto. Albert Einstein College of Medicine Petersen. Jennifer. National Institutes of Health Tumey. Stephen. Washington University Walsh. Mark. Washington University School of Medicine Course Assistants Chiu. Delia. Marine Biological Laboratory Nover. Harris. Marine Biological Laboratory Students Ang. Eugenius. Yale University Kettunen. Petronella. Karolinska Institute! Khabbaz, Anton. Princeton University/Lucent Technologies Livet. Jean, IBDM, Marseille Long, Michael, Brown University McKellar. Claire. Harvard University Misgeld, Thomas. Max-Planck-Institute of Neurobiology. Martinsried, Germany Nelson. Laura. National Institute for Medical Research. United Kingdom Ruta, Vanessa, The Rockefeller University Weissman. Tamily, Columbia University Yasuda, Ryohei, Teiko University Biotech Research Center Zhong. Haining, Johns Hopkins University Physiology: The Biochemical and Molecular Basis of Cell Signaling (June ll-July 22, 2000) Directors Garbers. David. University of Texas Southwestern Medical Center Reed. Randall. Johns Hopkins University School of Medicine Faculty Furlow. John. University of California, Davis Lockless, Steve. University of Texas Southwestern Medical Center Noel. Joseph. Salk Institute Prasad. Brinda. Johns Hopkins School of Medicine Quill, Timothy, University of Texas Southwestern Medical Center Ranganathan. Rama. University of Texas Southwestern Medical Center R24 Annual Report Verdecia, Mark, Salk Institute Wedel. Barbara, University of Texas Southwestern Medical Center Zhao. Haiqing. Johns Hopkins School of Medicine Zielinski. Raymond, University of Illinois, Urbana Isenberg Lecturer Hudspeth. A., James, Rockefeller University Lecturers Armstrong, Clay, University of Pennsylvania Buck, Linda, Harvard Medical School Clapham, David. Harvard Medical School Devreotes. Peter. Johns Hopkins University School of Medicine Dixon, Jack, University of Michigan Medical School Ehrlich. Barbara, Yale University Eraser, Scott, California Institute of Technology Freedman, Leonard. Memorial Sloan-Kettering Cancer Center Hilgemann, Donald W., University of Texas Southwestern Medical Center Huganir, Richard, Johns Hopkins University School of Medicine Jaffe. Lionel, Marine Biological Laboratory MacKinnon. Roderick, Rockefeller University Mangelsdorf, David, University of Texas Southwestern Medical Center Oprian, Daniel. Brandeis University Stamler, Jonathan S., Duke University Medical Center Wilkie. Thomas, University of Texas Southwestern Medical Center Course Coordinator Lemme, Scott, University of Texas Southwestern Medical Center Rossi. Kristen. University of Texas Southwestern Medical Center Students Brclid/.e. Tinatin. University of Miami School of Medicine Carroll, Michael. University of Newcastle upon Tyne, United Kingdom Colon-Ramos. Daniel. Duke University Cordeiro. Maria, Sofia Instituto Gulbenkian de Ciencia. Portugal Costa, Patricia, University of Rio de Janeiro Cotrufo, Tiziana, Scuola Normale Superiore Crespo-Barreto, Juan, University of Puerto Rico Cruz, Georgina, University of South Florida Dayel. Mark, University of California, San Francisco Fleegal. Melissa. University of Florida Fleischer, Jorg, University of Hohenheim Glater. Elizabeth. Brown University Jhaveri, Dhanisha, Tata Institute of Fundamental Research Johansson, Viktoria, Goteborg University Mah, Silvia, Scripps Institution of Oceanography Marrari, Yannick, Villefranche Sur Mer Meister, Jean-Jacques, Swiss Federal Institute of Technology Menna, Elisabetta, Institute of Neurophysiology, Pisa Nguyen, Anh, University of Kansas Petrie, Ryan, University of Calgary Rankin, Kathleen, Oberlin College Rodeheffer, Carey, Emory University Rodgers, Erin, University of Alabama, Birmingham Seipel, Susan, Rutgers University Sen, Subhojit. Tata Institute of Fundamental Research Shatkin-Margolis, Seth, Duke University Shilkrut, Mark, Technion-Israel Institute of Technology Takai, Erica, Columbia University Zeidner, Gil, Weizmann Institute of Science Special Topics Courses Analytical and Quantitative Light Microscopy {May 4-May 12, 2000) Directors Sluder, Greenfield, University of Massachusetts Medical School Wolf, David, BioHybrid Technologies Inc. Faculty' Amos, William B., Medical Research Council. United Kingdom Cardullo, Richard, University of California, Riverside Gelles. Jeff. Brandeis University Inoue, Shinya, Marine Biological Laboratory Oldenbourg. Rudolf, Marine Biological Laboratory Salmon, Edward, University of North Carolina, Chapel Hill Silver, Randi, Cornell University Medical College Spring, Kenneth, National Institutes of Health Straight, Aaron, Harvard Medical School Swedlow, Jason. University of Dundee Lecturer McCrone. Walter, McCrone Research Institute Teaching Assistants Grego, Sonia, University of North Carolina, Chapel Hill Hinchcliffe, Edward, University of Massachusetts Medical School Pollard, Angela, BioHybrid Technologies Course Coordinator Miller. Rick. University of Massachusetts Medical School Students Abraham, Clara, University of Chicago Alvarez, Xavier, N.E. Regional Primate Research Center, Harvard Medical School Andrews. Paul, University of Dundee Bonnet, Gregoire, Rockefeller University Bravo-Zanoguera, Miguel. University of California, San Diego Cohen, David, Cornell University Medical College Connett, Marie, Westvaco Forest Sciences Lab Crittenden, Sarah, University of Wisconsin, Madison Educational Programs R25 D'Onofrio, Terrence. Pennsylvania State University Faruki. Shamsa. Wadsworth Center Gasser. Susan. Swiss Cancer Institute Handwerger, Korie. Carnegie Institution of Washington Hunter, Edward. Q3DM Jansma. Patricia. University of Arizona Kraft. Catherine. University of Pittsburgh Lee, Michelle. Harvard Medical School Lowe. Christopher, University of California. Berkeley Lu. Bai. National Institutes of Health/NICHD Maldonado. Hector, Universidad Central del Carihe Matsumoto. Vutaka. University of Colorado McKinney. Leslie, Uniformed Services University Morelock. Maurice. Boehringer Ingelheim Pharmaceuticals Mundigl. Olaf. Roche Diagnostics Mycek. Mary-Ann. Dartmouth College Provencal. Bob. Los Alamos National Laboratory Sanabria. Priscila. Universidad Central del Caribe Sedwick. Caitlin. University of Chicago Tang. Jay. Indiana University Tirnauer, Jennifer. Harvard Medical School Xu. Fang. The Hospital for Special Surgery Frontiers in Reproduction: Molecular and Cellular Concepts and Applications (May 21-July I, 2000) Directors Hunt. Joan. University of Kansas Medical Center Mayo. Kelly. Northwestern University Schatten. Gerald. Oregon Health Sciences University Faculty Ascoli, Mario, University of Iowa College of Medicine Campbell, Keith, PPL Therapeutics Camper. Sally, University of Michigan Medical School Chan. Anthony. Oregon Health Sciences University Croy. Barbara Anne. University of Guelph. Canada Dominko. Tanja. Oregon Regional Primate Research Center Gibori. Geula. University of Illinois Hunt. Patricia A.. Case Western Reserve University Jaffe. Launnda. University of Connecticut Health Center Moore. Karen. University of Florida Morris. Patricia. The Rockefeller University Mukherjee, Abir, Northwestern University Nilson, John. Case Western Reserve Medical School Page. Ray. PPL Therapeutics Inc. Pedersen. Roger. University of California. San Francisco Shupnik. Margaret. University of Virginia Medical Center Smith, Lawrence, University of Montreal Terasaki. Mark. University of Connecticut Health Center Wakayama. Teruhiko, Rockefeller University Weigel. Nancy. Baylor College of Medicine Lecturers Balczon, Ronald. University of South Alabama Behringer. Richard, University of Texas Charo. Alta. University of Wisconsin, Madison Compton. Duane. Dartmouth Medical School Crowley, William, Massachusetts General Hospital De Sousa. Paul, Alexandre Roslin Institute Fazleabas, Asgerally. University of Illinois Hennighausen. Lothar, National Institutes of Health, NIDDK Mitchison. Timothy, Harvard Medical School Myles. Diana, University of California Ober. Carole. University of Chicago Orth, Joanne. Temple University School of Medicine Palazzo. Robert, University of Kansas Piedrahita. Jorge, Texas A&M University Reijo Pera. Renee, University of California Richards, Jo-Anne, Baylor College of Medicine Ruderman, Joan. Harvard Medical School Shenker, Andrew. Children's Memorial Hospital. CMIER Sluder. Greenfield. University of Massachusetts Medical School Stearns, Tim Strauss, Jerome, University of Pennsylvania Medical Center Tilly. Jonathan L., Massachusetts General Hospital Wall. Robert, U.S. Department of Agriculture Wessel, Gary. Brown University Woodruff. Teresa. Northwestern University Teaching Assistants Berard. Mark. University of Michigan Carroll. David. Florida Institute of Technology Giusti. Andrew, University of Connecticut Health Center Gray. Heather. University of Chicago Greenwood. Janice, University of Guelph Hmkle. Beth, University of Connecticut Health Center Hodges. Craig. Case Western Reserve University Jaquette, Julie, University of Iowa Malik. Nusrat, Baylor College of Medicine Miller, Michelle, Oregon Health Sciences University Payne, Christopher, Oregon Regional Primate Research Center Runft. Linda, University of Connecticut Health Center Saunders. Thomas, University of Michigan Takahashi, Diana, Oregon Regional Primate Research Center Voronina. Ekaterina. Brown University Week, Jennifer, Northwestern University Course Coordinators Burnett. Tim, University of Kansas Medical Center Marin Bivens, Carrie. University of Massachusetts McMullen, Michelle, Northwestern University Petroff, Margaret, University of Kansas Medical Center Simerly, Calvin, Oregon Regional Primate Research Center Students Alberio, Ramiro. Ludwig-Maximilian University, Germany Allegrucci, Cinzia, Perugia University, Italy Ashkar, Ali. University of Guelph Berkowitz. Karen. University of Pennsylvania Chong. Kowit-Yu, Oregon Regional Primate Research Center Diaz, Lorenza. INNSZ Graham, Kathryn. Oregon Health Sciences University Greenlee. Anne. Marshneld Medical Research Foundation Heifetz, Yael, Cornell University Keller. Dominique. Texas A&M University Lavoie. Holly, University of South Carolina Majumdar, Subeer, National Institute of Immunology Powell, Jacqueline, Morehouse School of Medicine Richard. Craig, Magee-Wornen's Research Institute Sahgal. Namita, Kansas University Medical Center Zhang, Gongqiao, University of Virginia R26 Annual Report Fundamental Issues in Vision Research (August 13-25, 2000) Directors Masur, Sandra K.. Mount Sinai School of Medicine Papermaster, David, University of Connecticut Health Center Faculty Barlow. Robert, Syracuse University Barres, Ben A., Stanford University School of Medicine Beebe, David C.. Washington University School of Medicine Berson, Eliot L., Harvard Medical School Bok, Dean, University of California, Los Angeles Dickersin, Kay, Brown University Dowling, John E., Harvard University Fisher, Richard, National Institutes of Health Gordon, Marion. Rutgers College of Pharmacy Hamm, Heidi E., Northwestern University Medical School Horton. Jonathan. University of California Horwitz. Joseph. University of California, Los Angeles Lang, Richard A.. New York University School of Medicine LaVail, Jennifer, University of California. San Francisco Lavker. Robert. University of Pennsylvania Lehrer. Robert, University of California, Los Angeles Leske, M. Cristina, State University of New York. Stony Brook Liberman. Ellen. National Institutes of Health Malchow, Robert. University of Illinois. Chicago Masland. Richard, Massachusetts General Hospital Nathans, Jeremy. Johns Hopkins University School of Medicine Niederkom, Jerry Y., University of Texas Southwestern Medical Center Overbeek, Paul A., Baylor College of Medicine Piatigorsky, Joram, National Institutes of Health Raviola, Elio. Harvard Medical School Shatz. Carla, Harvard Medical School Stambolian, Dwight. University of Pennsylvania Sugrue. Stephen P., University of Florida College of Medicine Wasson, Paul. Harvard Medical School Lecturers Assad. John, Harvard Medical School Hernandez, M. Rosario. Washington University School of Medicine Moses, Marsha, Children's Hospital, Boston Russell, Paul, National Institutes of Health Students Al-Khatib, Khaldun, University of Illinois, Chicago Bernstein, Audrey. Mount Sinai Medical School Birnbaum. Andrea, University of Illinois, Chicago Camelo. Serge, Institut Pasteur Cronin. Carolyn, University of Virginia Gaudio. Paul, Yale University Goh, Meilan Stephanie, University of Illinois, Chicago Hartford, April, University of Louisville Jessani. Nadim, Scripps Research Institute Jiang. Shunai. Emory University Kenyon, Kristy. Massachusetts Eye and Ear Infirmary Libby, Richard, Medical Research Council, United Kingdom Liu, Xiaorong. University of Virginia Mahajan, Vinit, University of California. Irvine Pennesi, Mark, Baylor College of Medicine Pittman, Kristi, North Carolina State University Rose, Linda, University of Maryland Ruttan, Gregory. University of Miami, Florida Sagdullaev. Botir, University of Louisville Shestopalov, Valery, Washington University Medical Informatics (May 28-June 3, 2000) Director Masys, Daniel, University of California, San Diego Faculty Canese, Kathi. National Library of Medicine Cimino, James, Columbia University Friedman, Charles, University of Pittsburgh Giuse. Nunzia, Vanderbilt University Medical Center Hightower, Allen, Centers for Disease Control and Prevention Kingsland, Lawrence, National Library of Medicine Lindberg, Donald, National Library of Medicine McDonald, Clement. Regenstrief Institute Miller. Randolph. Vanderbilt University Medical Center Nahin. Annette. National Library of Medicine Ozbolt, Judy, Vanderbilt University Medical Center Stead. William. Vanderbilt University Medical Center Wheeler. David. National Library of Medicine Students Athreya, Balu, DuPont Hospital for Children Barnes, Judith, Ingham Regional Medical Center Educational Programs R27 Belts. Eugene. Medical College of Georgia Blalt. Jod\. Health Care Financing Admiimiiation Brill, Peter. Trover Foundation Brown. Janis. University of Southern California Clintworth. William. University of Southern California Cohn. Wendy, University of Virginia Cowper, Diane. Hines VA Hospital Cooper. Natasha. Penn State College of Medicine Desai. Sundeep. Northwestern Medical Faculty Foundation Ebbeling. Kelly. University of Wisconsin. Madison Fulda. Pauline. Louisiana State University Halsted, Deborah. Houston Academy of Medicine Harris. Anthony. University of Maryland Levine. Alan. University of Texas. Houston Jenson, James, University of New Mexico Klingen, Donald, Riverside Regional Medical Center Kubal. Joseph. VA Information Resource Center Mcknight. Michelynn. Norman Regional Hospital Obijiofor, Chioma, Bioresources Development and Conservation Program Schwartz. Marilyn, Naval Medical Center, San Diego Smith. John. University of Alabama. Birmingham Sooho. Alan. Battle Creek Veterans Administration Stocking. John, University of Louisville Strachan. Dina. King/Drew Medical Center Thibodeau. Patricia, Duke University Vaidya. Vinay, University of Maryland Woeltje, Keith. Medical College of Georgia Yamamoto, David. University of California, Los Angeles Zick. Laura, Clarian Health Medical Informatics (October 1-7, 2000) Director Cimino. James. Columbia University Faculty Bakken. Suzanne. Columbia University Cimino, Chris, Albert Einstein College of Medicine Friedman. Charles. University of Pittsburgh Jenders, Robert. Columbia University Kingsland, Lawrence, National Library of Medicine Lindberg. Donald. National Library of Medicine Masys, Daniel. University of California. San Diego McCray, Alexa, National Library of Medicine Nahin, Annette. National Library of Medicine Perednia, Douglas, Association of Telehealth Providers Starren. Justin. Columbia University Wheeler. David. National Library of Medicine Students Amend. Clifford. Care First Blue Cross Blue Shield Babu. Ajit. St. Louis VA Medical Center Baer. Michael. Lebanon Veterans Admin. Medical Center Barclay. Allan, Indiana University School of Medicine Burke, Cynthia. Hampton University Byrd, Vetria, University of Alabama, Birmingham Dam. Steven, University of Western Ontario Davis. Wayne. University of Michigan Medical School DiPiro. Joseph. University of Georgia Fernandes, John, Chicago College Osteopathic Medicine Frank. Christine. Rush-Presbyterian-St. Luke's Medical Center Gallardo, Gladys, Universidad Central del Caribe Gamble, James. Maniilaq Health Center Gill, Jagjit. Mayo Clinic and Foundation Goodwin, Cheryl. Swedish Medical Center Guarcello. Catherine. St. Elizabeth's Medical Center Jones, Dixie. LSU Health Science Center Kelly, Catherine. Massachusetts General Hospital Mackowiak. Leslie. Duke University Health System McKoy. Karen. Lahey Clinic Moser, Stephen. University of Alabama. Birmingham Murray. Kathleen. University of Alaska Anchorage Pepper, David, University Medical Center Riesenberg, Lee, Ann Guthrie Healthcare System Sathe. Nila. Vanderbilt University Medical Center Sullivan, Eileen, University of New Mexico Taylor. Vera, Morehouse School of Medicine Wellik. Kay, Mayo Clinic Scottsdale Wiedermann. Bernhard. Children's National Medical Center, Washington Methods in Computational Neuroscience (July 30-August 26, 2000) Directors Bialek, William. NEC Research Institute de Ruyter, Rob. NEC Research Institute Faculty Abbott, Lawrence, Brandeis University Colby, Carol, University of Pittsburgh Collett, Thomas. University of Sussex Dan, Yang, University of California. Berkeley Delaney. Kerry, Simon Fraser University. Canada Doupe, Allison, University of California, San Francisco Ermentrout. Bard. University of Pittsburgh Ferster. David. Northwestern University Gelperin. Alan. Bell Laboratories Hopfield. John, Princeton University Johnston. Daniel. Baylor College of Medicine Kelley. Darcy. Columbia University Kleinfeld. David, University of California. San Diego Kopell. Nancy. Boston University Marder, Eve. Brandeis University Markram, H., University of California Miller. K. D.. University of California. San Francisco R28 Annual Report Mitra. Partha, AT&T Bell Laboratories Nemenman. Ilya, NEC Research Institute Rieke. Fred, University of Washington Seung. H. Sebastian, Massachusetts Institute of Technology Sigvardt, Karen. University of California. Davis Solla, Sara A., Northwestern University Medical School Sompolinsky, Maim, The Hebrew University. Israel Tank. David. AT&T Bell Laboratories Tishby, Naftali. The Hebrew University, Israel Tsodyks, Michail. Weizmann Institute of Science Zucker, Steven, Yale University Lab Instructor Jensen, Roderick, Wesleyan University Microinjection Techniques in Cell Biology (May 16-23, 2000) Director Silver, Robert. Marine Biological Laboratory Faculty Klaessig. Suzanne, Cornell University Kline, Douglas, Kent State University Shelden. Eric. University of Michigan Wilson, Susan, Cornell University Teaching Assistant Miller, Roy Andrew. Kent State University Lecturers Baylor, Denis, Stanford University Medical Center Berry. Michael, Princeton University Koberle. Roland. Universidade de Sao Paulo. Brasil Laughlin. Simon Barry. Cambridge University. United Kingdom Logothetis. Nikos, Max-Planck-Institute for Biological Cybernetics Srinivasan, Mandyam V., Australian National University. Australia Teaching Assistants Aguera y Areas, B., Princeton University Lewen, Geoffrey David. NEC Research Institute White, John, Boston University Course Assistants Jensen, Kate. Marine Biological Laboratory Purpura. Keith, Marine Biological Laboratory Students Cabot, Ryan. University of Missouri Caswell. Wayne, Lahey Clinic Combelles. Catherine. Tufts University Davies. Daryl, University of Southern California Dong. Lily. UT Health Science Center, San Antonio Geraci. Fabiana, University of Palermo Gilbert, Joanna, Harvard Medical School Gundersen-Rindal. Dawn, U.S. Department of Agriculture Harwood, Claire, University of Pennsylvania Hawash. Ibrahim. Purdue University Howe. Charles, Stanford University Kay, EunDuck, Doheny Eye Institute Kline-Smith, Susan. Indiana University Macdonald, Jennifer, Medical University of South Carolina Nguyen. Hong-Ngan, University of Louisiana of Lafayette Okusu. Akiko, Harvard University von Dassow, Peter, Scripps Institute of Oceanography Webb. Bradley. Queen's University Widelitz. Randall. University of Southern California Yang. Jin, Duke University, HHM1 Students Aharonov-Barki, Ranit, Hebrew University Bartlett, Edward, University of Wisconsin. Madison Bodekin. Clara, Boston University Boudreau. Christen (Beth), Baylor College of Medicine Feinerman, Ofer, Wiezmann Institute of Science Felsen, Gidon. University of California. Berkeley Globerson. Amir. Hebrew University Giitig. Robert. University of Freiburg Jin, Dezhe, University of California, San Diego Kang. Kukjin, Hebrew University Krishna, B. Suresh, New York University Lauritzen, Thomas. University of California. San Francisco Parthasarathy. Hemai. Nature America Paz. Ron. Hebrew University Petereit. Christian, Universitat Bielefeld Pierce, John. Vibration & Sound Sol. Ltd. Rokni. Uri, Hebrew University Schreiber, Susanne. Humboldt Universitat Berlin Shi, Songhai, Cold Spring Harbor Laboratory Sirota. Anton, Rutgers University Szalisznyo, Krisztina, Hungarian Academy of Science Taylor. Dawn. Arizona State Lmiversity Ulanovsky. Nachum, Hebrew University Werfel. Justin. Massachusetts Institute of Technology Modeling of Biological Systems (March 25-May 4, 2000) Director Silver, Robert. Marine Biological Laboratory Faculty Boston. Raymond C.. University of Pennsylvania Cheatham. Thomas E.. University of Utah Herzfeld. Judith, Brandeis University Hummel. John. Argonne National Laboratory Kollman. Peter. University of California. San Francisco Moate. Peter. University of Pennsylvania Pearson. John, Los Alamos National Laboratory Petsko, Greg A., Brandeis University Ponce Dawson. Silvina. Ciudad Universitaria. Argentina Students Genick. Ulrich. The Salk Institute Ginsberg. Tara, University of Texas, Houston Hershberg. Uri. Hebrew University Immerstrand. Charlotte. Linkoping University. Sweden Jiang, Yi. Los Alamos National Laboratory Educational Programs R29 Mosavi, Leila, University of Connecticut Health Center Quinteiro, Guillermo. University of Buenos Aires Teng. Ching-Ling. University of Virginia Uppal. Hirdesh. Punjab Veterinary Vaccine Institute, India Molecular Biology of Aging (August 12-18, 2000) Directors Guarente. Leonard P., Massachusetts Institute of Technology Wallace. Douglas, Emory University School of Medicine Faculty Austad. Steven, University of Idaho Beal, M. Flint, Cornell University Bohr. Vilhelm A., National Institutes of Health Campisi. Judith. Lawrence Berkeley National Laboratory Culotta. Valeria L., Johns Hopkins University de Lange. Titia, The Rockefeller University Hanawalt, Philip. Stanford University Johnson, Thomas. University of Colorado Jones. Dean P., Emory University Kenyon. Cynthia. University of California, San Francisco Kim, Stuart. Stanford, University School of Medicine Lithgow. Gordon J., University of Manchester Martin, George, University of Washington School of Medicine McChesney. Patricia, University of Texas Southwestern Medical Center Price, Donald L., Johns Hopkins University School of Medicine Richardson, Arlan, University of Texas Health Science Center, San Antonio Ruvkun. Gary, Massachusetts General Hospital Tanzi. Rudolph E , Harvard Medical School Tower. John, University of Southern California Van Voorhies, Wayne, University of Arizona, Tucson Wright. Woodnng E., University of Texas Southwestern Medical Center Lecturers Finch. Celeb. LIniversity of Southern California Hekimi. Siegfried, McGill University Wemdruch, Richard H., Veterans Administration Hospital Teaching Assistants Coskun. Elif Pinar, Emory University School of Medicine Ford, Ethan, Massachusetts Institute of Technology Kerstann, Keith, Emory University School of Medicine Kokoszka, Jason, Emory University Levy, Shawn, Vanderbilt-Ingram Cancer Center Marcimak. Robert, Massachusetts Institute of Technology McVey, Mitch, Massachusetts Institute of Technology Murdock, Deborah, Emory University Course Coordinator Burke. Rhonda E., Emory University School of Medicine Course Assistant Ament. Nell, Marine Biological Laboratory Students Bailey, Adina, University of California, Berkeley Baur. Joe. UT Southwestern Medical Center, Dallas Bordone. Laura, University of Minnesota Cui. Wei, Roslin Institute, Edinburgh Cypser. James, University of Colorado Filosa. Stefania, 1IGB-CNR Furfaro, Joyce. Pennsylvania State University Harper. James. University of Idaho Huang. Xudong. Massachusetts General Hospital Johnson. Kristen, Purdue University Konigsberg, Mina, Universidad Autonoma Metropolitana Kostrominova, Tatiana, University of Michigan Luo, Yuan, University of Southern Mississippi Munoz, Denise, University of Buenos Aires/UC Berkeley Peel, Alyson, The Buck Center for Research in Aging Podlutsky, Andrej, National Institute on Aging Radulescu, Andreea. Albert Einstein College of Medicine Srivivsan, Chandra, University of California, Los Angeles Tong, Jiayuan (James), Cold Spring Harbor Zaid, Ahmed, Stockholm University Molecular Mycology: Current Approaches to Fungal Pathogenesis (August 7-25. 2000) Directors Edwards, John, Jr., Harbor-UCLA Medical Center Magee. Paul T., University of Minnesota Mitchell, Aaron P.. Columbia University Faculty Filler, Scott, Harbor-UCLA Medical Center Heitman, Joseph. Duke University Medical Center Rhodes, Judith, University of Cincinnati Medical Center White, Theodore. Seattle Biomedical Research Institute Lecturers Cushion. Melanie, University of Cincinnati Doering, Tamara. Washington University School of Medicine Fink, Gerald, Whitehead Institute Kozel, Thomas, University of Nevada School of Medicine Kwon-Chung, June. National Institutes of Health Levitz, Stuart. Boston University Magee. Beatrice. University of Minnesota Puziss, John, Proteome, Inc. Quinn, Cheryl, Pharmacia & Upjohn Scherer, Stewart, Rosetta Inpharmatics Teaching Assistants Flenniken, Michelle, Montana State University Johnston, Douglas, Harbor-UCLA Medical Center Lengeler, Klaus B., Duke University Medical Center Course Assistant Martin, Sam, Marine Biological Laboratory Students Askew, David. University of Cincinnati Austin, W. Lena, Howard University Blankenship. Jill. Duke University Burr, Ian, Pfizer Central Research Francis, Susan. University of Washington Hochstenbach. Frans, University of Amsterdam Ibrahim, Ashraf, Harbor-UCLA Medical Center R30 Annual Report Lo, Hsiu-Jung, National Health Research Institutes Mol, Pietemella, University of Amsterdam Munro, Carol, University of Aberdeen Perea, Sofia. University of Texas Spellberg. Brad, Harbor-UCLA Medical Center Spreghini, Elisabetta. Yale University Toenjes, Kurt. University of Vermont Wasylnka. Julie, Simon Fraser University Neural Development and Genetics of Zebrafish (August 13-26, 2000) Directors Dowling, John E., Harvard University Hopkins, Nancy, Massachusetts Institute of Technology Faculty Chien, Chi-Bin. University of Utah Medical Center Collazo. Andres, House Ear Institute Eisen, Judith S., University of Oregon Fetcho, Joseph, State University of New York, Stony Brook Hanlon, Roger, Marine Biological Laboratory Houart, Corrine. University College London, United Kingdom Kimmel. Charles. University of Oregon Lin. Shuo. Medical College of Georgia Neuhauss, Stephan, Max-Planck-Institut fur Entwicklungsbiologie, Germany Talbot, William S., Stanford University School of Medicine Wilson, Stephen. University College London, United Kingdom Lecturers Astrofsky, Keith, Massachusetts Institute of Technology Fraser, Scott, California Institute of Technology Teaching Assistants Amacher, Sharon, University of California. Berkeley Clarke. Jon, University College London. United Kingdom Fadool, James, Florida State University Granato. Michael. University of Pennsylvania Lyons, David. University College London Mazanec, April, University of Oregon Mullins, Mary. University of Pennsylvania Perkins, Brian. Harvard University Pomrehn, Andrea, Stanford University Wagner. Daniel, University of Pennsylvania Medical School Walker-Durchanck, Charline. University of Oregon Waterbury. Julie. University of Pennsylvania Course Coordinator Schmitt. Ellen. Harvard University Facility Technician Linnon. Beth. Marine Biological Laboratory Course Assistant Bradley, Margaret, Marine Biological Laboratory Students Challa, Anil Kumar, Ohio State University Croall, Dorothy, University of Maine Darimont, Beatrice, University of Oregon Kaneko, Maki, University of Houston Leung, Fung Ping, Hong Kong University Levkowitz, Gil. Weizmann Institute of Science Lupo, Giuseppe. University of Pisa Maldonado. Ernesto. Massachusetts Institute of Technology Mangoli. Maryam. University College London. United Kingdom Meyer. Martin. Stanford University Naco, Grace. Johns Hopkins School of Medicine Nelson. Ralph. National Institutes of Health Niell. Cristopher, Stanford University Schneider, Valerie, Harvard Medical School Starr, Catherine, The Rockefeller University Yvon, Anne-Marie, University of Massachusetts, Amherst Neurobiology & Development of the Leech (August 13-September 1, 2000) Directors Calabrese. Ronald L.. Emory University Sahley, Christine. Purdue University Shankland, Martin, University of Texas, Austin Faculty Ali. Declan. Hospital for Sick Children Baader. Andreas. Universitat Bern, Switzerland Bissen, Shirley. University of Missouri Blackshaw, Susanna. University of Oxford. United Kingdom Brodfuehrer. Peter, Bryn Mawr College Carbonetto, Salvatore, Montreal General Hospital, Canada Drapeau, Pierre, McGill University, Canada Fernandez de Miguel, Francisco, Universidad Nacional Autonoma de Mexico Masino, Mark. Emory University Modney. Barbara, Cleveland State University Muller. Kenneth. University of Miami School of Medicine Nicholls. John. SISSA. Italy Weisblat. David. University of California, Berkeley Lecturer Macagno, Eduardo. Columbia LIniversity Course Assistant Johnson, Ben, Marine Biological Laboratory Educational Programs R31 Students Carrasco. Rosa. Purdue University Duan. Yuanli, University of Miami Kuo. Dian-Hun. University of Texas. Austin Kwon. Hyung-wook, University of Arizona Rela. Lorena. University of Buenos Aires Scimemi. Annalisa. SISSA. Italy Song. Mi Hye. University of California. Berkeley Trueta. Citlali. UNAM Weber, Douglas. Arizona State University West. Morris, University of Florida Yashina, Irene. University of Illinois at Chicago Zoccolan, Davide, SISSA, Italy Optical Microscopy and Imaging in the Biomedical Sciences (October 11-19, 2000) Director Izzard, Colin, State University of New York. Albany Faculty DePasquale, Joseph. New York State Department of Health Hard. Robert. State University of New York. Buffalo Inoue. Shinya. Marine Biological Laboratory Maxfield. Frederick. Cornell University Medical College Murray. 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University of, Urbana-Champaign Indiana University Indiana University School of Medicine Ingham Regional Medical Center Institute for Genomic Research Iowa University College of Medicine Iowa State University Iowa, University of Johns Hopkins University Johns Hopkins University School of Medicine Joint Genome Institute Dartmouth College Dartmouth Medical School Deaconess Medical Center Kansas University Medical Center Kansas, University of Kent State University R40 Annual Report Kentucky University Medical Center Kentucky, University of Kewalo Marine Laboratory King/Drew Medical Center Knight Ridder Newspapers Lahey Clinic Lawrence Berkeley National Laboratory Lehman College, CUNY Leica, Inc. Lilly Research Labs Los Alamos National Laboratory Louisiana State University Louisiana State University Health Sciences Center Louisiana, University of, Lafayette Louisville. University of Loyola University of Chicago Lucent Technologies Magee-Women's Research Institute Maine, University of Maniilaq Health Center Marine Biological Laboratory Marquette University Marshlield Medical Research Foundation Maryland. University of, Baltimore County Massachusetts Eye and Ear Infirmary Massachusetts General Hospital Massachusetts Institute of Technology Massachusetts, University of, Amherst Massachusetts. University of. Medical School Maxygen. Inc. Mayo Clinic and Foundation McCrone Research Institute Medical College of Georgia Medical University of South Carolina Meharry Medical College Memorial Sloan-Kettering Cancer Center Merck & Co. Miami, University of Miami University School of Medicine Michigan State University Michigan University Medical School Michigan. University of Midwestern University Minnesota University Medical School Minnesota. University of Missouri, University of, Rolla Montana State University Monterey Bay Aquarium Research Institute Morehouse School of Medicine Morgan State University Mount Holyoke College Mount Sinai School of Medicine Murdoch Institute National Aeronautics and Space Administration National Institute of Mental Health National Institute on Aging. NIH National Institutes of Health National Library of Medicine Nature America Naval Medical Center. San Diego Nebraska. University of. Lincoln NEC Research Institute Neuroscience Institute Nevada University School of Medicine New England Regional Primate Research Center New Mexico, University of New York Health Science Center, State University of New York State Department of Health New York State Institute for Basic Research New York, State University of, Albany New York, State University of, Buffalo New York. State University of. Stony Brook New York University New York University Medical Center New York University School of Medicine Norman Regional Hospital North Carolina State University North Carolina, University of. Chapel Hill Northwestern Medical Faculty Foundation Northwestern University Northwestern University Medical School Notre Dame, University of Oberlin College Ohio State Llniversity Ohio University Oregon Health Sciences University Oregon Regional Primate Research Center Oregon State University Oregon, University of PE Biosystems Penn State University Pennsylvania State University College of Medicine Pennsylvania University Medical Center Pennsylvania. University of Pennsylvania University School of Medicine Pfizer Central Research Pharmacia & Upjohn Pittsburgh, University of Pomona College Princeton University Proteome. Inc. Puerto Rico. University of Purdue University Purdue University Cancer Center Q3DM, Inc. Regenstrief Institute Rensselaer Polytechnic Institute Riverside Regional Medical Center Robert Wood Johnson Medical School Roche Diagnostics Rochester. University of Rockefeller University, The Rosetta Inpharmatics Rush-Presbyterian-St. Luke's Medical Center Rutgers College of Pharmacy Rutgers University Saint Peter's College Salk Institute San Francisco State University Scripps Institution of Oceanography Summer Research R41 Scripps Research Institute Seattle Biomedical Research Institute Smith College Smithsonian Institution Solomon Schechter Day School SoundVision Productions South Alabama. University of South Carolina. University of South Florida. University of Southampton University Southern California. University of Southern Mississippi, University of St. Elizabeth's Medical Center St. Joseph's Hospital St. Louis VA Medical Center St. Mary's College of Maryland Stanford University Stanford University Medical Center Stanford University School of Medicine Stevens Institute of Technology Swarthmore College Swedish Medical Center Syracuse University Temple University School of Medicine Tennessee Depanment of Health Tennessee State University Texas A&M University Texas Tech Medical School Texas University Health Science Center Texas University Medical School Texas. University of. Austin Texas, University of. Houston Texas. University of. San Antonio Texas University Southwestern Medical Center Toledo. University of Trover Foundation Tufts University Tufts University School of Medicine Tulane University U.S. Department of Agriculture U.S. News & World Report Uniformed Services University Union College University of Medicine and Dentistry of New Jersey Utah University Medical Center Utah. University of VA Information Research Center VA Maryland Health Care System Vanderbilt University Vanderbilt University Medical Center Vanderbilt-Ingram Cancer Center Vermont. University of Veterans Administration Hospital Veterans Affairs Medical Center Virginia University Health Sciences Center Virginia University Medical Center Virginia. University of Wadsworth Center Wake Forest University Wake Forest University School of Medicine Washington University Washington, University of Washington University School of Medicine Weill Medical College of Cornell University Wellesley College Wesleyan University Western Reserve Medical School Westvaco Forest Sciences Lab Whitehead Institute Whitney Laboratory. University of Florida Williams College Winston-Salem Journal Wisconsin, University of, Madison Woods Hole Oceanographic Institution Wyeth-Ayerst Research Yale University Yale University School of Medicine Yeshiva University Zeiss Optical Systems Foreign Institutions Represented Aberdeen, University of. Scotland Albert-Ludwigs-Universitat Freiburg, Germany Alfred Wegener Institute, Germany Amsterdam. University of. The Netherlands Australian National University, Australia Basel Institute for Immunology, Switzerland Basel. University of. Switzerland Bern, University of. Switzerland Bielefeld. University of. Germany Biomedical Primate Research Centre. The Netherlands Boehringer Ingelheim Pharmaceuticals, Inc.. Germany Buenos Aires. University of. Argentina Calgary. University of. Canada Cambridge University, United Kingdom Cape Town. University of. South Africa Centre de Genetique Moleculaire, France Centre National de la Recherche Scientifique CNRS, France Centro de Pesquisas "Rene Rachou." Brazil Charles University. Prague, Czech Republic Comision Nacional de Energia Atomica, Argentina Copenhagen, University of. Denmark Dalhousie University. Canada Dundee. University of. Scotland Edinburgh. University of. Scotland European Molecular Biology Laboratory, Germany Friedrich Miescher Institute. Switzerland Freie Universitat. Berlin. Germany Gartnaval Royal Hospital, Scotland Genoa, University of, Italy Glasgow, University of, Scotland Goteborg University, Sweden Guelph, University of. Canada R42 Annual Report Haaedorn Research Institute. Denmark Hebrew University. Israel Hebrew University Medical School, Israel Hohenheim. University of. Germany Hong Kong. University of Hong Kong University of Science and Technology Hospital for Sick Children. Canada Humboldt Universitat Berlin. Germany Hungarian Academy of Sciences, Hungary IBDM. Marseille. France Imperial College of Science, Technology and Medicine, United Kingdom Innsbruck. University of, Austria Institut fur Biologische Informationsverarbeitung, Germany Institut Pasteur, France Institute of Cell. Animal, and Population Biology. Scotland Institute of Neurophysiology, Pisa. Italy Institute of Parasitology ASCR, The Czech Republic Institute of Protein Research, Russia Institute de Investigacion Medica "Mercedes y Martin Ferreyra,' Argentina Institute de Investigaciones Biomedicas, Spain Institute Gulbenkian de Ciencia, Portugal Institute Nacional de la Nutricion. Mexico Institute Venezolano Investigaciones Cientificas, Venezuela Istituto Intemazionale di Genetica e Biofisica. Italy Karolinska Institute, Sweden Koln, University of. Germany Konstanz. University of, Germany Kyoto University. Japan Kyunghee University. Korea Leeds, University of. United Kingdom Leicester. University of. United Kingdom Leiden University Medical Centre. The Netherlands Lethbridge, University of. Canada Liege. University of. Belgium Linkoping University, Sweden Ludwig-Maximilian University. Germany Manchester. University of. United Kingdom Marine Biology Station, France Max-Planck-Institute for Biological Cybernetics. Germany Max-PUmck-Institute for Medical Research. Germany McGill University, Canada McMaster University, Canada Medical Research Council, United Kingdom Melbourne, University of, Australia Montreal General Hospital. Canada Montreal. University of. Canada Naples. University of. Italy National Institute for Medical Research. United Kingdom Netherlands Cancer Institute New Brunswick, University of, Canada Newcastle-upon-Tyne. University of. United Kingdom Niigata University Brain Research Institute, Japan Nobel Institute for Neurophysiology. Sweden Oldenburg, University of, Germany Ottawa. University of. Canada Oxford University, United Kingdom Palermo, University of, Italy Perugia. University of. Italy Pisa, University of. Italy Porto, University of, Portugal PPL Therapeutics Inc.. Scotland Punjab Agricultural University, India Rayne Institute, United Kingdom Rio de Janeiro. University of. Brazil Roslin Institute. Edinburgh, Scotland Sao Paulo, University of. Brazil Sars Centre, Norway Saskatchewan. University of. Canada Scuola Intemazionale Superiore di Studi Avanzati (SISSA). Italy Scuola Normale Superiore, Italy Shirshov Institute of Oceanology, Russia Simon Fraser University. Canada Sofia Institute Gulbenkian de Ciencia. Portugal St. Andrews, University of, Scotland Stirling, University of. Scotland Stockholm University. Sweden Surrey, University of. United Kingdom Sussex, University of. United Kingdom Swiss Federal Institute of Technology, Switzerland Swiss Institute for Experimental Cancer Research, Switzerland Sydney, University of. Australia Tata Institute of Fundamental Research, India Technion-Israel Institute of Technology. Israel Teikyo University Biotechnology Research Center. Japan Tokyo University School of Medical and Dental. Japan Universidad Autonoma Metropolitana. Mexico Universidad Nacional Autonoma de Mexico Universidad Nacional de Cuyo. Argentina Universidade Federal de Minas Gerais. Brazil Universitat Freiburg, Germany Universite Libre de Bruxelles, Belgium Universite Paris-Sud. France Uppsala University, Sweden Veterinary Vaccine Institute. India Weizmann Institute of Science. Israel Western Ontario. University of, Canada Zurich, University of, Switzerland Year-Round Research Programs Architectural Dynamics in Living Cells Program Established in 1992. this program focuses on architectural dynamics in living cells the timely and coordinated assembly and disassembly of macromolecular structures essential for the proper functioning, division, motility. and differentiation of cells; the spatial and temporal organization of these structures: and their physiological and genetic control. The program is also devoted to the development and application of powerful new imaging and manipulation devices that permit such studies directly in living cells and functional cell-free extracts. The Architectural Dynamics in Living Cells Program promotes interdisciplinary research carried out by resident core and visiting investigators. Ki'Milcin Cure Investigators Inoue, Shinya. Distinguished Scientist Oldenbourg. Rudolf, Associate Scientist Shribak. Michael. Staff Scientist Staff Knudson. Robert, Instrumental Development Engineer Baraby. Diane. Laboratory Assistant MacNeil. Jane. Executive Assistant Visiting Investigators Desai, Arshad. EMBL. Heidelburg. Germany Fukui. Yoshio. Northwestern University Medical School Coda. Makoto. Kyoto University, Japan Keefe. David. Rhode Island Women and Infants Hospital Liu. Lin. Rhode Island Women and Infants Hospital Maddox. Paul, University of North Carolina-Chapel Hill Mitchison. Timothy J.. Harvard Medical School Salmon. Edward D.. University of North Carolina-Chapel Hill Tran. Phong. Columbia University The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution This Center employs the latest advances in phylogenetic theory, computational biology, and high-throughput genome sciences to study evolutionary processes that trace back to the first life forms on earth. Through the application of high-powered statistical techniques, scientists in the Josephine Bay Paul Center investigate how the evolution of genes and genomes has driven phenotypic change at all levels of biological organization. This holistic approach provides tools to quantify and assess biodiversity and to identify genes and genetic mechanisms of biomedical and environmental importance. We study all kinds of microbes, their evolutionary history, their interactions with each other and macroscopic forms of life, and how members of diverse microbial communities contribute and respond to environmental change. Examples of current research include: 1 ) a project supported by the National Science Foundation to study the co-evolution of genomes for symbiotic bacteria and their hosts; 2) investigations supported by the National Institutes of Health to study expression and the complete genome sequence of Giardia lamblia. a water-borne human pathogen that attacks the intestinal tract and exacts a terrible toll on public health worldwide; 3) a computational biology program funded by the NIH. NASA, and private corporations to integrate evolutionary theory with the functional annotation of protein coding regions in bacterial genomes; and 4) an interdisciplinary study supported by NASA and NSF to study life in extreme environments on the planet earth in search of general principles that can guide the quest for living forms elsewhere in the universe. The Center encourages studies of genotypic diversity across all phyla and promotes the use of modem molecular genetics and phytogeny to gain insights into the evolution of molecular structure and function. Our research activities are complemented by an active education program. In addition to training postdoctoral fellows, the Josephine Bay Paul Center offers the internationally recognized Workshop in Molecular Evolution at the Marine Biological Laboratory, a workshop for secondary educators titled Living in the Molecular World, and several comprehensive web sites: 1 ) a description of research and education associated with our membership in the Astrobiology Institute at the Marine Biological Laboratory; 2) the interactive EcoCyc Project (an interactive program that describes the metabolism of E. coli as well as the identity and location of functional genes in the E. coli genome); 3) the Giardia lamblia genome page (which provides annotated analyses and current progress summaries from the MBL's Giardia lamblia genome project); and 4) the Workshop in Molecular Evolution site (which offers descriptions, information, and advice about sophisticated software packages for phylogenetic inferences and analyses of population biology data). A generous gift from the Bay Paul Foundation and continuing annual support from the G. Linger Vettlesen Foundation provided initial funding in 1997 to form The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution. The Center has excellent resources for studies of molecular biology and evolution, including well-equipped research laboratories and a powerful computational facility. With a grant from the W.M. Keck Foundation in 2000, the center established a technology-rich Ecological and Evolutionary Genetics Facility. This advanced laboratory provides a full range of high-throughput. DNA- sequencing equipment, a DNA microarray facility and high-performance computers. Several adjunct appointments and collaborative projects strengthen research activities in the center. These activities include interdisciplinary investigations of microbial diversity with scientists at the Woods Hole Oceanographic Institution, molecular ecology studies at the MBL Ecosystem Center's Plum Island LTER site, physiology R43 R44 Annual Report studies of acidophilic protists with the MBL BioCurrents Research Center, and collaborative efforts to study mechanisms and patterns of evolution with faculty of Harvard University, the Harvard School of Public Health, and the University of Sydney, Australia. Future expansion in the Josephine Bay Paul Center will focus upon molecular evolution of global infectious disease and genome sciences. Resident Core Investigators Sogin, Mitchell. Director and Senior Scientist Cornell, Neal, Senior Scientist Cummings, Michael, Assistant Scientist McArthur, Andrew, Staff Scientist II Morrison, Hilary. Staff Scientist II Riley. Monica. Senior Scientist Wernegreen, Jennifer, Assistant Scientist Adjunct Scientists Halanych. Ken, Woods Hole Oceanographic Institution Meselson, Matthew, Harvard University Patterson, David, University of Sydney Teske, Andreas, Woods Hole Oceanographic Institution Laboratory of Neal Cornell Dr. Neal Cornell, a senior scientist at the Marine Biological Laboratory, played a key role in designing and attracting new faculty to the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution. Dr. Cornell passed away in 2000. but all of us who knew him cherish fond memories and harbor a deep gratitude for his contributions to science and the MBL community. Research in Dr. Cornell's laboratory, which continued to pursue his research goals through the end of 2000, was concerned with the comparative molecular biology of genes that encode the enzymes for heme biosynthesis. These efforts placed particular emphasis on 5-aminolevulinate synthase, the first enzyme in the pathway. Because the ability to produce heme from common metabolic materials is a near universal requirement for living organisms, these genes provide useful indicators of molecular aspects of evolution. For example. 5-aminolevulinate synthase in vertebrate animals and simple eukaryotes such as yeast and Plasmodium falciparum have high sequence similarity to the enzyme from the alpha-purple subgroup of eubacteria. This supports the suggestion that alpha-purple bacteria are the closest contemporary relatives of the ancestor of eukaryotic mitochondria. The analysis also raises the possibility that plant and animal mitochondria had different origins. Aminolevulinate synthase genes in mitochondria-containing protists are currently being analyzed to obtain additional insight into endosymbiotic events. Also, genes of primitive chordates are being sequenced to gain information about the large-scale gene duplication that played a very important role in the evolution of higher vertebrates. Other studies in the laboratory have been concerned with the effects of environmental pollutants on heme biosynthesis in marine fish, and it has been shown that polychlorinated hiphenyls (PCBs) enhance the expression of the gene for aminolevulinate synthase. Laboratory of Michael P. Cummings Our research is in the area of molecular evolution and genetics and includes examination of patterns and processes of sequence evolution. We use methods from molecular biology, population genetics, systematics, statistics, and computer science. The basis for much of the research is comparative; it includes several levels of biological organization, and involves both computer-based and empirical studies. A major research focus is analysis genotype-phenotype relationships using tree-based statistical models (decision trees) and extension of this methodology. Current investigations in this area examine how gene sequence data can be used to understand and predict drug resistance in tuberculosis, variation in color vision, and basic immune system functions at the molecular level. For example, using drug resistance in M\cobacterium tuberculosis as a model system, we are investigating how well phenotype (level of drug resistance) can be predicted with genotype information (DNA sequence data). Understanding evolution of drug resistance, and developing accurate methods for its prediction using DNA sequence data, can help in assessing potential resistance in a more timely fashion and circumvent the need for culturing bacteria. More generally, the relationship of genotype to phenotype is a fundamental problem in genetics, and through these investigations we hope to gain insight. The primary empirical work in the laboratory involves examination of opsins, proteins involved in color vision, from local ' species of Odonata (dragonflies and damselflies). Other projects include a review of genetic diversity in plants using coalescence-based analyses and the genetic consequences of reserve designs in conservation. Suff Cornell. Neal W.. Senior Scientist Faggart. Maura A., Research Assistant Staff Cummings, Michael P.. Assistant Scientist Mclnemey, Laura A., Research Assistant II Year-Round Research R45 Visiting Investigators Clegg. Michael T., University of California, Riverside Clegg. Janet, University of California. Riverside Neel, Maile C., University of California, Riverside Visiting Graduate Students Church, Sheri A., University of Virginia Garcia- Verela, Martin, Universidad Nacional Autonoma de Mexico Undergraduates Myers. Daniel S., Pomona College Waring. Molly E.. Harvey Mudd College Laboratory of Monica Riley The genome of the bacterium Escherichia coli contains all of the information required for a free-living chemoautotrophic organism to live, adapt, and multiply. The information content of the genome can be dissected from the point of view of understanding the role of each gene and gene product in achieving these ends. The many functions of E. coli have been organized in a hierarchical system representing the complex physiology and structure of the cell. In collaboration with Dr. Peter Karp of SRI International, an electronic encyclopedia of information has been constructed on the genes, enzymes, metabolism, transport processes, regulation, and cell structure of E. coli. The interactive EcoCyc program has graphical hypertext displays, including literature citations, on nearly all of E. call metabolism, all genes and their locations, a hierarchical system of cell functions and some regulation and transport processes. In addition, the E. coli genome contains valuable information on molecular evolution. We are analyzing the sequences of proteins of E. coli in terms of their evolutionary origins. By grouping like sequences and tracing back to their common ancestors, we learn not only about the paths of evolution for all contemporary E. coli proteins, but we extend even further back before E. coli, traversing millennia to the earliest evolutionary times when a relatively few ancestral proteins served as ancestors to all contemporary proteins of all living organisms. The complete genome sequence of E. coli and sophisticated sequence analysis programs permit us to identify evolutionarily related protein families, determining ultimately what kinds of unique ancestral sequences generated all of present-day proteins. The data developed in the work has proven to be valuable to the community of scientists sequencing other genomes. E. coli data serve as needed reference points. Staff Riley. Monica. Senior Scientist Liang. Ping. Staff Scientist I McCormack, Tom, Postdoctoral Scientist Nahum, Laila, Postdoctoral Scientist Pelegrini-Toole. Alida, Research Assistant II Serres. Margerethe. Postdoctoral Scientist Laboratory of Mitchell L. Sogin This laboratory employs comparative phylogenetic studies of genes and genomes to define patterns of evolution that gave rise to contemporary biodiversity on the planet earth. The laboratory is especially interested in discerning how the eukaryotic cell was invented as well as the identity of microbial groups that were ancestral to animals, plants, and fungi. The laboratory takes advantage of the extraordinary 1 conservation of ribosomal RNAs to define phylogenetic relationships that span the largest of evolutionary distances. These studies have overhauled traditional eukaryotic microbial classifications systems. The laboratory has discovered new evolutionary assemblages that are as genetically diverse and complex as plants, fungi, and animals. The nearly simultaneous separation of these eukaryotic groups (described as the eukaryotic "Crown") occurred approximately one billion years ago and was preceded by a succession of earlier diverging protist lineages, some as ancient as the separation of the prokaryotic domains. Many of these early branching life forms are represented by parasitic protists including Giardia lamklia, which is a significant human parasite. Because of its medical importance and relevance to understanding the evolutionary history of eukaryotes. we have initiated a project to determine the entire genome sequence of Giardia lainMia. In addition to identifying other genes that will be of value for unraveling sudden evolutionary radiations that cannot be resolved by rRNA comparisons, this project will provide insights into the presence or absence of important biochemical properties in the earliest ancestors common to all eukaryotic species. Finally, this project has revealed important features of genome architecture that require a reconsideration of available mechanisms for controlling gene expression in eukaryotes. A second research theme is the study of microbial life in extreme environments and molecular-based investigations of diversity and gene expression in microbial communities. Using the ribosomal RNA database and nucleic acid-based probe technology, it is possible to detect and monitor microorganisms, including those that cannot be cultivated in the laboratory. This strategy has uncovered new habitats and major revelations about geographical distribution of microorganisms. We are particularly interested in protists that thrive in acid mine drainages and the characterization of physiological mechanisms that allow their growth at extraordinarily low (<2.0) pH levels. Our investigations of gene expression in microbial communities is based upon the premise that microorganisms are the primary engines of our biosphere. They orchestrate all key processes in geochemical cycling, biodegradation. and in the protection of entire ecosystems from environmental insults. They are responsible for most of the primary production in the oceans. Microbial creatures of untold diversity have complex chemistries, physiologies, developmental cycles, and behaviors. With the powerful tools of high-throughput DNA sequencing and DNA microarrays for massive parallel expression studies, we can directly measure how microbial gene expression patterns in an entire ecosystem respond to changing chemical and physical parameters. We will employ an experimental paradigm that links biogeochemical processes with ever-changing temporal and spatial distributions of microbial populations and their metabolic properties. The concurrent measurement of biogeochemical parameters, community-wide gene expression patterns, and spatial descriptions of microbial populations offers a means to understand the structure and function of biogeochemical machinery at different levels of biological organization. We seek to discover the links between biological diversity and the resilience and stability of biogeochemical transformations. Staff Sogin. Mitchell L.. Director and Senior Scientist Amaral Zettler. Linda. Postdoctoral Scientist Beaudoin, David. Research Assistant Bressoud. Scott. Laboratory Technician Eakin. Nora, Research Assistant Edgcomb. Virginia. Postdoctoral Scientist Fair, Rebecca, Research Assistant Gao. Lingqiu, Research Assistant II Kim. Ulandt. Research Assistant Kysela, David. Research Assistant Laan. Maris. Research Assistant II R46 Annual Report Lim, Pauline, Executive Assistant Luders. Bruce. Research Assistant McArthur. Andrew. Postdoctoral Scientist Medina, Monica, Postdoctoral Scientist Morrison. Hilary G., Postdoctoral Scientist Nixon. Julie. Postdoctoral Scientist Sansone. Rebecca. Executive Assistant Schlichter. Mimi, Executive Assistant Shulman, Laura, Senior Research Assistant Shakir, Muhammed Afaq. Postdoctoral Scientist Visiting Investigators Bahr. Michele. The Ecosystems Center Campbell, Robert, Serono Laboratories. Inc. Crump, Byron. The Ecosystems Center Laboratory of Jennifer Wernegreen The work in this lab focuses on the evolution of bacteria that complete their life cycles within or closely related with eukaryotic host cells. These symbiotic prokaryotes include well-known parasites as well as obligately mutualistic bacteria that provide nutritional or other benefits to their hosts. By virtue of their host associations, endosymbionts may have smaller population sizes and experience different selective forces than their free-living bacterial relatives. These changes in population size and selection may each contribute to the features shared by many endosymbiont genomes, such as low genomic G + C (guanine + cytosinel contents, small genome sizes, and elevated rates of DNA sequence evolution. Our research explores the molecular and evolutionary mechanisms that shape these characteristics of endosymbiont genomes, with a focus on mutualistic endosymbionts of insects and obligate pathogens of animals. One aim of this lab is to differentiate the effects of genetic drift, directional mutation pressure, and natural selection on molecular evolution of symbiotic and free-living bacteria. Our primary approach has been to compare patterns of DNA sequence divergence at homologous loci across symbiotic and related free-living bacterial species. These comparisons have revealed a strong effect of genetic drift and directional mutational pressure on sequence evolution in Buclmera aphidicola. the vertically transmitted endosymbiont of aphids, compared to its close free-living relative, Escherichia cn/i. Recently, our molecular phylogenetic analyses have shown that Buclmera lacks horizontal gene transfer that is typical of many free-living bacterial groups. On-going and future work will explore the molecular evolution of other insect endosymbionts in the gamma-3 Proteobacteria. including the obligate bacterial associates of carpenter ants (Camponotus). We also employ full genome comparisons to identify genes that have been lost in small endosymbiont genomes, and to compare patterns of genome reduction in mutualistic and pathogenic lineages. Of particular interest is the substantial loss of proof-reading and DNA repair loci from several symbiont genomes, which may elevate mutational rates and biases in these species. Staff Wernegreen. Jennifer. Assistant Scientist BioCurrents Research Center The BioCurrents Research Center (BRC) is a national resource of the National Institutes of Health, part of the Biomedical Technology Resource Program of the NCRR. As with all such resources it has two main goals: 1 1 to research and develop new biomedical technologies, and 2) to make specialized technologies available to visiting biomedical investigators. The emphasis of the BRC is on the physiology of cellular transport mechanisms, particularly as they influence the boundary conditions in the media adjacent to the plasma membrane. To this end we develop new microsensor technologies that operate in a self- referencing mode. We offer access to ion-selective, electrochemical, and biosensor devices, coupled to advanced imaging techniques and electrophysiological approaches combinations unique to the BRC. The BRC has seen a marked expansion in year 2000 after a successful competitive renewal in December of 1999. This resulted in an increase in staff, which included the appointment of two Assistant Scientists: Stefan McDonough and Orian Shirihai. Two new postdoctoral researchers also joined the group in 2000: Sung-Kwon Jung and Andreas Hengstenberg. as did Laurel Moore and Robert Lewis in support roles. Towards the end of 2000 we added Mark Messerli, who works with both the BRC and Bay Paul Center. The current structure of the resource comprises the core support facility and three independent laboratories, as well as a number of affiliate endeavors where the members of the Center work closely with colleagues in the MBL and the regional medical schools. In particular, we have strong links with the MBL program in Architectural Dynamics in Living Cells, the Laboratory for Reproductive Medicine, and the Bay Paul Center. Our involvement with regional hospitals includes Boston Medical Center (diabetes). Massachusetts General Hospital (protein trafficking), and Women and Infants (reproductive biology). In summary, the core in-house research emphasis is on biophysics of calcium transport and regulation (S. McDonough), the molecular biology Year-Round Research R47 of transport processes (O. Shirihail. and sensor development and the biology of transport mechanisms (P.J.S. Smith). In addition, the BRC is developing an online database of pharmacological compounds. The database has made considerable progress over the past year and should be openly available by the summer of 2001. It will be accessible through our web page at . The Center supports an extensive outreach program to regional and national universities, medical schools, and hospitals, and publishes extensively in the field of cellular transport. Over the past year we have continued to host a diverse group of visiting investigators whose studies have ranged from ion transport and metabolic studies at the single cell level to mapping ion flux associated with the olfactory sensilla of the intact blue crab. Overall our emphasis remains on biomedical studies using the specialized microsensors available, particularly those designed to measure flux of calcium, potassium, hydrogen, oxygen, nitric oxide, and ascorbate. Under development are the newer biosensors and electro- optical probes. The Center also maintains other core support facilities, such as a fully equipped cell culture facility, electrode manufacture, and microinjection systems which, as available, we also open to the general scientific community. Staff Smith, Peter J.S.. Director and Senior Scientist Hammar. Kasia. Research Assistant III Hengstenberg, Andreas, Visiting Postdoctoral Fellow Jung. Sung-Kwon, Postdoctoral Researcher Lewis, Robert, Electronic Support McDonough, Stefan. MBL Assistant Scientist McLaughlin. Jane A.. Research Assistant HI Messerli. Mark. NASA Research Fellow Moore. Laurel. Database Development Sanger. Richard H.. Research Assistant III Shirihai, Orian, MBL Assistant Scientist Laboratory of Stefan McDonough Calcium ions trigger many cellular functions including neurotransmission, muscle contraction, regulation of cell membrane excitability, and the activation of enzymatic cascades. A major route of calcium entry into a cell is through voltage-gated calcium ion channels, proteins found in the plasma membrane of every excitable cell and many nonexcitable cells. These proteins form channels that open to allow a selective influx of calcium ions into the cell when the cell fires an electrical spike. Calcium channels are current or potential targets for clinical drugs treating cardiac arrhythmia, epilepsy, hypertension, pain, diabetes, and brain damage after stroke. Research in this laboratory focuses on the channels that conduct calcium entry, the mechanisms controlling calcium levels within the cell, and the tools to distinguish among different types of calcium channels. Experiments are carried out using patch-clamp electrophysiology on mammalian neurons, mammalian cardiac myocytes. or cloned calcium channels expressed in nonexcitable cells. One effort, in collaboration with the laboratories of Bruce and Barbara Furie and of Alan Rigby. is to identify and characterize conotoxins that target voltage-gated ion channels. Other experiments use the self-referencing ion-selective and oxygen sensors of the BioCurrents Center, in collaboration with the Laboratory of Peter Smith. Current areas of research include the effects of zinc ions on calcium channels, a possible cause of ischemic neuronal damage; calcium channel biophysics during the cardiac ventricular action potential; the metabolic cost to the heart of maintaining calcium homeostasis during resting and excited states; and the mechanisms of activation of alternatively spliced forms of neuronal N-type calcium channels. Laboratory of Orian Shirihai Erythroid differentiation involves expression of a set of unique transport and enzymatic systems to support a robust induction of hemoglobin synthesis. Active communication between the mitochondria! matrix and cytosol is essential for heme biosynthesis. The first step, production of aminolevulinic acid (ALA), occurs in the inner matrix. ALA is transported to the cytosol and eventually converted to coproporphynnogen III. which reenters the mitochondrion and. upon further modifications, is joined with iron to form heme. This product is then transported out of the inner matrix for assembly of cytochromes or hemoglobin. Thus, at least four mitochondria! transport steps are required. Although the enzymatic steps in heme synthesis are well characterized, little is known about how biosynthetic intermediates are shuttled across mitochondria] membranes. While malfunctioning of these transporters most probably underlie hematologic and neurologic diseases, their substrates are photoactivated toxic molecules used in photo-dynamic therapy for cancer; the mechanism of transport into the target organelle is of major interest. A novel mitochondria! transporter, discovered by Dr. Shirihai. has been the focus of research in the lah. This protein, named ABC-me (for ATP-binding cassette-mitochondrial erythroid). localizes to the mitochondria! inner membrane and is expressed at particularly high levels in erythroid tissues of embryos and adults. ABC-me is induced during erythroid maturation in cell lines and primary hematopoietic cells, and its over-expression enhances hemoglobin synthesis in erythroleukemia cells. Members of the ABC transporter superfamily have been implicated in numerous human diseases, including cystic fibrosis (CFTR), adrenoleukodystrophy (ALDP). Zellweger's syndrome R48 Annual Report (PMP70), progressive familial intrahepatic cholestasis (SPGP). and Stargardt macular dystrophy (ABCR). To explore the functional role of this transporter, the lab is generating a knockout mouse and cell line, which would serve as a tool to study the biophysics and biochemistry of this transporter as well as the phenotype appearing in the absence of this gene. ABC-me represents a novel member of the ABC superfamily with a potentially important role in erythroid development. In collaboration with Dr. Weiss from the University of Pennsylvania and Dr. Orkin from Harvard, we have recently cloned and sequenced the human homologue of ABC-me and started screening multiple samples from candidate patients send to us by physicians from the United States, Italy, and England. Laboratory of Peter J.S. Smith The activities of this laboratory center on instrument development, providing new insights into cellular transport mechanisms, and applying these devices to biomedical problems. Much of the biological work is done in collaboration with visiting investigators to the BRC. Over the past year an increasing body of work has been undertaken using the new amperometric microsensors capable of measuring single cell movement of gases such as oxygen and nitric oxide. We continue to investigate the metabolic cost of ion regulation in single cultured neurons. In collaboration with Mitch Sogin of the Bay Paul Center, a new research area was launched, investigating the transport physiology of extremophilic organisms. The emphasis is to understand how membrane- borne transport proteins continue to regulate a near neutral cytosol while being exposed to acidic conditions of pH 1 or 2. This project is funded through the NSF LEXEN program, attracting Mark Messerli to the group, first as an MBL summer fellow but now on a full-time basis funded by a NASA Fellowship. In sensor design, we have made great progress with the new generation of amperometric sensors, incorporating an immobilized enzyme. Our attempts have focused on glucose and, thanks to the efforts of Sung-Kwon Jung, our first single cell glucose flux measurements have been achieved. Hybrid, electro-optical sensors have also been a focus over the past year, where, working with visiting fellow Andreas Hengstenberg, we have successfully built a micro-oxygen sensor on the surface of a single mode fiber optic capable of stimulating a preloaded cellular reporter molecule. In collaboration with Stefan McDonough, this technology has been successful in imaging calcium activity while recording oxygen uptake from a single cardiac myocyte. Boston University Marine Program Faculty Atema. Jelle. Professor of Biology, Director Dionne, Vincent, Professor of Biology Golubic. Stjepko, Professor of Biology Kaufman, Les. Associate Professor of Biology Lobel. Phillip. Associate Professor of Biology Voigt, Rainer. Research Associate Professor Ward, Nathalie. Lecturer Staff Decarie. Linette. Senior Staff Coordinator DiNunno. Paul, Research Assistant, Dionne Lab Hall, Sheri. Program Manager McCafferty. Michelle. Administrative Assistant Gilbert, Niki. Program Assistant Postdoctoral In vestigators Grasso. Frank. Atema Laboratory Kaatz. Ingrid, Lobel Laboratory Trott. Thomas, Atema Laboratory Visiting Fucitltv and Investigators Hanlon, Roger. Marine Biological Laboratory Hecker, Barbara, Hecker Consulting Moore. Michael. Woods Hole Oceanographic Institution Simmons, Bill. Sandia National Laboratory Wamwright. Norman, Marine Biological Laboratory Graduate Students PhD Students Existing Cole. Marci Dale, Jonathon Dooley. Brad Hauxwell, Jennifer Herrold. Ruth Kroeger, Kevin Ma. Diana Miller, Carolyn Oliver. Steven Stieve. Erica Tomasky. Gabrielle York, Joanna Zettler, Erik New Frenz. Christopher Skomal. Gregory Masters Students Existing Allen, Christel Atkinson, Abby Bentis. Christopher Bowen, Jennifer Casper. Brandon Cavanaugh. Joseph Chichester. Heather D'Ambrosio. Alison Errigo, Michael Evgenidou. Angeliki Fredland. Inga Frenz. Christopher Grable. Melissa Grebner. Dawn Kollaros. Maria Konkle, Anne Lamb. Amy Lawrence, David Lever. Mark Levine, Michael Malley. Vanessa Martel. David Neviackas, Justin Oweke. Ojwang William Perez. Edmundo Pugh. Tracy Year-Round Research R49 Ramon, Marina Ripley, Jennifer Roycrot't. Karen Smith. Spence Stueckle, Todd Sweeny. Melissa Tuohy-Sheen, Elizabeth Watson, Elise Weiss. Erica Wright, Dana New Bogomolni. Andrea Bonacci. Lisa deHart. Pieter Estrada. James Rice. Aaron Rutecki. Deborah Shriver, Andrea Summers. Erin Wittenmyer. Robert Undergraduate Students Spring 00 Kwong. Grace Loewensteiner. David Fall 00 Batson. Melissa Bergan. Michael Boynton. Seth Burke, Alexandra Buynevitch, Artem Christie. Mark Combellick. Lindsay Darrell. Andrea De Falco, Tomaso Dewey. Hollis Faloon, Kristine Feeney, Brett Hendricks. Amber Hunt. Tamah Kavountzis, Erol Kim, Joanne Kowalchuk. Lynn Linehan, Candace Lynch. Michael Mattei, Bethany McKay. Breda McOwen. Micah Miller, Jessica Morano. Janelle Newville. Melinda Nichols, Dominica Nguyen, Jean O'Neil. Diane Rohrbaugh, Lynne Sorocco, Debra Tubbs. Mollie Wezensky, Eryn Yopak, Kara Zink. Rachel Summer 2000 Interns O'Connell. Timmy Laboratory of Jelle Ate in a Many organisms and cellular processes use chemical signals as their main channel of information about the environment. All environments are noisy and require some form of filtering to detect important signals. Chemical signals are transported by turbulent currents, viscous flow, and molecular diffusion. Receptor cells extract chemical signals from the environment through various filtering processes. In our laboratory, fish, marine snails, and Crustacea have been investigated for their ability to use chemical signals under water. Currently, we use the lobster and its exquisite senses of smell and taste as our major model to study the signal-filtering capabilities of the whole animal and its narrowly tuned chemoreceptor cells. Research in our laboratory focuses on amino acids, which represent important food signals for the lobster, and on the function and chemistry of pheromones used in lobster courtship. We examine animal behavior in the sea and in the lab. This includes social interactions and chemotaxis. To understand the role of chemical signals in the sea we use real lobsters and untethered small robots. Our research includes measuring and computer modeling odor plumes and the water currents lobsters generate to send and receive chemical signals. Other research interests include neurophysiology of receptor cells and anatomical studies of receptor organs and pheromone glands. Laboratory of Vincent Dionne Odors are powerful stimuli. They can focus the attention, elicit behaviors (or misbehaviors), and even resurrect forgotten memories. These actions are directed by the central nervous system, but they depend upon the initial transduction of chemical signals by olfactory receptor neurons in the nasal passages. More than just a single process appears to underlie odor transduction, and the intracellular pathways that are used are far more diverse than once thought. Hundreds of putative odor receptor molecules have been identified that work through several different second messengers to modulate the activity of various types of membrane ion channels. Our studies are being conducted with aquatic salamanders using amino acids and other soluble chemical stimuli that these animals perceive as odors. Using electrophysiological and molecular approaches, the research examines how these cellular components produce odor detection, and how odors are identified and discriminated. R50 Annual Report Laboratory of Les Kaufman Current research projects in the laboratory deal with speciation and extinction dynamics of haplochromine fishes in Lake Victoria. We are studying the systematics, evolution, and conservation genetics of a species flock encompassing approximately 700 very recently evolved taxa in the dynamic and heavily impacted landscape of northern East Africa. In the lab we are studying evolutionary morphology, behavior. and systematics of these small, brightly colored cichlid fishes. Another area of study is developmental and skeletal plasticity in fishes. We are studying the diversity of bone tissue types in fishes, differential response to mineral and mechanical challenge, and matrophic versus environmental effects in the development of coral reef fishes. We also study the biological basis for marine reserves in the New England fisheries. We are involved in collaborative research with NURC. NMFS, and others studying the relative impact on groundfish stocks of juvenile habitat destruction versus fishing pressure. Laboratory of Phillip Label Fishes are the most diverse vertebrate group and provide opportunities to study many aspects of behavior, ecology, and evolution. We primarily study 1) how fish are adapted to different habitats, and 2) behavioral ecology of species interactions. Current research focuses on fish acoustic communications. We are also conducting a long-term study of the marine biology of Johnston Atoll, Central Pacific Ocean. Johnston Atoll has been occupied continuously by the military since the 1930s and has proven to be a unique opportunity for assessing the biological impacts of island industrialization and its effects on reefs. Johnston Atoll is the site of the U.S. Army's chemical weapons demilitarization facility, JACADS. Laboratory of Ivan Valiela A focus of our work is the link between land use on watersheds and consequences in the receiving estuarine ecosystems. The work examines how landscape use and urbanization increase nutrient loading to groundwater and streams. Nutrients in groundwater are transported to the sea, and. after biogeochemical transformation, enter coastal waters. There, increased nutrients bring about a series of changes on the ecological components. To understand the coupling of land use and consequences to receiving waters, we study the processes involved, assess ecological consequences, and define opportunities for coastal management. A second long-term research topic is the structure and function of salt marsh ecosystems, including the processes of predation. herbivory. decomposition, and nutrient cycles. Center for Advanced Studies in the Space Life Sciences In 1^45. the NASA Life Sciences Division and the Marine Biological Laboratory established a cooperative agreement with the formation of the Center for Advanced Studies in the Space Life Sciences (CASSLS at MBL). The Center's overall goals are to increase awareness of the NASA Life Sciences Program within the basic science community and to examine and discuss potential uses of microgravity and other aspects of spaceflight as probes to provide new insights to fundamental processes important to basic biology and medicine. Through symposia, workshops and seminars, CASSLS advises NASA and the biological science community on a wide variety of topics. Through fellowships. CASSLS supports summer research for investigators in areas pertinent to the aims of NASA life sciences. Since the Center began its operations in July 1995. more than 400 people have attended eight CASSLS workshops. Typically these workshops last for two to four days and feature an international array of scientists and NASA/International space agency staff. In many cases, workshop chairs have a long-time association with the MBL. Workshop schedules incorporate many opportunities for interaction and discussion. A major outcome for workshops is the publication of proceedings in a peer-reviewed journal. Moreover, our meetings introduce outstanding biologists to research questions and prominent scientists involved in gravitational biology and the NASA Life Sciences Program. The Center sponsored one workshop in 2000: "Invertebrate Sensory Information Processing: Implications for Biologically Inspired Autonomous Systems," chaired by Dr. Frank Grasso. The Center sponsored one Fellow during the summer of 2000: Dr. Mark Messerli, Biology Department. Purdue University. He conducted research in reaulation of cytoplasmic pH in eucaryotic acidophiles in collaboration with Dr. Peter J.S. Smith and Dr. Mitchell Sogin of the Marine Biological Laboratory. In addition, two scholars-in-residence worked with the Center in 2000: Dr. Richard Wassersug of Dalhousie University and Dr. Lawrence Schwartz of the University of Massachusetts, Amherst. Finally, the Center worked with colleagues in Astrobiology and the Josephine Bay Paul Center to offer a stimulating lecture series. Staff Blazis, Diana E.J., Director Oldham. Pamela A., Administrative Assistant Scholars-in-residence Schwartz, Lawrence Wassersug. Richard The Ecosystems Center The Ecosystems Center carries out research and education in ecosystems ecology. Terrestrial and aquatic scientists work in a wide variety of ecosystems ranging from the streams, lakes and tundra of the Alaskan Arctic (limits on plant primary production) to sediments of Massachusetts Bay (controls of nitrogen cycling), to forests in New England (effects of soil warming on carbon and nitrogen cycling), and South America (effects on greenhouse gas fluxes of conversion of rain forest to pasture) and to large estuaries in the Gulf of Maine (effects on plankton and benthos of nutrients and organic matter in stream runoff). Many projects, such as those dealing with carbon and nitrogen cycling in forests, streams, and estuaries, use the stable isotopes I3 C and L N to investigate natural processes. A mass spectrometer facility is available. Data from field and laboratory research are used to construct mathematical models of whole-system responses to change. Some ot these models are combined with geographically referenced data to produce estimates of how environmental changes affect key ecosystem indexes, such as net primary productivity and carbon storage, throughout the world's terrestrial biosphere. The results of the Center's research are applied, wherever possible, to the questions of the successful management of the natural resources ot the earth. In addition, the ecological expertise of the staff is made available to public affairs groups and governmental agencies who deal with problems such as acid rain, coastal eutrophication, and possible carbon dioxide-caused climate change. The Semester in Environmental Science was offered again in Fall Year-Round Research R51 2000. Fifteen students from seven colleges participated in the program. The center also offers opportunities for postdoctoral fellows. Administrative Staff Hobble, John E., Co-Director Melillo. Jerry M., Co-Director Foreman, Kenneth H.. Associate Director. Semester in Environmental Studies Berthel, Dorothy J.. Administrative Assistant Donovan. Suzanne J.. Executive Assistant Moniz. Priscilla C.. Administrative Assistant, Semester in Environmental Studies Nunez, Guillermo. Research Administrator Scanlon, Deborah G., Executive Assistant Seifert. Mary Ann, Administrative Assistant Scientific Staff Hobbie. John E.. Senior Scientist Melillo. Jerry M.. Senior Scientist Deegan, Linda A.. Associate Scientist Giblin. Anne E.. Associate Scientist Herbert. Darrell A.. Staff Scientist Holmes. Robert M., Staff Scientist Hopkinson. Charles S.. Senior Scientist Hughes. Jeffrey E.. Staff Scientist Nadelhoffer. Knute J., Senior Scientist Neill, Christopher, Assistant Scientist Peterson. Bruce J., Senior Scientist Rastetter. Edward B., Associate Scientist Shaver, Gaius R., Senior Scientist Steudler. Paul A., Senior Research Specialist Tian, Hanqin. Staff Scientist Vallino, Joseph J., Assistant Scientist Williams. Mathew. Assistant Scientist Educational Staff Appointments Buzby. Karen. Postdoctoral Scientist Cieri. Matthew D.. Postdoctoral Scientist Crump, Byron. Postdoctoral Scientist Garcia-Montiel. Diana C., Postdoctoral Scientist LeDizes-Maurel, Severine, Postdoctoral Scientist Kappel-Schmidt, Inger, Postdoctoral Scientist Nordin. Annika, Postdoctoral Scientist Raymond. Peter. Postdoctoral Scientist Sommerkom. Martin. Postdoctoral Scientist Tobias. Craig R., Postdoctoral Scientist Williams, Michael R., Postdoctoral Scientist Technical Staff Ahrens, Toby. Research Assistant Bahr. Michele P.. Research Assistant Bettez. Neil D., Research Assistant Burnette, Donald W.. Research Assistant Claessens. Lodevicus H.J.M., Research Assistant Colman. Benjamin P.. Research Assistant Eldridge. Cynthia. Research Assistant Fox, MaryKay. Research Assistant Garritt. Robert H., Senior Research Assistant Gay, Marcus O., Research Assistant Goldstein. Joshua H.. Research Assistant Jablonski. Sarah A., Research Assistant Jillson. Tracy A., Research Assistant Kelsey, Samuel. Research Assistant Kicklighter, David W., Senior Research Assistant Kwiatkowski. Bonnie L., Research Assistant Laundre. James A.. Senior Research Assistant Lezberg, Ann. Research Assistant Lux, Heidi, Research Assistant Merson. Rebekah. Research Assistant Micks, Patricia, Research Assistant Morriseau, Sara. Research Assistant Nolin. Amy L.. Research Assistant Nowicki, Genevieve, Research Assistant O'Brien. Kathenne A.. Research Assistant Otter, Marshall L., Research Assistant Pan, Shufen, Research Assistant Peterson, G. Gregory, Research Assistant Regan. Kathleen M., Research Assistant Ricca, Andrea. Research Assistant Schwamb, Carol. Laboratory Assistant Slavik. Karie A.. Research Assistant Thieler, Kama K., Research Assistant Tholke, Kristin S.. Research Assistant Thomas. Suzanne M.. Research Assistant Tucker, Jane, Senior Research Assistant Vasiliou, David S., Research Assistant Weston. Nathaniel B., Research Assistant Wright. Amos, Research Assistant Wyda. Jason C.. Research Assistant Consultants Bowles. Francis P., Research Systems Consultant Bowles, Margaret C.. Administrative Consultant Visiting Scientists and Scliolars DeStasio. Bart, SES Faculty Fellow, Lawrence College Koba. Keisuke, Graduate School of Informatics, Kyoto University. Japan Laboratory of Aquatic Biomedicine Our laboratory has developed the Spisula solidissima embryo model to study mechanisms of neurotoxicology. We have shown that polychlonnated biphenyls (PCBs) selectively target the nervous system during development. We are now linking up and down regulation of the p53 family of genes with neuronal cell development using new probes developed by this laboratory. In the second line of research, we are using the clam leukemia model to investigate how environmental chemicals influence the progression of leukemia. Further, we are studying whether mutations in p53 alter the pathogenesis of leukemia in populations of Mya arenaria. Field work to Nova Scotia showed that leukemia in Mya was also detected in Sydney. N.S., which is heavily polluted with a variety of industrial chemicals. Staff Reinisch. Carol L., Senior Scientist Cox, Rachel. Postdoctoral Scientist Jessen-Eller. Kathryn, Postdoctoral Scientist Kreiling. Jill. Postdoctoral Scientist Stephens, Ray, Adjunct Scientist R52 Annual Report Visiting Scientists Shohel, Stephen, University of California, San Francisco Walker, Charles. University of New Hampshire Student Miller. Jessica. Boston University Laboratory of Cell Communication completed pollen and stratigraphic analyses, now being prepared for publication, of the first transglacial lake core from a forested site (Maicuru inselberg) in the eastern Amazon lowlands. Our collaborators at the Florida Institute of Technology and the University of Cincinnati identified chemical changes in the early sedimentary history of Lake Pata in the western Amazon lowlands that show a strong synchroneity with insolation changes associated with the precessional component of astronomical climate forcing back to marine oxygen isotope stage 7, this being the first such signal from the equatorial lowlands. In 2000 we also concluded a paleoenvironmental reconnaissance of the Lake Nicaragua region and are developing plans for raising a long core from the lake. Established in 1994, this laboratory is devoted to the study of intercellular communication. The research focuses on the cell-to-cell channel, a membrane channel built into the junctions between cells. This channel provides one of the most basic forms of intercellular communication in organs and tissues. The work is aimed at the molecular physiology of this channel, in particular, at the mechanisms that regulate the communication. The channel is the conduit of growth- regulating signals. It is instrumental in a basic feedback loop whereby cells in organs and tissues control their number; in a variety of cancer forms it is crippled. This laboratory has shown that transformed cells lacking communication channels lost the characteristics of cancer cells, such as unregulated growth and tumorigenicity. when their communication was restored by insertion of a gene that codes for the channel protein. Work is now in progress to track the channel protein within the cells from its point of synthesis, the endoplasmic reticulum. to its functional destination in the plasma membrane, the cell-to-cell junction, by expressing a fluorescent variant of the channel protein in the cells. Knowledge about the cellular regulation of this process will aid our understanding of what goes awry when a cell loses the ability to form cell-to-cell channels and thus to communicate with its neighbors, thereby taking the path towards becoming cancerous. Another line of work is taking the first steps at applying information theory to the biology of cell communication. Here, the intercellular information spoor is tracked to its source: the macromolecular intracellular information core. The outlines of a coherent information network inside and between the cells are beginning to emerge. Staff Loewenstein. Werner, Senior Scientist Rose, Birgit, Senior Scientist Jillson, Tracy, Research Assistant Laboratory of Paul Colinvaux Staff Colinvaux. Paul. Adjunct Scientist Laboratory of Ayse Dosemeci The laboratory investigates molecular processes that underlie synaptic modification. The main project is to clarify how the frequency of activation at a synapse can determine whether the synapse will he potentiated (strengthened) or depressed (weakened) through the participation of an enzyme called CaM kinase II. This enzyme is regulated by autophosphorylation on distinct sites in the presence and absence of calcium. Biochemical studies with isolated postsynaptic density fractions are conducted to clarity functional consequences of CaMKII autophosphorylation in response to sequential exposure to calcium-containing and calcium-free media at different temporal patterns. In a related project, a new affinity-based method is being developed for the preparation of postsynaptic density fractions of high purity. In collaboration with Dr. Lucas Pozzo-Miller (University of Alabama. Birmingham), we are studying changes in the activity of CaMKII in hippocampal slices following high-frequency and low-frequency electrical stimulation to generate long-term potentiation and long-term depression, respectively. Related projects in collaboration with Dr. Thomas Reese (NIH. NINDS) include studies on the redistribution of CaMKII and structural changes in the post-synaptic density in response to excitatory stimuli. Staff Dosemeci. Ayse. Adjunct Scientist Visiting Invcstigatur Pozzo-Miller. Lucas, University of Alabama We have shown that accumulated pollen data now leave little doubt that the Amazon lowlands remained forested without fragmentation throughout glacial cycles. Changes in relative abundance of trees within the highly diverse forests can be seen in the pollen record, however, particularly in response to changing temperature. The pollen vocabulary for the Amazon on which this conclusion is based has been codified in our Amazon Pol/en Manual and Atlas with text in Portuguese as well as English for the benefit of Brazilian researchers. We show that the Amazon ecosystems yield two kinds of pollen signals: what might be called the "classical" signal by wind-blown pollen, which allows separation of biomes and many edaphically constrained facies of Ama/on forests such as var-ea or igapo; and a species-rich signal from animal-pollinated trees washed from the immediate watershed or catchment of the sedimentary basin. With our collaborators in Brazil and the University of Florida we Laboratory of Barbara Furie and Bruce Furie y-Carboxyglutamic acid is a calcium-binding amino acid that is found in the conopeptides of the predatory marine cone snail, Conus. This laboratory has been investigating the biosynthesis of this amino acid in Conus and the structural role of y-carboxyglutamic acid in the conopeptides. This satellite laboratory relates closely to the main laboratory, the Center for Hemostasis and Thrombosis Research, on the Harvard Medical School campus in Boston: the main focus of the primary laboratory is the synthesis and function of y-carboxyglutamic acid in blood-clotting proteins and the role of vitamin K. Cone snails are obtained from the South Pacific and maintained in the Marine Resources Center. Until recently, the marine cone snail had been the sole invertebrate known to synthesize y-carboxyglutamic acid (Gla). Year-Round Research R53 The venomous cone snail produces neurotoxic conopeptides, some rich in Gla, which it injects into its prey to immobilize it. To examine the biosynthetic pathway for Gla. we have studied the Comix carboxylase which converts glutamic acid to y-carboxyglutamic acid. This activity has an absolute requirement for vitamin K. The Conux carboxylase substrates contain a carboxylation recognition site on the conotoxin precursor. Given the functional similarity of mammalian vitamin K- dependent carboxylases and the vitamin K-dependent carboxylase from Conns textile, we hypothesized that structurally conserved regions would identity sequences critical to this common functionality. Furthermore, we examined the diversity of animal species that maintain vitamin K-dependent carboxylation to generate y-carboxyglutamic acid. We have cloned full-length carboxylase homologs from the beluga whale (Delphinaptenis leitcas) and toadfish (Opsanus tail}. In addition, we have partially cloned the carboxylase gene from chicken (Gal/ns gallus), hagfish (\l\\inc glutinosa), horseshoe crab (Limulus polyphemus), and cone snail (Conns textile) in order to compare these structures to the known bovine, human, rat. and mouse cDNA sequences. Comparison of the predicted amino acid sequences identified a highly conserved 32-amino acid residue region in all of these putative carboxylases. In addition, this amino acid motif is also present in the Drosophila genome and identified a Drosophihi homolog of the y-carboxylase. Assay of hagfish liver and Drosophila demonstrated carboxylase activity in these non-vertebrates. More recently, we hu\e cloned the entire vitamin K-dependent carboxylase gene from the cone snail. Conns textile. The predicted amino acid sequence shows most region-, are similar to the mammalian sequence, and that there is about 40% sequence identity overall. These results demonstrate the broad distribution of the vitamin K-dependent carboxylase gene, including a highly conserved motif that is likely critical for enzyme function. The vitamin K-dependent biosynthesis of y-carboxyglutamic acid appears to be a highly conserved function in the animal kingdom. Novel y-carboxyglutamic acid-containing conopeptides have been isolated from the venom of Conns textile. The amino acid sequence, amino acid composition, and molecular weights of these peptides have been determined. For several peptides. the cDNA encoding the precursor conotoxin has been cloned. The three-dimensional structure of some of these Gla-containing conopeptides are being determined by 2D NMR spectroscopy. Complete resonance assignments of conotoxin P14.1 were made from 2D 'H NMR spectra via identification of intraresidue spin systems using 'H-'H through-bond connectivities. NOESY spectra provided d N , d NN . and d N NOE connectivities and vicinal spin-spin coupling constants 3 J HNu were used to calculate torsion angles. Structure determination is nearing completion. The goal of this project is to determine the structural role of y-carboxyglutamic acid in the Gla-containing conotoxins and other y-carboxyglutamic acid-containing proteins. Staff Fune. Barbara C. Adjunct Scientist Furie. Bruce. Adjunct Scientist Begley. Gail, Scientist I Czerwiec. Eva, Postdoctoral Fellow Rigby, Alan. Adjunct Scientist Stenflo. Johan. Visiting Scientist Laboratory of Shiny a I none Scientists in this laboratory study the molecular mechanism and control of mitosis, cell division, cell motility, and cell morphogenesis, with emphasis on biophysical studies made directly on single living cells, especially developing eggs in marine invertebrates. Development of biophysical instrumentation and methodology, such as the centrifuge polarizing microscope, high-extinction polarization optical and video microscopy, digital image processing techniques including dynamic stereoscopic imaging, and exploration of their underlying optical theory are an integral part of the laboratory's efforts. Staff Inoue, Shinya, Distinguished Scientist Burgos. Mario, Visiting Scientist Goda. Makoto. Visiting Scientist Baraby. Diane. Laboratory Assistant Knudson. Robert. Instrument Development Engineer MacNeil, Jane. Executive Assistant Laboratory of Rudolf Oldenbourg The laboratory investigates the molecular architecture of living cells and of biological model systems using optical methods for imaging and manipulating these structures. For imaging cell architecture non- invasively and non-destructively. dynamically and at high resolution, we have developed a new polarized light microscope (Pol-Scope). The Pol- Scope combines microscope optics with new electro-optical components, video, and digital image processing for fast analysis of specimen birefringence over the entire viewing field. Examples of biological systems currently investigated with the Pol-Scope are microtubule-based structures (asters, mitotic spindles, single microtubules); actin-based structures (acrosomal process, stress fibers, nerve growth cones); zona pellucida of vertebrate oocytes; and biopolymer liquid crystals. Staff Oldenbourg. Rudolf, Associate Scientist Shribak. Michael. Staff Scientist Knudson. Robert. Instrument Development Engineer Baraby, Diane. Laboratory Assistant Laboratory of Michael Rabinowitz This laboratory investigates environmental geochemistry and epidemiology. Areas of recent activity include modeling lead bioavailability, writing a history of lead biokinetic models, performing a case control survey of tea drinking and oral cancer in Taiwan, quantifying the transport and fate of various sources of residential lead exposure, and serving on several advisory boards of Superfund research projects in Boston and New York. Current activity focuses on characterizing lead paints and pigments. Hundreds of lead poisoning lawsuits are filed every year against landlords, but no compensation has ever been paid by the half dozen companies that made lead pigments, because it has not been possible to identify the specific manufacturer. This research has been funded by the Eagle Picher Trust. Other activity, sponsored by HLID. involves using stable isotopes of lead to determine the relative importance of various household surfaces (doors, floors, windows, walls) as sources of indoor dust lead levels. Dust lead is the major predictor of childhood lead exposure and poisoning. This would allow for more focused deleading. Another effort has been using historical fire insurance maps to locate and identify unrecognized hazardous waste sites. Staff Rabinowitz. Michael. Associate Scientist R54 Annual Report Laboratory for Reproductive Medicine, Brown University and Women and Infants Hospital, Providence Work in this laboratory centers on investigating cellular mechanisms underlying female infertility. Particular emphasis is placed on the physiology of the oocyte and early embryo, with the aim of assessing developmental potential and mitochondria dysfunction arising from mtDNA deletions. The studies taking place at the MBL branch of the Brown Laboratory use some of the unique instrumentation available through the resident programs directed by Rudolf Oldenbourg and Peter J.S. Smith. Most particularly, non-invasive methods for oocyte and embryo study are being sought. Of several specific aims, one is to use the Pol-Scope to analyze the dynamic birefringence of meiotic spindles. An additional aim is to study transmembrane ion transport using non- invasive electro-physiological techniques available at the BioCurrents Research Center. The newly developed oxygen probe offers the possibility of looking directly at abnormalities in the mitochondria arising from accumulated mtDNA damage. Our laboratory has also focused on studying the mechanism underlying age-associated infertility in terms of oocyte quality and has attempted to rescue developmentally compromised oocytes or embryos through nuclear-cytoplasmic transfer technology. We have characterized oxidative stress-induced mitochondrial dysfunctions, developmental arrest, and cell death in early embryos using animal models. Ultimately, this laboratory aims to produce clinical methods for assessing preimplantation embryo viability. an advance that will significantly contribute to the health of women and children. Staff Keefe, David. Director Liu. Lin, Adjunct Scientist Trimarchi, James, Adjunct Scientist Laboratory of Osainu Shimomitra Aequorin, from the jellyfish Aequorea aequorea, was the first calcium-sensitive photoprotein discovered by us in 1961. Because of its high sensitivity to Ca 2+ and biological harmlessness. the protein has been widely used as a probe to monitor intracellular free calcium levels. Aequorin is a unique protein that contains a high level of energy for light emission in the molecule, and its structure has been the target of many studies in the past. The complete 3-dimensiona] structure of aequorin was finally obtained by X-ray crystallography 38 years after its discovery, in collaboration with three other laboratories. Aequorin is found to be a globular molecule having four helix-loop-helix "EF-hand" domains, of which three can bind Ca 2 + . The molecule contains coelenterazine-2-hydroperoxide in its hydrophobic core cavity, as the chromophoric ligand which decomposes into coelenteramide and carbon dioxide accompanied by the emission of blue light. Staff Shimomura. Osamu. Senior Scientist, MBL, and Boston University School of Medicine Shimomura. Akemi. Research Assistant Laboratory of Robert B. Silver The members of this laboratory study how living cells make decisions. The focus of the research, typically using marine models, is on two main areas: the role of calcium in the regulation of mitotic cell division (sea urchins, sand dollars, etc.) and structure and function relationships of hair cell stereociliary movements in vestibular physiology (oyster toadfish). Other related areas of study, i.e. synaptic transmission (squid), are also, at times, pursued. Tools include video light microscopy, multispectral, subwavelength, and very high-speed (sub-millisecond frame rate) photon counting video light microscopy, telemanipulation of living cells and tissues, and modeling of decision processes. A cornerstone of the laboratory's analytical efforts is high performance computational processing and analysis of video light microscopy images and modeling. With luminescent, fluorescent, and absorptive probes, both empirical observation and computational modeling of cellular, biochemical, and biophysical processes permit interpretation and mapping of space-time patterns of intracellular chemical reactions and calcium signaling in living cells. A variety of in vitro biochemical, biophysical, and immunological methods are used. In addition to fundamental biological studies, the staff designs and fabricates optical hardware, and designs software for large video image data processing, analysis, and modeling. Staff Silver. Robert, Associate Scientist Visiting Investigators Hummel, John, Argonne National Laboratory Jiang, Yi, Los Alamos National Laboratory Keller, Bruce. SUNY Upstate Medical University Kriebel, Mahlon, SUNY Upstate Medical University Pappas, George, University of Illinois School of Medicine Pearson. John. Los Alamos National Laboratory Laboratory of Norman Wainwright The mission of the laboratory is to understand the molecular defense mechanisms exhibited by marine invertebrates in response to invasion by bacteria, fungi, and viruses. The primitive immune systems demonstrate unique and powerful strategies for survival in diverse marine environments. The key model has been the horseshoe crab Limitliis polyphemus. tinnitus hemocytes exhibit a very sensitive LPS- triggered protease cascade which results in blood coagulation. Several proteins found in the hemocyte and hemolymph display microbial binding proteins that contribute to antimicrobial defense. Commensal or symbiotic microorganisms may also augment the antimicrobial mechanisms of macroscopic marine species. Secondary metabolites are being isolated from diverse marine microbial strains in an attempt to understand their role. Microbial participation in oxidation of the toxic gas hydrogen sulfide is also being studied. Staff Wainwright. Norman. Senior Scientist Child, Alice. Research Assistant Williams. Kendra, Research Assistant Visiting Investigator Anderson, Porter. University of Rochester Year-Round Research R55 Laboratory of Seymour Ziginan This laboratory is investigating basic mechanisms of photooxidative stress to the ocular lens due to environmentally compatible UVA radiation. This type of oxidative stress contributes to human cataract formation. Other studies are the search for and use of chemical antioxidants to retard the damage that occurs. Cultured mammalian lens epithelial cells and whole lenses in vitro are exposed to environmentally compatible UVA radiation with or without previous antioxidant feeding. The following parameters of lens damage are examined: molecular excitation to singlet states via NADPH (the absorber): cell growth inhibition and cell death; catalase maclivauon: cytoskeletal description (of actin. tubulin. integrins); and cell membrane damage (lipid oxidation, loss of gap junction integrity and intercellular chemical communications). Thus far. the most successful antioxidant to reduce these deficiencies is alpha-tocopherol ( 10 ^ig/ml) and tea polvphenols (especially from green tea). The preliminary phases of the research are usually carried out using marine animal eyes (i.e., smooth dogfish) as models. Our goal is to provide information that will suggest means to retard human cataract formation. Staff Zigman. Seymour. Laboratory Director, Professor of Ophthalmology. Boston University Medical School Rafferty. Keen, Research Associate, Boston University Medical School Rafferty. Nancy S.. Research Associate. Boston University Medical School Zigman. Bunnie R., Laboratory Manager, Boston University Medical School The Marine Resources Center The Marine Resources Center (MRC) a modern, 32.000-square-foot structure features advanced facilities for maintaining and culturing aquatic organisms essential to advanced biological, biomedical. and ecological research. In addition to research, the MRC provides a variety of important, complementary services to the MBL community through its Aquatic Resources Division, its Aquaculture and Engineering Division, and its administrative division. The MRC and its life support systems have increased the ability of MBL scientists to conduct research and have inspired new concepts in scientific experiments. Vigorous research programs focusing on basic biological and biomedical aquatic models are currently being developed at the Center, including the Program in Scientific Aquaculture and the Program in Sensory Biology and Neuroethology. Research and educational opportunities for established investigators, postdoctoral fellows, and graduate and undergraduate students are available at the MRC. Investigators and students find that the MRC's unique life support and seawater engineering systems make this a favorable environment in which to conduct research using a variety of aquatic organisms and flexible tank space for customized experimentation on live animals. Staff Hanlon. Roger. Director and Senior Scientist Carroll. James. Life Support Technical Assistant Enos, Edward. Aquatic Resources Division Superintendent Gilland, Edwin. Research Associate Grossman, William, Marine Specimen Collector/Diving Safety Officer Hanley. Janice. Water Quality and Animal Health Technician Klimm. William. Licensed Boat Captain R/V Gemma Kuzirian, Alan. Associate Scientist Linnon. Beth. Special Projects Coordinator Mebane. William. Aquaculture and Engineering Division Superintendent Santore. Gabrielle. Executive Assistant Sexton, Andrew. Marine Organism Shipper Smolowitz. Roxanna, MBL Veterinarian Sullivan. Daniel, Boat Captain Tassinari, Eugene. Senior Biological Collector Whelan. Sean. Diver/Marine Specimen Collector Summer and Full Employees and Volunteers Buynevich. Artem, Work-study Student. Boston University Carroll. Amanda, Volunteer Dimond, Jay, Diver/Collector Douton, Kate, AmeriCorps Assistant Faloon. Kristine. Work-study Student. Boston University Gudas. Chris. Diver/Collector Kavountzis. Erol. Work-study Student, Boston University Miraglia, Valentina. Volunteer. Universita di Napoli "Federico II." Italy Potter. Chris, Diver/Collector Reynolds, Justin, Diver/Collector Robhins, Gillian. Volunteer Rohrbaugh. Lynne. Work-study Student. Boston University Tubbs, Mollie. Work-study Student. Boston University Zucchini, Mossimo, Volunteer. Universita di Napoli "Federico II," Italy Laboratory of Roger Hanlon This laboratory investigates the behavior of cephalopods and other marine organisms with an integrative biology approach focused at the organismal level. Molecular, cellular, and ecological approaches are used to complement this organismal approach, and there is emphasis on sensory biology and behavioral ecology. Laboratory studies on the mechanisms and functions of polarized light sensiiivity in cephalopods are underway. Olfactory sensing by Nautilus (which functions in food detection and location as well as mate choice) is being studied in the laboratory. Visual features that octopuses use for maze learning are also being investigated. Lab experiments in large indoor seawater tanks are being conducted to determine how male squids. Laligo pealeii, use visual, then contact, chemical cues in egg capsules to initiate highly robust agonistic behavior. The functional morphology and neurobiology of the chromatophore system of cephalopods are studied on a variety of cephalopod species, and image analysis techniques are being developed to study crypsis and the mechanisms that enable cryptic body patterns to be neurally regulated by visual input. Various aspects of predation, antipredator defenses, and reproduction are conducted in field sites worldwide. Sexual selection theory is being tested using squid and cuttlefish. Field and laboratory studies focus on mechanisms of agonistic behavior. R56 Annual Report female mate choice, and sperm competition. The latter studies involve DNA Fingerprinting to determine paternity and help assess alternative mating tactics. Population structure and reproductive success in several highly valuable squid fisheries (Loligo vulgaris reynaudii in South Africa, Loligo pealeii in the N.E. United States. Loligo opalescens in California) are being assessed for fishery management and conservation. We also culture species of commercial and biomedical importance. For example, the toadfish Opsanus beta is used in vestibular research related to human medicine, yet the species is difficult to obtain from nature. Thus, we are performing the first mariculture experiments to culture toadfish through the life cycle to provide the biomedical community with high-quality experimental animals. Such an approach lightens the impact of collecting toadfish from the natural environment. Staff Hanlon. Roger, Senior Scientist Ament, Seth. Summer Research Assistant, Harvard University Boal. Jean. Adjunct Scientist Buresch. Kendra, Research Assistant Conroy. Lou-Anne. Summer Research Assistant. Dartmouth College Gilles, Nicole, REU Intern, University of Minnesota, Duluth Lee. Tony. REU Intern. Duke University Richmond, Hazel, Research Assistant Shashar. Nadav. Adjunct Scientist Sussman, Raquel, Investigator Vaughan. Katrina. Summer Research Assistant, University of Wales. Swansea Visiting Investigators Baddeley. Roland, University of Sussex, England Baker. Robert. New York University Cavanaugh. Joseph. Boston University Marine Program Chiao, Chuan-Chin. Grass Fellow. University of Maryland. Baltimore County Cronin, Thomas. University of Maryland, Baltimore County Grable, Melissa, Boston University Marine Program Hall. Karina. University of Adelaide, Australia Hatfield, Emma, FRS Marine Laboratory, Aberdeen. Scotland Karson. Miranda. Michigan State University Kier, William. University of North Carolina Mensinger. Allen. University of Minnesota, Duluth Messenger, John, University of Cambridge. England Osorio, Daniel. Investigator. University of Sussex, England Saidel, William, Rutgers University Schmolesky, Matthew, Grass Fellow. University of Utah Laboratory of Alan M. Ktizirian Research in the laboratory explores the functional morphology and ultrastructure of various organ systems in molluscs. The program includes mariculture of the nudibranch, Hemrissenda crassicomis, with emphasis on developing reliable culture methods for rearing and maintaining the animal as a research resource. The process of metamorphic induction by natural and artificial inducers is being explored in an effort to understand the processes involved and as a means to increase the yield of cultured animals. Morphologic studies stress the ontogeny of neural and sensory structures associated with the photic and vestibular systems which have been the focus of learning and memory studies, as well as the spatial and temporal occurrence of regulatory and transmitter neurochemicals. Concurrent studies detailing the toxic effects of lead on Hennissendu learning and memory. feeding, and the physiology of cultured neurons are also being conducted. New studies include cytochemical investigations of the Ca 2+ /GTP binding protein, calexcitin, and its modulation with learning and lead exposure. Collaborative research includes histochemical investigations on strontium's role in initiating calcification in molluscan embryos (shell and statoliths), iminunoeytochemical labelling of cell-surface antigens, neurosecretory products, second messenger proteins involved with learning and memory, as well as intracellular transport organelles using mono- and polyclonal antibodies on squid (Loligo pealeii) giant axons and Hermissenda sensory and neurosecretory neurons. Additional collaborations involve studying neuronal development of myelin, myelination defects, as well as nerve regeneration and repair in phylogenetically conserved nervous systems. Additional collaborative research includes DNA fingerprinting using RAPD-PCR techniques in preparation for isogenic strain development of laboratory-reared Hennissenda and hatchery produced bay scallops (Argopectin irradians) with distinct phenotypic markers for rapid field identification and stock assessments. Recently obtained funding will expand this research to perform population genetic analyses of currently designated yellowtail flounder ( Limanda ferruginea I stocks occurring in the Northeast Fisheries Region. Systematic and taxonomic studies of nudibranch molluscs, to include molecular phylogenetics, are also of interest. Staff Kuzirian. Alan M., Associate Scientist Kozlowski. Robbin. Research Technician Visiting Investigators Chikarmane, Hemant, Investigator Clay. John R.. NINDS/NIH Gould. Robert. NYS Institute of Basic Research Summer Intents Kingston, Margaret. REU Intern, Wake Forest University Kuzirian. Mark, REU Intern. University of Rhode Island Lee, Tony. REU Intern. Duke University Laboratory of Roxanna Smolowitz This laboratory investigated the pathogenesis of aquatic animal diseases using traditional pathological methods combined with in situ molecular methods. Research conducted during 2000 included 1) examination of hard-clam-strain susceptibility to a protistan disease agent named Quahog Parasite Unknown, and the methods of transmission of that organism between infected and uninfected animals; 2) detection of disease-causing, protozoan organisms (MSX and SSO) in eastern oysters using PCR and in situ hybridization techniques; and 3) evaluation of inbred strains of oysters for resistance to disease vs. productivity as commercial aquaculture stock. Work began on the determination of possible causes of lobster shell disease in the northeast. Staff Smolowitz, Roxanna. MBL Veterinarian Brothers, Christine, Laboratory Assistant Cavanaugh. Joseph. Laboratory Assistant Marks, Ernie. AmeriCorps member Stukey. Jetley, Laboratory Assistant Summers, Erin, Laboratory Assistant Tirrell, Kerri-Ann. AmeriCorps member Honors Friday Evening Lectures June 16 June 23 June 30 July 7 July 14 July 20-21 July 28 August 4 August 1 1 Edward Pearce, Cornell University "Life-long Enemies The Relationship Between Schistosomes and Their Hosts" Stephen Farrand, University of Illinois at Champaign-Urbana "Agrobacterium lumefaciens: Nature's Own Genetic Engineer" Judith Eisen. University of Oregon "From Lobster to Zebrafish: Development of Identified Neurons" (Lang Lecture) David Anderson, California Institute of Technology "Stem Cells from the Mammalian Nervous System: Basic Biology and Implications for Tissue Repair" Sallie Chisholm (Penny). Massachusetts Institute of Technology "The Invisible Forest: Marine Phytoplankton and Climate" Eve Marder. Brandeis University 1) "Activity-dependent Timing of Neurons and Synapses in Adult and Developing Circuits" 2) "Neurotransmitter Modulation of Neural Networks" (Forbes Lectures) Jean-Pierre Changeux, Institut Pasteur "Chemical Communications in the Brain: Nicotine, Receptors, and Learning" (Classman Lecture) Susan Middleton/David Liittschwager "Paradise Up Close: Hawaii Endangered Eden" Titia de Lange. The Rockefeller University "At the Ends of Our Chromosomes: the Key to Immortality" Fellowships and Scholarships In 2( Hill, the MBL awarded research fellowships to 22 scientists from around the world. The MBL awarded scholarships to 77 students in the MBL's summer courses as well as 4 post-course research awards. Donors provided gifts for endowed and expendable funds amounting to $256.090 in support of the research fellowships program and an additional $738.107 to provide scholarships to students in MBL courses. Those funds that received donations in 2000 are listed below. The individuals who received fellowships and scholarships are listed beginning on p. R58. Robert Day Allen Fellowship Fund Drs. Joseph and Jean Sanger The American Society for Cell Biology Scholarships The American Society for Cell Biology Frederik B. Bang Fellowship Fund Mrs. Betsy G. Bang Max Burger Endowed Scholarship for the Embryology Course Dr. Max M. Burger Jean and Katsuma Dan Fellowship Fund Drs. Joseph and Jean Sanger Mrs. Eleanor Steinbach Bernard Davis Fellowship Fund Mrs. Elizabeth M. Davis The Mac V. Edds, Jr. Endowed Scholarship Fund Dr. and Mrs. James D. Dr. and Mrs. Kenneth T. Edds Dr. Louise M. Luckenbill-Edds Gerald D. and Ruth L. Fischbach Endowed Scholarship Fund Drs. Gerald and Ruth Fischbach R57 Thomas B. Grave and Elizabeth F. Grave Scholarship Estate of Elizabeth F. Grave Daniel S. and Edith T. Grosch Scholarship Fund Mr. Gustav Grosch Ms. Laura Grosch and Mr. Herb Jackson Aline D. Gross Scholarship Fund Dr. and Mrs. Paul R. Gross Dr. and Mrs. Benjamin Kaminer Technic, Inc. E. E. Just Endowed Research Fellowship Fund The Cole Memorial Family Fund R58 Annual Report Fred Karush Endowed Library Readership Dr. and Mrs. Laszlo Lorand Dr. and Mrs. Arthur M. Silverstein Keffer Hartline Fellowship Fund Mrs. Elizabeth K. Hartline Dr. and Mrs. Edward F. MacNichol. Jr. Dr. William H. Miller Dr. Torsten Wiesel and Ms. Jean Stein Dr. and Mrs. Stephen Yeandle Kuffler Fellowship Fund Dr. and Mrs. Edward A. Kravitz MBL Associates Endowed Scholarship Fund MBL Associates Mrs. Anne L. Meigs-Brown James A. and Faith Miller Fellowship Fund Drs. David and Virginia Miller Frank Morrell Endowed Memorial Scholarship Dr. Leyla deToledo-Morrell Mountain Memorial Fund Dr. and Mrs. Dean C. Allard. Jr. Ms. Brenda J. Bodian Dr. and Mrs. Benjamin Kaminer Mr. and Mrs. Thomas H. Roberts Dr. and Mrs. R. Walter Schlesinger Neural Systems and Behavior Scholarship Fund Anonymous ( 1 ) Mr. Srdjan D. Antic Drs. Mary Atkisson and Joel White Bristol-Myers Squibb Corporation Dr. and Mrs. John Byrne Ms. Lu Chen Dr. Warren M. Grill Dr. Anya C. Hurlbert Dr. Eve Marder Dr. Mark W. Miller Fellowships Awarded Mr. Rex R. Robison Ms. M. Jade Zee Nikon Fellowship Nikon Instruments. Inc. The Plum Foundation John E. Dowling Fellowship Fund The Plum Foundation William Townsend Porter Scholarship Fund for Minority Students William Townsend Porter Foundation Phillip H. Presley Scholarship Fund Carl Zeiss, Inc. Science Writing Fellowships Program Support American Society for Biochemistry and Molecular Biology American Society for Cell Biology American Society for Photobiology FASEB NASA (Astrobiology Institute) National Institutes of Health Office of Science Education National Institutes of Health National Cancer Institute National Science Foundation Biological Sciences National Science Foundation Office of Polar Programs Society for Integrative and Comparative Biology Times Mirror Foundation Waksman Foundation for Microbiology The Washington Post Company 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 Evelyn and Melvin Spiegel Fellowship Fund The Sprague Foundation Drs. Joseph and Jean Sanger H. B. Steinbach Fellowship Fund Mrs. Eleanor Steinbach Eva Szent-Gyorgyi Scholarship Fund Dr. and Mrs. Laszlo Lorand Drs. Joseph and Jean Sanger Dr. Andrew and Ms. Ursula Szent-Gyorgyi Universal Imaging Fellowship Fund Universal Imaging Corporation The Irving Weinstein Endowed Scholarship The Irving Weinstein Foundation, Inc. Walter L. Wilson Endowed Scholarship Dr. Paul N. Chervm Dr. Jean R. Wilson Mr. and Mrs. Ross A. Wilson Young Scholars/Fellows Program Drs. Harriet and Alan Bernheimer Dr. and Mrs. Francis P. Bowles Dr. and Mrs. Sherwin J. Cooperstein Mrs. Elizabeth M. Davis Mrs. James R. Glazebrook Ms. Jeannie Leonard Mrs. Barbara C. Little Dr. and Mrs. Anthony Liuzzi Drs. Luigi and Elaine Mastroianni Drs. Matthew and Jeanne Meselson Dr. and Mrs. Philip Person Drs. Dorothy Skinner and John Cook Drs. Ann Stuart and John Moore Mr. and Mrs. Richard Yoder MBL Summer Research Fellows Srdjan Antic. M.D., is a post-doctoral fellow in the Department of Cellular and Molecular Physiology at Yale University School of Medicine. New Haven, CT. The title of his project is "Selective modulation of the dendritic membrane potential." Dr. Antic is funded by the Baxter Postdoctoral Fellowship, the Charles R. Crane Fellowship Fnntl, the MBL A\\Oi'i(ift's Felltni'shift Fund, and the .liiine A. and Fuitli Miller Memorial Fund. Honors R59 Roberto Bru//one. Ph.D.. is ;in Associate Professor at the Institut Pasteur in Paris. France. The title of this research is "Molecular analysis of the biophysical properties of connexin channels that mediate cell- cell communication between neurons of the vertebrate retina and CNS." Dr. Bruzzone is funded by the Erik R. Fries Endowed Fellowship, the MBL Associates Fellowship Fund, and the H. B. Steinbach Fellowship Fund. Mario H. Burgos. M.D., is an Emeritus Professor of the Medical School at the Universidad Nacional de Cuyo and Director of the Institute de Histologia y Embriologia. National Council of Research (CONICET). Argentina. His research project is titled. "Mechanism of release of spermatozoa from the Serroli cells." He is also collaborating with Dr. Shinya Inoue in the identification of the birefringent zones in Arbacia eggs after centrifuge polarizing microscopy. Dr. Burgos is funded by the Chairman 's Fellowship. Jean-Pierre Changeux is a Professor at the College de France and Director of the Unit of Molecular Neurobiology at the Institut Pasteur in Paris. He is the author of Neunmal Man: The Biology of Mind (1990). Dr. Changeux has been awarded a Herbert W. Rand Fellowship for his research. Debra Ann Fadool. Ph.D.. is an Associate Professor in the Biomedical Research Facility at Florida State University. Tallahassee, FL. The title of her research project is "Chemosensory transduction in the vomero-nasal organ." Dr. Fadool is funded by the Frederik B. Bang Fellowship Fund, the Ann E. Kammer Memorial Fellowship Fund, and the MBL Associates Fellowship Fund. Mariano A. Garcia-Bhnco, M.D.. Ph.D.. is Associate Professor of Genetics. Microbiology, and Medicine at Duke University Medical Center. Durham. NC. He is a Raymond and Beverly Sackler Scholar and is a member of the Biochemistry Study Section of the National Institutes of Health. The Josiah Macy. Jr. Foundation is funding his research. George G. Holz. Ph.D.. has been appointed Associate Professor at New York University School of Medicine to establish a Diabetes Research Laboratory at New York University Medical Center. His summer research project is "Spatial distribution of second messengers in pancreatic fi-cells." Dr. Holz is funded by the Erik B. Fries Endowed Fellowship, the Frank R. Lillie Fellowship Fund, and the MBL Associates Fellowship Fund. Peter Koulen. Ph.D.. is a postdoctoral associate in the Department of Pharmacology at Yale University School of Medicine, New Haven. CT. The title of his research project is "Calcium signaling in zebrafish neurons mediated by differentially distributed intracellular calcium channels." Dr. Koulen has received funding from the Erik B. Fries Endowed Fellowship and the Litc\ B. Lemann Fellowship Fund. George Langford. Ph.D., is the Ernest Everett Just Professor of Natural Sciences and Professor of Biological Sciences at Dartmouth College. Hanover. NH. His research project is titled "Actin-based vesicle transport in the squid giant axon." Dr. Langford is funded by the Josiah Macy, Jr. Foundation. Jennifer LaVail. Ph.D.. is Professor of Anatomy/ Ophthalmology at the University of California. San Francisco. She is spending her second summer at the MBL. Her research project is tilled "HSV tegument proteins in axonal transport and microtubule architecture." Dr. LaVail is funded by an MBL Research Fellowship and the Evelyn and Melvin Spiegel Fellowship Fund. Carolyn Lesser has published eight children's books and numerous articles. She has also served as a consultant and lecturer. Ms. Lesser was awarded a Science Writing Fellowship in 1999. and is a Desk Reader at the MBLAVHO1 Library in 2000. Ms. Lesser is funded by the Fred Karu.sh Endowed Library Readership. Jeffrey Magee. Ph.D., is an Assistant Professor in the Neuroscience Center at Louisiana State University Medical Center. New Orleans. Louisiana. The title of his research is "Mechanisms of Ca~* entry into hippocampal neurons." Dr. Magee is funded by the MBL Associates Fellowship Fund and the Lnc\ B. Lemann Fellowship f- mid Antonio Malgaroli. Ph.D.. is a Professor in the Unit of Neurobiology of Learning at the University of San Rafaele. Milan. Italy. The title of his summer research is "Presynaptically silent synapses in the hippocampus." Dr. Malgaroli is funded by the Herbert W. Rand Fellowship and the Frank R. Lillie Fellowship Fund. * Mark Messerli. Ph.D.. is a Research Associate in the Department of Biological Sciences at Purdue University. West Lafayette. IN. The title of his research project is "Regulation of cytoplasmic pH in eucaryotic acidophiles." Dr. Messerli is funded by a NASA Life Sciences Program Fellowship. * Timothy Mitchison. M.D., is a Professor in the Department of Cell Biology at Harvard Medical School, Boston. MA. His research project is titled "Optical Approaches to Cell Division." The Universal Imaging Corporation is funding Dr. Mitchison. David Ogden. Ph.D.. is a Principal Investigator at the National Institute for Medical Research in London. England. The title of his research is "Central electrosensory processing in the skate." Dr. Ogden is funded by an MBL Associates Fellowship. ' Oladele A. Ogunseitan, Ph.D.. is an Associate Professor in the Department of Environmental Analysis and Design at the University of California, Irvine. Dr. Ogunseitan returns to the MBL to study "Toxic metal resistance, swarming phenotype. and enzyme polymorphism in Vibrio alginolyticus." Dr. Ogunseitan is funded by the Josiah Macy. Jr. Foundation. ' David Paydarfar, Ph.D.. is an Associate Professor at the Department of Neurobiology at the University of Massachusetts Medical School in Worcester. The title of his research project is "Can noise regulate oscillatory state? In nurnero and in vitro analysis of squid axon membrane." Dr. Paydartar is funded by the Frederick B. Bang Fellowship Fund, the M. G. F. Fuortes Memorial Fellowship Fund, the MBL Associates Fellowship Fund, and the John O. Crane Fellowship Fund. * Peter Saggau. Ph.D.. is an Associate Professor in the Division of Neuroscience at Baylor College of Medicine. Houston. Texas. The title of his summer research project is "Transmission and plasticity at single hippocampal synapses." Dr. Saggau received the Nikon Fellowship. Miduturu Srinivas. Ph.D.. is a Research Associate at the Albert Einstein College of Medicine, Bronx, New York. His research project for the summer is titled "Biophysical properties of gap junctions in retinal neurons." Dr. Srinivas is lunded by the Erik B. Fries Endowed Fellowship and the H. Keffer Hartline Fellowship Fund. Thomas W. White, Ph.D., is an Instructor in the Department of Neurobiology at Harvard Medical School. Boston, MA. His research title is "Gap junctional communication in the retina." Dr. White is funded by the H. Kefter Hartline Fellowship Fund, the Stephen W. Kuffler Fellowship Fund, and the Frank R. Lillie Fellowship Fund. * Iain Stuart Young. Ph.D.. is a Research Associate in the Department of Biology at the University of Pennsylvania. Philadelphia. The title of Dr. Young's summer research is "The molecular mechanisms of relaxation in superfast muscles." Dr. Young is funded by the Robert Day Allen Fellowship Fund, the MBL Research Fellowship Fund, the H. Burr Steinbach Memorial Fellowship Fund, and the Lucy B. Lemann Fellowship Fund. Grass Fellows Leonardo Belluscio. Ph.D.. Duke University Medical Center. Project: "The role of spontaneous activity in the olfactory system." Chuan-Chin Chiao. University of Maryland, Baltimore County. R60 Annual Report Project: "Camouflage in cephalopods: visual control and effectiveness when viewed by predators." Melina Hale. Ph.D.. State University of New York at Stony Brook. Project: "The neural basis of startle behavior and its development in the toadfish (Opsainis tan}." Johann Hofmann. Ph.D.. Stanford University. Project: "The consequences of socially induced differential growth on the retina." Thomas Kuner. M.D.. Duke University Medical Center. Project: "The timing of NSF action in neurotransmitter release probed with photolysis of caged peptides." Brian Link, Ph.D., Harvard University. Project: "Time-lapse analysis of zebrafish retinal cells during development: investigation of lamination mutants." Matthew B. McFarlane, Ph.D.. New York University Medical Center. Project: "Central pathways mediating the horizontal vestibulo- ocular reflex in an elasmobranch, Scyliorliinus canicula." ' Matthew T. Schmolesky, University of Utah. Project: "Visual stimulus encoding in the optic lobe of squid Loligo pealei." Ava J. Udvadia. Ph.D., Duke University Medical Center. Project: "Investigation of signaling pathways that activate axon growth- associated gene expression in regenerating spinal neurons." Jing W. Wang. Ph.D.. Lucent Technologies. Project: "Optical imaging and electrophysiological recording of Drosophila central nervous system: A search for the significance of synchrony." Zachary P. Wills. Harvard Medical School. Project: "The function of dAbl pathway components in neuronal outgrowth and growth cone turning in vitro." MBL Science Writing Fellowships Program Fellows Begos, Kevin, Winston-Sulein Journal Ben-Ari. Elia. BioScience Berger, Cynthia. Finger Lakes Productions Borenstein. Seth. Knight Ridder Newspapers Enright, Leo, BBC Fagin, Dan, Newsday Garber, Ken, Freelance Gorman, Jessica. Discover magazine Hathaway, William, Hartford Courant Helmuth, Laura, Science magazine Mansur. Mike. Kansas City Star Martin, Roger, University of Kansas Marx. Vivien, Freelance Milano, Gianna, Mondadori Publishing Company Nemecek. Sasha. Scientific American Poulson, David, Booth Newspapers Senkowsky, Sonya. Anchorage Daily News Scott, Bari. SoundVision Productions Tangley, Laura, U.S. News and World Report Program Staff Goldman, Robert D.. Northwestern University, Co-Director Hinkle, Pamela Clapp, Marine Biological Laboratory. Administrative Director Rensberger. Boyce, Knight Science Journalism Program. Co-Director Hwids-On Laboratory' Fiwulty Chisholm, Rex, Northwestern University, Biomedical Hands-On Laboratory Director Hobbie, John E., Marine Biological Laboratory, Environment Hands-On Laboratory Co-Director Melillo. Jerry, Marine Biological Laboratory, Environment Hands-On Laboratory Co-Director Palazzo, Robert, University of Kansas, Biomedical Hands-On Laboratory Associate Director Scholarships Awarded The Bruce and Betty Alberts Endowed Scholarship in Physiology Fleegal. Melissa. University of Florida American Society for Cell Biology Colon-Ramos, Daniel, Duke University Bradford. Yvonne. University of Oregon Crespo-Barreto. Juan, University of Puerto Rico Espinoza, Nora, University of Chicago Glater. Elizabeth, Brown University Greenlee, Anne, Marshfield Medical Research Foundation Hubhard. Aida, University of Texas, San Antonio Mah, Silvia, Scripps Institution of Oceanography Powell, Jacqueline, Morehouse School of Medicine Triggs, Veronica, University of Wisconsin, Madison Biology Club of the College of the City of New York Konur. Sila. Columbia University C. Lalor-Burdick Scholarship Powell. Jacqueline, Morehouse School of Medicine Burroughs Wellcome Fund Biology of Parasitism Course Andersson. John. Karolinska Institute D'Angelo, Maximiliano, University of Buenos Aires Dolezal, Pavel, Charles University Ferreira. Ludmilu. Universidade Federal de Minas Gerais Figueiredo. Luisa. University of Porto Gilk, Stacey. University of Vermont Lowell, Joanna, Rockefeller University Martins, Gislaine, Faculdade de Medicina de Ribeirao Preto Murta, Silvane, Centra e Pesquisas Rene Rachou-FIOCRUZ Sehgal, Alfica, Tata Institute of Fundamental Research Ulbert, Sebastian. Netherlands Cancer Institute Villarino. Alejandro, University of Pennsylvania Burroughs Wellcome Fund Frontiers in Reproduction Course Alberio, Ramiro, Ludwig-Maximilian University Allegrucci. Cinzia, Universita degli Studi di Perugia Honors R61 Ashkar, All. University of Guelph Chong. Kowit-Yu. Oregon Regional Primate Research Cenler Heit'etz. Yael. Cornell University Lavoie, Holly. University of South Carolina Majumdar, Subeer, Primate Research Center Powell, Jacqueline, Morehouse School of Medicine Richard, Craig. University of Pittsburgh Sahgal, Namita, Kansas University Medical Center Zhang, Gongqiao, University of Virginia Burroughs Wellcome Fund Modeling of Biological Systems Course Ginsberg, Tara, University of Texas Medical School Immerstrand, Charlotte. Linkoping University Quinteiro, Guillermo, Comision Nacional de Energia Atomica Teng, Ching-Ling. University of Virginia Uppul. Hirdesh. Punjab Agricultural University Genick, Ulrich, The Salk Institute for Biological Studies Hershberg, Uri, Hebrew University Mosavi. Leila. University of Connecticut Health Center Burroughs Wellcome Fund Molecular Mycology Course Austin. W. Lena. Howard University School of Medicine Ibrahim, Ashraf. Harbor-UCLA Medical Center Mol, Pieternella, University of Amsterdam Munro. Carol, University of Aberdeen Perea, Sofia, The University of Texas Health Science Center Spellberg. Brad, Harbor-UCLA Medical Center Spreghini, Elisabetta. Yale University School of Medicine Toenjes, Kurt. University of Vermont Wasylnka. Julie. Simon Eraser University The Ellison Medical Foundation Molecular Biology of Aging Course Bailey. Adina, University of California, Berkeley Baur. Joe. University of Texas Southwestern Bordone. Laura, University of Minnesota Medical School Cui, Wei. Roslin Institute Cypser. James. University of Colorado Filosa, Stefania, IIGB-CNR Furfaro, Joyce. Pennsylvania State University College of Medicine Harper, James, University of Idaho Huang, Xudong, Massachusetts General Hospital-East Johnson, Kristen, Purdue University Konigsberg. Mina. Universidad Autonoma Kostrominova, Tatiana, University of Michigan Luo, Yuan, University of Southern Mississippi Munoz. Demse, University of California Peel, Alyson, The Buck Center for Research in Aging Podlutsky. Andrej. National Institute on Aging. NIH Radulescu, Andreea. Albert Einstein College of Medicine Srivivsan, Chandra. University of California, Los Angeles Tong. Jiayuan (James), Cold Spring Harbor Laboratory Zaid, Ahmed. Stockholm University Caswell Grave Scholarship Fund Bates. Damien, Murdoch Institute Brelidze, Tinatin, University of Miami School of Medicine Cordeiro, Maria Sofia, Instituto Gulbenkian de Ciencia Gong. Ying. California Institute of Technology Menna, Elisabetta, Institute of Neurophysiology. Pisa Prud'homme. Benjamin, Centre de Genetique Moleculaire Daniel S. Grosch Scholarship Fund Neretin, Lev, Shirshov Institute of Oceanography Gary N. Calkins Memorial Scholarship Fund Ellertsdottir. Eh'n. University of Freiburg Aline D. Gross Scholarship Fund Cordeiro, Maria Sofia, Instituto Gulbenkian de Ciencia Johansson. Viktoria. Gtiteborgs Universitet Edwin Grant Conklin Memorial Fund Jhaveri. Dhanisha. Tata Institute of Fundamental Research William F. and Irene C. Diller Memorial Scholarship Fund Jhaveri. Dhanisha. Tata Institute of Fundamental Research Menna. Elisabetta. Institute of Neurophysiology. Pisa The Ellison Medical Foundation Biology of Parasitism Course Gilk, Stacey. University of Vermont Lowell, Joanna, Rockefeller University O'Donnell, Rebecca, University of Melbourne Ralph, Stuart. University of Melbourne Triggs, Veronica, University of Wisconsin, Madison Villarino. Alejandro, University of Pennsylvania William Randolph Hearst Foundation Scholarship Rankin, Kathleen. Oberlin College Rodeheffer, Carey, Emory University Rodgers, Erin, University of Alabama, Birmingham Shatkin-Margolis. Seth. Duke University Takai, Erica. Columbia University Howard Hughes Medical Institute Akay, Turgay. KSIn University Barak. Yoram. Hebrew University Ding. Long. University of Pennsylvania Globerson, Amir. Hebrew University Imai. Kazushi. Columbia University P&S Konur, Sila. Columbia University Krishna. B. Suresh. New York University Kyrkjebo, Vibeke, Sars International Centre Lauritzen, Thomas. University of California. San Francisco Lin, Li-hung, Princeton University McCance. James, University of Leicester Menna, Elisabetta. Institute of Neurophysiology, Pisa R62 Annual Report Nasevicius. Aidas. University of Minnesota Paz. Ron, Hebrew University Medical School Petereit, Christian. Universitat Bielefeld Prud'homme. Benjamin. Centre de Genetique Moleculaire Rela. Lorena, Universidad de Buenos Aires Rokni. Uri. Hebrew University of Jerusalem Schreiber. Susanne. Humboldt Universitat Berlin Shi. Songhai, Cold Spring Harbor Laboratory Singh. Brajesh, Imperial College at Silwood Park Skromne, Isaac, Princeton University Szalisznyo, Krisztina, Hungarian Academy of Sciences Warkman, Andrew, University of Western Ontario Zaar, Annette. Universitat Freiburg International Brain Research Organization Challa, Anil Kumar, Ohio State University Leung. Fung Ping, Hong Kong University of Science and Technology Lupo, Giuseppe, University of Pisa Maldonado. Ernesto, Massachusetts Institute of Technology Arthur Klorfein Scholarship and Fellowship Fund Aspock. Gudrun, University of Basel Gong. Ying, California Institute of Technology Imai. Kazushi, Columbia University P&S KyrkjebO. Vibeke. Sars International Centre Lee, Vivian, Oregon Health Sciences University Prud'homme, Benjamin, Centre de Genetique Moleculaire Skromne, Isaac, Princeton University Frank R. Lillie Fellowship and Scholarship Fund Carroll, Michael. University of Newcastle Costa, Patricia. Universidad Federal do Rio de Janeiro Cotrufo. Tiziana. Institute of Neurophysiology Dayel. Mark, University of California, San Francisco Fleegal. Melissa, University of Florida Marrari. Yannick. Observatoire Oceanographique Petrie, Ryan. University of Calgary Rankin. Kathleen. Oberlin College Shatkin-Margolis. Seth. Duke University Takai. Erica. Columbia University Jacques Loeb Founders' Scholarship Fund Fleegal. Melissa, University of Florida Petrie, Ryan, University of Calgary Shilkrut. Mark. Technion-Israel Institute of Technology Massachusetts Space Grant Consortium Barbour. Jason, University of California. San Francisco Handley. Scott. Centers for Disease Control and Prevention Holland. Brenden. University of Hawaii Longnecker. Krista, Oregon State University McMahun, Katherine, University of California. Berkeley Munroe, Stephen, Marquette University Nepokroelt. MolK. Smithsonian Institution Stone. Karen. University of Alaska Museum. Fairbanks Xie. Gary. 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Co-Vice Chair Anderson. Porter W. li.nK'u. Robert B. Bloom. Kerry S. Burris. John E. Conrad, Mary B., Treasurer Mainer. Robert E. O'Hanley. Ronald P. Speck. William T. \\ cissmann, Gerald Science Council Barlow. Robert B.. Chair (2001 ) Bloom, Kerry S., Chair (2000) Armstrong. Clay M. (2002) Armstrong, Peter (2002) Atema. Jelle (2001) Burris. John E.* Dawidowicz. E. A.* Haimo. Leah (2001) Hopkinson, Charles (2002) Jaffe. Laurinda (2001) Smith. Peter J. S. (2001) Sogin, Mitchell (2002) Speck. William T.* Weeks. Janis C. (2002) Standing Committees of the Board of Trustees Development Speck. William, Chair Anderson. Porter W, Barlow. Robert W. Bay, Frederick Conrad. Mary B. Cox, Martha W. Dowling, John Ebert. James D. Grant. Philip Lakian, John R. Langford. George Lee, Burton J. Miller. G. William Pierce. Jean Steere. William C. Weld, Christopher M. Wiesel. Torsten Facilities and Capital Equipment Anderson, Porter W.. Chair Bay. Frederick Boyer. Barbara Langford. George Pros. 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Woods Hole, MA 02543 (deceased 2000] Adelberg, Edward A., 204 Prospect Street, New Haven, CT 0651 1- 2107 Afzelius, Bjorn, University of Stockholm. Wenner-Gven Institute. Department of Ultrastructure Research. Stockholm. SWEDEN Amatniek, Ernest, (address unknown) Arnold, John M., 329 Sippewissett Road, Falmouth. MA 02540 Copeland, D. Eugene, Marine Biological Laboratory, Woods Hole. MA 02543 Corliss, John O., P.O. Box 2729, Bala Cynwyd. PA 19004-21 16 Costello, Helen M., 137 Carolina Meadows, Chapel Hill, NC 27514-8512 Crouse, Helen, Rte. 3, Box 213, Hayesville. NC 28904 DeHaan. Robert L., Emory University School of Medicine, Department of Anatomy & Cell Biology, 1648 Pierce Drive, Room 108, Atlanta. GA 30322 Dudley, Patricia L., 3200 Alki Avenue SW, #401, Seattle. WA 981 16 Bang. Betsy G., 76 F. R. Lillie Road. Woods Hole. MA 02543 Bartlett, James H., University of Alabama, Department of Physics, Box X70324. Tuscaloosa. 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FL 33149-1322 Cooperstein, Sherwin J., University of Connecticut, School of Medicine. Department of Anatomy, Farmington, CT 06030-3405 Edwards, Charles, 3429 Winding Oaks Drive. Longboat Key. FL 34228 Elliott, Gerald F., The Open University Research Unit, Foxcombe Hall. Berkeley Road, Boars Hill, Oxford OX1 5HR, UK Failla, Patricia M., 2149 Loblolly Lane, Johns Island, SC 29455 Frazier, Donald T., University of Kentucky Medical Center. Department of Physiology and Biophysics, MS501 Chandler Medical Center. Lexington, KY 40536 Gabriel, Mordetai L., Brooklyn College, Department of Biology. 2900 Bedford Avenue. Brooklyn. NY 1 1210 Glusman, Murray, New York State Psychiatric Institute. 722 W. 168th St.. Unit #70, New York. NY 10032 Graham, Herbert, 36 Wilson Road, Woods Hole, MA 02543 Hamburger, Viktor, Washington University, Department of Biology, 740 Trinity Avenue. St. Louis. MO 63130 (deceased 2001) Hamilton, Howard L., University of Virginia, Department of Biology, 238 Gilmer Hall. Charlottesville. VA 22901 Harding, Clifford V.. Jr., 100 Saconesset Road, Falmouth. MA 02540 Haschemeyer, Audrey E. V., 21 Glendon Road. Woods Hole. MA 02543-1406 Hayashi, Teru, 15 Gardiner Road. Woods Hole, MA 02543-1 1 13 Hisaw, Frederick L., (address unknown) Hoskin, Francis C. G., do Dr. John E. Walker. U.S. Army Natick RD&E Center. SAT NC-YSM, Kansas Street, Natick, MA 01760- 5020 R71 R72 Annual Report Hubbard, Ruth, Harvard University, Biological Laboratories, Cambridge. MA 02138 Hunter, W. Bruce, 305 Old Sharon Road. Peterborough, NH 03458- 1736 Hurwitz, Charles, Stratton VA Medical Center, Research Service, Albany, NY 12208 Katz, George, 1636 Brookhouse Drive. Apt. BR131. Sarasota, FL 34731 Kingsbury, John M., Cornell University, Department of Plant Biology, Plant Science Building, Ithaca. NY 14853 K li iiilini/. Lewis, Reed College, Department of Biology. 3203 SE Woodstock Boulevard. Portland. OR 97202 Kusano, Kiyoshi, National Institutes of Health, Building 36, Room 4D- 20, Bethesda. MD 20892 Laderman, Ezra, Yale University. New Haven, CT 06520 LaMarche, Paul H., Eastern Maine Medical Center, 489 State Street, Bangor, ME 04401 Lauffer, Max A., Penn State University Medical Center. Department of Biophysics & Physiology, Hershey. PA 17033 Levitan, Herbert, National Science Foundation, 4201 Wilson Boulevard. Room 835, Arlington, VA 22230 Lochhead, John H., 49 Woodlawn Road, London SW6 6PS, UK Loewus, Frank A., Washington State University. Institute of Biological Chemistry. Pullman, WA 99164 Loftfield, Robert B., University of New Mexico. School of Medicine, 915 Stanford Drive, Albuquerque, NM 87131 Lorand, Laszlo, Northwestern University Medical School, CMS Biology, Searle 4-555, 303 East Chicago Avenue, Chicago. 1L 60611- 3008 Mainer, Robert E., The Boston Company, Inc., One Boston Place. OBP-15-D. Boston, MA 02108 Malkiel, Saul, 174 Queen Street, #9A, Falmouth, MA 02540 Marsh, Julian B., 9 Eliot Street. Chestnut Hill, MA 02467-1407 Martin, Lowell V., 10 Buzzards Bay Avenue, Woods Hole. MA 02543 Mathews, Rita W., East Hill Road, P.O. Box 237. Southfield. MA 01259-0237 Metuzals, Janis, University of Ottawa Faculty of Medicine, Department of Pathology, 451 Smyth Road, Ottawa, ON K1H 8M5. Canada Moore, John A., University of California, Department of Biology, Riverside, CA 92521 Moore, John W., Duke University Medical Center, Department of Neurobiology. Box 3209, Durham. NC 27710 Moscona, Aron A., 221 West 82 nd Street, #8C, New York, NY 10024 Musacchia, X. J., P.O. Box 5054, Bella Vista, AR 72714-0054 Nasatir, Maimon, P.O. Box 379, Ojai, CA 93024 Passano, Leonard M., University of Wisconsin. Department of Zoology, Birge Hall. Madison. WI 53706 Price, Carl A., 20 Maker Lane. Falmouth, MA 02540 Prosser, C. Ladd, University of Illinois. Department of Physiology. 524 Bumll Hall. Urbana, IL 61801 Prytz, Margaret McDonald, (Address unknown) Renn, Charles E., (Address unknown) Reynolds, George T., Princeton University, Department of Physics. Jadwin Hall, Princeton, NJ 08544 Rice, Robert V., 30 Burnham Drive, Falmouth, MA 02540 Rockstein, Morris, 600 Biltmore Way. Apt. 805. Coral Gables. FL 33134 Roth, Jay S., 26 Huettner Road, P.O. Box 692. Woods Hole. MA 02543-0692 Ronkin, Raphael R., 32 1 2 McKinley Street, NW. Washington, DC 20015-1635 Roslansky, John D., 57 Buzzards Bay Avenue, Woods Hole, MA 02543 Roslansky, Priscilla F., Associates of Cape Cod, Inc., P.O. Box 224. Woods Hole. MA 02543-0224 Sanders, Howard L., Woods Hole Oceanographic Institution, Woods Hole. MA 02543 (deceased 2001) Sato, Hidemi, Nagova University, 3-24-101. Oakimshi Machi. Toba Mie 517-0023, JAPAN Schlesinger, R. Walter, 7 Langley Road, Falmouth. MA 02540-1809 Scott, Allan C., Colby College. Waterville. ME 04901 Silverstein, Arthur M., Johns Hopkins University, Institute of the History of Medicine, 1900 E. Monument Street, Baltimore. MD 21205 Sjodin, Raymond A., 3900 N. Charles Street, Apt. #1301, Baltimore, MD 21218-1719 Smith, Paul F., P.O. Box 264, Woods Hole, MA 02543-0264 Speer, John W., 293 West Main Road. Portsmouth. RI 0287 1 Sperelakis, Nicholas, University of Cincinnati, Department of Physiology/Biophysics. 231 Bethesda Avenue, Cincinnati. OH 45267- 0576 Spiegel, Evelyn, Dartmouth College. Department of Biological Sciences, 204 Oilman, Hanover, NH 03755 Spiegel, Melvin, Dartmouth College. Department of Biological Sciences, 204 Oilman. Hanover, NH 03755 Stephens, Grover C., University of California. School of Biological Sciences, Department of Ecology and Evolution/Biology, Irvine. CA 92717 Strehler, Bernard L., 31561 Crystal Sands Drive. Laguna Niguel, CA 92677 Sussman, Maurice, 72 Carey Lane, Falmouth. MA 02540 Sussman. Raquel B., Marine Biological Laboratory. Woods Hole. MA 02543 Szent-Gyorgyi, Gwen P., 45 Nobska Road, Woods Hole, MA 02543 Thorndike, W. Nicholas, Wellington Management Company. 200 State Street, Boston, MA 02109 Trager, William, The Rockefeller University, 1230 York Avenue. New York. NY 10021-6399 Trinkaus, J. Philip, Yale University, Department of Molecular. Cellular and Developmental Biology. Osborne Memorial Laboratory. New Haven. CT 06520 Villee, Claude A., Jr., Harvard Medical School. Carrel L, Countway Library. 10 Shattuck Street. Boston, MA 021 15 Vincent, Walter S., 16 F.R. Lillie Road. Woods Hole, MA 02543 Waterman, Talbot H., Yale University, Box 208103, 912 KBT Biology Department, New Haven. CT 06520-8103 Wigley, Roland L., 35 Wilson Road, Woods Hole, MA 02543 Witkovsky, Paul, NYU Medical Center, Department of Ophthalmology. 550 First Avenue, New York. NY 10016 Members Abt, Donald A., Aquavet. University of Pennsylvania. School of Veterinary Medicine. 230 Main Street, Falmouth. MA 02540 Members of the Corporation R73 Adams, James A., 3481 Paces Ferry Road, Tallahassee, FL 32308 Adelman. William J., 160 Locust Street. Falmouth. MA 02540 Alkon, Daniel L., National Institutes of Health, Laboratory of Adaptive Systems. 36 Convent Drive, MSC 4124, 36/4A21, Bethesda, MD 20X92-4124 Allen, Garland E., Washington University, Department of Biology, Box 1 137, One Brookings Drive, St. Louis, MO 63130-4899 Allen. Nina S., North Carolina State University. Department of Botany. Box 7612. Raleigh. NC 27695 Alliegro, Mark C., Louisiana State University Medical Center. Department of Cell Biology and Anatomy. 1901 Perdido Street, New Orleans. LA 70112 Anderson, Everett, Harvard Medical School. Department of Cell Biology, 240 Longwood Avenue, Boston, MA 021 15-6092 Anderson, John M., 1 10 Roat Street, Ithaca, NY 14850 Anderson, Porter W., 914 Grande Avenue, Key Largo, FL 33037 Armett-Kibel, Christine, University of Massachusetts. Dean of Science Faculty, Boston, MA 02125 Armstrong, Clay M., University of Pennsylvania School of Medicine, B701 Richards Building, Department of Physiology, 3700 Hamilton Walk, Philadelphia, PA 19104-6085 Armstrong, Ellen Prosser, 57 Millfield Street, Woods Hole, MA 02543 Armstrong, Peter B., University of California. Section of Molecular and Cell Biology. 149 Bnggs Hall. Davis, CA 95616-8755 Arnold, William A., Oak Ridge National Laboratory, Biology Division, 102 Balsalm Road, Oak Ridge, TN 37830 Ashton, Robert W., Bay Foundation. 1 7 West 94th Street. New York, NY 10025 Atema, Jelle, Boston University Marine Program, Marine Biological Laboratory. Woods Hole. MA 02543 Baccetti, Baccio, University of Sienna. Institute of Zoology, 53100 Siena. Italy Baker, Robert G., New York University Medical Center, Department Physiology and Biophysics, 550 First Avenue, New York, NY 10016 Baldwin, Thomas O., University of Arizona. Department of Biochemistry. P.O. Box 210088, Tucson, AZ 85721-0088 Baltimore. David, California Institute of Technology. 1200 East California Boulevard. Pasadena. CA 91 125 Barlow, Robert B., SUNY Upstate Medical University, Center for Vision Research. 750 East Adams Street, Syracuse, NY 13210 Barry, Daniel T., National Aeronautics and Space Administration, Lyn B. Johnson Space Center, 2101 NASA Road 1, Houston, TX 77058 Barry, Susan R., Mount Holyoke College, Department of Biological Sciences, South Hadley, MA 01075 Bass. Andrew H., Cornell University, Department of Neurobiology and Behavior, Seely Mudd Hall, Ithaca, NY 14853 Battelle, Barbara-Anne, University of Florida. Whitney Laboratory, 9505 Ocean Shore Boulevard. St. Augustine, FL 32086 Bay, Frederick, Bay Foundation, 17 W. 94th Street, First Floor, New York. NY 10025-7116 Baylor, Martha B., P O Box 93. Woods Hole, MA 02543 Bearer, Elaine L., Brown University, Division of Biology and Medicine, Department of Pathology, BMC 518. Providence. RI 02912 Beatty, John M., University of Minnesota, Department of Ecology and Behavioral Biology, 1987 Gortner. Street Paul, MN 55108 Beauge, Luis Alberto, Institute de Investigacion Medica. Department of Biophysics. Casilla de Correo 389, Cordoba 5000. Argentina Begenisich, Ted, University of Rochester, Medical Center, Box 642, 601 Elmwood Avenue, Rochester, NY 14642 Begg, David A., University of Alberta. Faculty of Medicine. Department of Cell Biology and Anatomy, Edmonton. Alberta T6G 2H7, Canada Bell, Eugene, 305 Commonwealth Avenue, Boston, MA 02115 Benjamin, Thomas L., Harvard Medical School, Pathology. D2-230, 200 Longwood Avenue, Boston. MA 021 15 Bennett, Michael V. L., Albert Einstein College of Medicine. Department of Neuroscience, 1300 Morris Park Avenue, Bronx, NY 10461 Bennett, Miriam F., Colby College. Department of Biology, Waterville, ME 04901 Bennett, R. Suzanne, Albert Einstein College of Medicine, Department of Neuroscience, 1410 Pelham Parkway South. Bronx, NY 10461 Berg, Carl J., Jr., P.O. Box 681, Kilauea, Kauai, HI 96754-0681 Berlin, Suzanne T., 87 Payneton Hill Road, York, ME 03909-5401 Bernstein, Norman, Columbia Realty Venture, 5301 Wisconsin Avenue, NW, #600, Washington, DC 20015-2015 Bezanilla, Francisco, Health Science Center, Department of Physiology, 405 Hilgard Avenue, Los Angeles, CA 90024 Biggers, John D., Harvard Medical School, Department of Physiology, Boston. MA 02115 Bishop. Stephen H., 2609 Eisenhower, Ames, I A 50010 Blaustein, Mordecai P., University of Maryland. School of Medicine, Department of Physiology, Baltimore, MD 21201 Blazis, Diana E. J., Marine Biological Laboratory. Center for Advanced Studies in the Space Life Sciences, Woods Hole, MA 02543 Blennemann, Dieter, 1117 East Putnam Avenue, Apt. #174, Riverside, CT 06878-1333 Bloom, George S., The University of Texas Southwestern Medical Center. Department of Cell Biology and Neuroscience, 5323 Harry Hines Boulevard, Dallas, TX 75235-9039 Bloom, Kerry S., University of North Carolina. Department of Biology, 623 Fordham Hall CB#3280, Chapel Hill, NC 27599-3280 Bodznick, David A., Wesleyan University. Department of Biology, Lawn Avenue. Middletown, CT 06497-0170 Boettiger, Edward G., P.O. Box 48, Rochester, VT 05767-0048 Boolootian, Richard A., Science Software Systems, Inc., 3576 Woodcliff Road, Sherman Oaks, CA 91403 Borgese, Thomas A., Lehman College, CUNY. Department of Biology, Bedford Park Boulevard. West, Bronx, NY 10468 Borst, David W., Jr., Illinois State University. Department of Biological Sciences. Normal, IL 61790-4120 Bowles, Francis P., Marine Biological Laboratory, Ecosystems Center. Woods Hole. MA 02543 Boyer, Barbara C., Union College, Biology Department, Schenectady, NY 12308 Brandhorst, Bruce P., Simon Eraser University, Institute of Molecular Biology/Biochemistry, Barnaby, B.C. V5A 1S6, Canada Brinley, F. J., Jr., NINCDS/NIH, Neurological Disorders Program, Room 812 Federal Building, Bethesda, MD 20892 Bronner-Fraser, Marianne, California Institute of Technology, Beckman Institute Division of Biology, 139-74, Pasadena, CA 91125 Brown, Stephen C., SUNY, Department of Biological Sciences, Albany, NY 12222 Brown, William L., 80 Black Oak Road, Weston, MA 02193 Browne, Carole L., Wake Forest University, Department of Biology, Box 7325 Reynolda Station, Winston-Salem, NC 27109 Browne, Robert A., Wake Forest University. Department of Biology. Box 7325. Winston-Salem. NC 27109 Bucklin, Anne C., University of New Hampshire. Ocean Process Analysis Laboratory, 142 Morse Hall, Durham, NH 03824 Bullis, Robert A., Oceanic Institute of Applied Aquaculture, 41-202 Kalanianaole Highway, Waimanalo, HI 96795 Burger, Max M., Friedrich Miescher-Institute, P.O. Box 2543. CH- 4002 Basel, Switzerland R74 Annual Report Burgess, David R., Boston College. Department of Biology. Higgins Hall, 140 Commonwealth Avenue, Chestnut Hill, MA 02167 Burgos, Mario H., IHEM Medical School. UNC Conicet. Casilla de Correo 56. 5500 Mendoza, Argentina Burky, Albert, University of Dayton, Department of Biology. Dayton, OH 45469 Burris, John E., Beloit College. 700 College Street, Beloit. WI 5351 1 Burstyn, Harold Lewis, Air Force Research Laboratory (IFOJ), Office of the Staff Judge Advocate, 26 Electronic Parkway, Rome. NY 13441-4514 Bursztajn, Sherry, LSU Medical Center, 1501 Kings Highway, Building BRIF 6-13. Shreveport, LA 71 130 Calabrese, Ronald L., Emory University, Department of Biology. 1510 Clifton Road. Atlanta, GA 30322 Cameron, R. Andrew, California Institute of Technology. Division of Biology 156-29, Pasadena, CA 91 125 Campbell, Richard H., Bang-Campbell Associates, Eel Pond Place, Box 402. Woods Hole, MA 02543 Candelas, Graciela C., University of Puerto Rico, Department of Biology. P.O. Box 23360. UPR Station. San Juan. PR 00931-3360 Cariello, Lucio, Stazione Zoologica "A. Dohrn," Villa Comunale, 80121 Naples, Italy Case, James F., University of California, Marine Science Institute. Santa Barbara, CA 93106 Cassidy, Father Joseph D., Providence College. Priory of St. Thomas Aquinas, Providence, RI 02918-0001 Cavanaugh, Colleen M., Harvard University. Biological Laboratories, 16 Divinity Avenue, Cambridge, MA 02138 Chaet, Alfred B., University of West Florida. Department of Cell and Molecular Biology. 11000 University Parkway, Pensacola, FL 32514 Chambers, Edward L., University of Miami School of Medicine, Department of Physiology and Biophysics P.O. Box 016430, Miami, FL 33101 Chang, Donald C., Hong Kong University, Science and Technology, Department of Biology, Clear Water Bay. Kowloon. Hong Kong Chappell, Richard L., Hunter College. CUNY, Department of Biological Sciences, 695 Park Avenue, New York, NY 10021 Child, Frank M., 28 Lawrence Farm Road. Woods Hole. MA 02543- 1416 Chisholm, Rex Leslie, Northwestern University, Medical School. Department of Cell Biology. Chicago, IL 6061 1 Citkowitz, Elena, Hospital of St. Raphael, Lipid Disorders Clinic, 1450 Chapel Street, New Haven. CT 065 1 1 Clark, Eloise E., Bowling Green State University. Biological Sciences Department, Bowling Green, OH 43403 Clark, Hays, 150 Gomez Road, Hobe Sound. FL 33455 Clark, Wallis H., Jr., 12705 NW 1 12th Avenue, Alachua, FL 32615 Claude, Fhilippa, University of Wisconsin. Department of Zoology, Zoology Research Building 125. 1 1 17 W Johnson Street, Madison, WI 53706 Clay, John R., National Institutes of Health, NINDS, Building 36. Room 2-CO2. Bethesda. MD 20892 Clowes. Alexander W., University of Washington. School of Medicine, Department of Surgery, Box 356410, Seattle, WA 98195-6410 Cobb, Jewel Plummer, California State University, 5151 University Drive. Health Center 205. Los Angeles. CA 90032-8500 Cohen, Carolyn, Brandeis University. Rosenstiel Basic Medical. Sciences Research Center. Waltham, MA 02254 Cohen, Lawrence B., Yale University School of Medicine, Department of Physiology. 333 Cedar Street. New Haven. CT 06520 Cohen, Maynard M., Rush Medical College, Department of Neurological Sciences. 600 South Paulina. Chicago, IL 60612 Cohen, William D., Hunter College. Department Biological Sciences, 695 Park Avenue. New York. NY 10021 Coleman, Annette W., Brown University, Division of Biology and Medicine. Providence, RI 02912 Colinvaux, Paul, Marine Biological Laboratory, Woods Hole, MA 02543 Collier, Jack R., 3431 Highway. #107, P.O. Box 139, Effie, LA 71331 Collier, Marjorie McCann, 3431 Highway 107. P.O. Box 139, Effie. LA 71331 Collier, R. John, Harvard Medical School. Department of Microbiology and Molecular Genetics, 200 Longwood Avenue, Room 356, Boston, MA 02 1 1 5 Cook, Joseph A., Edna McConnell Clark Foundation, 250 Park Avenue, New York, NY 10177-0026 Cornwall, Melvin C., Jr., Boston University School of Medicine. Department of Physiology L714, Boston. MA 021 18 Corson, D. Wesley, Jr., Storm Eye Institute. Room 537. 171 Ashley Avenue, Charleston, SC 29425 Corwin. Jeffrey T., University of Virginia, School of Medicine, Department Otolaryngology and Neuroscience. Box 396, Charlottesville. VA 22908 Couch, Ernest F., Texas Christian University, Department of Biology. TCU Box 298930, Fort Worth, TX 76129 Cox. Rachel Llanelly, Woods Hole Oceanographic Institute, Biology Department, Woods Hole. MA 02543 Crane, Sylvia E., c/o Mr. Thomas Crane, 40 Chestnut Street, Weston, MA 02493 Cremer-Bartels, Gertrud, Horstmarer Landweg 142. 48149 Muenster. Germany Crow, Terry J., University of Texas Medical School. Department of Neurobiology and Anatomy. Houston. TX 77225 Crowell, Sears, Indiana University, Department of Biology, Bloomington. IN 47405 Crowther, Robert J., Shriners Hospitals for Children. 51 Blossom Street. Boston. MA 02114 Cummings, Michael P., Marine Biological Laboratory, Bay Paul Center. Woods Hole. MA 02543 Cunningham, Mary-Ellen, 62 Cloverly Road, Grosse Pointe Farms, MI 48236-33 13 (deceased 2000) Cutler, Richard D., Marine Biological Laboratory. Woods Hole. MA 02543 Davidson, Eric H., California Institute of Technology. Division of Biology 156-29. 391 South Holliston. Pasadena, CA 91125 Davison, Daniel B., Bristol-Myers Squibb PR1. Bioinformatics Department. 5 Research Parkway. Wallingford. CT 06492 Daw, Nigel W., 5 Old Pawson Road. Brunford. CT 06405 Dawidowicz, Eliezar A., Marine Biological Laboratory. Office of Research Administration and Education. Woods Hole, MA 02543 De Weer. Paul J., University of Pennsylvania, B400 Richards Building. Department of Physiology. 3700 Hamilton Walk. Philadelphia. PA 19104-6085 Deegan, Linda A., Marine Biological Laboratory. The Ecosystems Center. Woods Hole, MA 02543 DeGroof, Robert C., 145 Water Crest Drive, Doylestown. PA 18901- 3267 Denckla, Martha Bridge. Johns Hopkins University. School of Medicine. Kennedy-Krieger Institute. 707 North Broadway. Baltimore. MD 2 1 205 DePhillips, Henry A., Trinity College, Department of Chemistry, 300 Summit Street. Hartford. CT 06106 Members of the Corporation R75 DeSimone, Douglas \V., University of Virginia. Department of Cell Biology. Box 439. Health Sciences Center. Charlottesville, VA 22908 Dettbarn, Wolf-Dietrich, 4422 Wayland Drive, Nashville. TN 37215 Dionne. Vincent E., Boston University Marine Program. Marine Biological Laboratory. Woods Hole, MA 02543 Dow ling, John E., Harvard University, Biological Laboratories, 16 Divinity Street, Cambridge. MA 02138 Drapeau, Pierre, Montreal General Hospital, Department of Neurology, 1650 Cedar Avenue, Montreal, Quebec H3G IA4. Canada DuBois, Arthur Brooks, John B. Pierce Foundation Laboratory, 290 Congress Avenue. New Haven, CT 06519 Duncan, Thomas K., Nichols College, Environmental Sciences Department. Dudley, MA 01571 Dunham, Philip B., Syracuse University. Department of Biology, 130 College Place, Syracuse. NY 13244-1220 Dunlap, Paul V., University of Michigan, Department of Biology. 830 North University Avenue, Ann Arbor, MI 48109-1048 Ehert, James D., The Johns Hopkins University. Department of Biology. Homewood. 3400 North Charles Street. Baltimore. MD 21218-2685 (deceased 2001) Eckberg, William R.. Howard University. Department of Biology. P.O. Box 887, Administration Building, Washington. DC 20059 Edds, Kenneth T., R & D Systems, Inc., Hematology Division, 614 McKinley Place, NE. Minneapolis, MN 55413 Eder, Howard A., Albert Einstein College of Medicine. 1300 Morris Park Avenue. Bronx, NY 10461 Edstrom, Joan, 53 Two Ponds Road, Falmouth, MA 02540 Egyud, Laszlo G., Cell Research Corporation, P.O. Box 67209, Chestnut Hill, MA 02167-0209 Ehrlich, Barbara E., Yale University Medical School, Department of Pharmacology, Sterling Hall of Medicine, B207, 333 Cedar Street. New Haven, CT 06520-8066 Eisen, Arthur Z., Washington University, Division of Dermatology, St. Louis. MO 63110 Eisen, Herman N., Massachusetts Institute of Technology. Center for Cancer Research, El 7- 128, 77 Massachusetts Avenue. Cambridge, MA 02139-4307 Elder, Hugh Young, University of Glasgow. Institute of Physiology, Glasgow G12 8QQ. Scotland Knglund, Paul T., Johns Hopkins Medical School, Department of Biological Chemistry, 725 North Wolfe Street, Baltimore, MD 21205 Epel, David, Stanford University, Hopkins Marine Station, Ocean View Boulevard, Pacific Grove, CA 93950 Epstein, Herman T., 18 Lawrence Farm Road, Woods Hole. MA 02543 Epstein, Ray L., 701 Winthrop Street. #311, Taunton, MA 02780-2187 Farb, David H., Boston University School of Medicine. Department of Pharmacology L603, 80 East Concord Street, Boston, MA 021 18 Earmanfarmaian, A. Verdi, Rutgers University, Department of Biological Sciences, Nelson Biology Laboratory FOB 1059, Piscataway. NJ 08855 Feldman, Susan C., University of Medicine and Dentistry. New Jersey Medical School. 10(1 Bergen Street. Newark, NJ 07103 Festoff, Barry William, VA Medical Center, Neurology Service (151). 4801 Linwood Boulevard, Kansas City, MO 64128 Fink, Rachel D., Mount Holyoke College, Department of Biological Sciences, Clapp Laboratories, Room 215, South Hadley, MA 01075 Finkelstein, Alan, Albert Einstein College of Medicine. 1300 Morris Park Avenue, Bronx. NY 10461 Fischbach, Gerald D., Columbia College of Physicians and Surgeons, 630 West 168 th Street. R 2-401. New York. NY 10032 Fishman, Harvey M., University of Texas Medical Branch. Department of Physiology and Biophysics, 301 University Boulevard, Galveston. TX 77555-0641 Flanagan. Dennis, 12 Gay Street, New York, NY 10014 Fluck, Richard Allen, Franklin and Marshall College. Department of Biology, Box 3003, Lancaster, PA 17604-3003 Foreman, Kenneth H., Marine Biological Laboratory, Woods Hole, MA 02543 Fox, Thomas Oren, Harvard Medical School, Division of Medical Sciences, MEC 435, 260 Longwood Avenue, Boston. MA 021 15 Franzini-Armstrong, Clara, LIniversity of Pennsylvania. School of Medicine. 330 South 46th Street, Philadelphia, PA 19143 Fraser, Scott, California Institute of Technology. Beckman Institute 139-74, 1201 East California Boulevard, Pasadena, CA 91 125 Frazier, Donald T., University of Kentucky Medical Center. Department of Physiology and Biophysics, MS501 Chandler Medical Center. Lexington, KY 40536 French, Robert J., University of Calgary, Health Sciences Centre. Alberta, T2N 4N1. CANADA Fulton, Chandler M., Brandeis University. Department of Biology. MS 008, Waltham. MA 02454-91 10 Furie, Barbara C., Beth Israel Deaconess Medical Center, BIDMC Cancer Center, Kirstein 1, 330 Brookline Avenue, Boston. MA 02215 Furie, Bruce, Beth Israel Deaconess Medical Center, BIDMC Cancer Center, Kirstein 1. 330 Brookline Avenue, Boston. MA 02215 Furshpan, Edwin J., Harvard Medical School, Department of Neurobiology, 220 Longwood Avenue, Boston. MA 021 15 Futrelle, Robert P., Northeastern University. College of Computer Science, 360 Huntington Avenue, Boston, MA 021 15 Gabr, Howaida, Suez Canal University. Department of Marine Science. Faculty of Science. Ismailia. Egypt Gadsby, David C., The Rockefeller University, Laboratory of Cardiac Physiology, 1230 York Avenue. New York, NY 10021-6399 Gainer, Harold, National Institutes of Health. NINDS.BNP.DIR. Neurochemistry. Building 36. Room 4D20, Bethesda, MD 20892- 4130 Galatzer-Levy, Robert M., 534 Judson Avenue, Evanston, IL 60202 Gall, Joseph G., Carnegie Institution, 1 15 West University Parkway, Baltimore, MD 21210 Gallo, Michael A., UMDNJ-Robert Wood Johnson Medical School, EOHSI, Room 408, 170 Frelinghuysen Road, Piscataway, NJ 08854- 8020 Garber, Sarah S., Allegheny University of the Health Sciences, Department of Physiology. 2900 Queen Lane. Philadelphia. PA 19129 Gelperin, Alan, Bell Labs Lucent. Department Biology Comp., Rm 1C464. 600 Mountain Avenue. Murray Hill. NJ 07974 German, James I.., Ill, Weill Medical College of Cornell University, 1300 York Avenue. New York. NY 10021 Gibbs, Martin, Brandeis University. Institute for Photobiology of Cells and Organelles. Waltham. MA 02254 (iiblin. Anne E., Marine Biological Laboratory. The Ecosystems Center. Woods Hole. MA 02543 Gibson, A. Jane, Cornell LIniversity. Department of Biochemistry. Biotech Building. Ithaca. NY 14850 Gifford, Prosser, Library of Congress, Madison Building LM605, Washington DC 20540 Gilbert, Daniel L., National Institutes of Health. Biophysics Sec.. BNP, Building 36, Room 5A-27. Bethesda, MD 20892 R76 Annual Report Giudice, Giovanni, Universita di Palermo, Dipartimento di Biologia. Cellulare e Dello Sviluppo, 1-90123 Palermo. Italy Giuditta, Antonio, University of Naples, Department of General Physiology, Via Mezzocannone 8, Naples. 80134, Italy Glynn, Paul, P.O. Box 369, Hampton Falls, NH 03844 Golden, William T., Chairman Emeritus. American Museum of Natural History, 500 Fifth Avenue. 50* Floor. New York, NY 10110 Goldman, Robert D., Northwestern University Medical School, Department of Cell and Molecular Biology, 303 E. Chicago Avenue, Chicago, IL 60611-3008 Goldsmith, Paul K., National Institutes of Health, 9000 Rockville Pike, Building 10, Room 8C206, Bethesda. MD 20892 Goldsmith, Timothy H., Yale University, Department of Biology, New Haven. CT06510 Goldstein, Moise H., Jr., The Johns Hopkins University, ECE Department, Barton Hall, Baltimore, MD 21218 Gould, Robert Michael, NYS Institute of Basic Research, Department of Pharmacology, 1050 Forest Hill Road, Staten Island, NY 10314- 6399 Govind. C. K., Scarborough College, Life Sciences Division, 1265 Military Trail, West Hill, Ontario MIC 1A4, Canada Grace, Dick, Doreen Grace Fund, The Brain Center, Promontory Point, New Seabury, MA 02649 Graf, Werner M., College of France, 1 1 Place Marcelin Berthelot, 75231 Paris Cedex 05, France Grant, Philip, National Institutes of Health. NINDS\BN\DIR- Neurochemistry, Building 36, Room 4D20, Bethesda, MD 20892- 4130 Grass, Ellen R., The Grass Foundation, 77 Reservoir Road. Quincy. MA 02170-3610 (deceased 2001 ) Grassle, Judith P., Rutgers University, Institute of Marine and Coastal Studies, 71 Dudley Road, New Brunswick. NJ 08901-8521 Graubard, Katherine G., University of Washington. Department of Zoology, NJ-15, Box 351800, Seattle, WA 98195-1800 Greenberg, Everett Peter, University cf Iowa, College of Medicine, Department of Microbiology, Iowa City, IA 52242 Greenberg, Michael J., University of Florida, The Whitney Laboratory. 9505 Ocean Shore Boulevard, St. Augustine, FL 32080-8610 Greer, Mary J., 176 West 87th Street. #12A, New York, NY 10024- 2902 Griffin, Donald R., Harvard University, Concord Field Station, Old Causeway Road, Bedford, MA 01730 Gross, Paul R., 123 Perkins Street, Jamaica Plain, MA 02130 Grossman, Albert, New York University Medical Center, 550 First Avenue, New York, NY 10016 Grossman, Lawrence, The Johns Hopkins University, Hygien Building, Room W8306, Baltimore, MD 21205 Gruner, John A., Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380-4245 Gunning, A. Robert, P. O. Box 165, Falmouth, MA 02541 Gwilliam, G. Francis, Reed College. Department of Biology. Portland. OR 97202 Haimo, Leah T., University of California. Department of Biology. Riverside. CA 92521 Hajduk, Stephen L., University of Alabama, School of Medicine/Dentistry. Department of Biochemistry/Molecular Genetics. University Station. Birmingham. AL 35294 Hall. Linda M., Shriners Hospital for Children. 2425 Stockton Boulevard. Sacramento, CA 95817 Halvorson, Harlyn O., University of Massachusetts, Policy Center for Marine Biosciences and Technology. 100 Morrissey Boulevard, Boston, MA 02125-3393 Haneji, Tatsuji, The University of Tokushima. Department of Histology & Oral Histology, School of Dentistry, 18-15, 3 Kuramoto-cho, Tokushima 770-8504, Japan Hanlon, Roger T., Marine Biological Laboratory. Woods Hole. MA 02543 Harosi, Ferenc, New College of the USF. Division of Natural Sciences. 5700 North Tamiami Trail, Sarasota, FL 34243-2197 Harrigan, June F.. 7415 Makaa Place, Honolulu. HI 96825 Harrington, Glenn W., Weber State University. Department of Microbiology, Ogden, UT 84408 Harrington, John P., University of South Alabama, Department of Chemistry, Mobil. AL 36688 Harrison, Stephen C., Harvard University. Department of Molecular and Cell Biology, 7 Divinity Avenue, Cambridge. MA 02138 Haselkorn, Robert, University of Chicago, Department of Molecular Genetics and Cell Biology, Chicago. IL 60637 Hastings, J. Woodland, Harvard University, The Biological Laboratories, 16 Divinity Avenue, Cambridge, MA 02138-2020 Hayes, Raymond L., Jr., Howard University. College of Medicine, 520 W Street. NW. Washington, DC 20059 Heck, Diane E., Rutgers University, Department of Pharmacology/Toxicology, 681 Frelinghuysen Road. Piscataway, NJ 08855 Henry, Jonathan Joseph, University of Illinois. Department of Cell and Structural Biology. 601 South Goodwin Avenue #B107, Urbana, IL 61801-3709 Hepler, Peter K., University of Massachusetts. Department of Biology. Morrill 111. Amherst. MA 01003 Herndon, Walter R., University of Tennessee, Department of Botany. Knoxville, TN 37996-1100 Hershko, Avram, Technion-Israel Institute of Technology, Unit of Biochemistry, The Bruce Rappaport Faculty of Medicine, Haifa 31096, Israel Herskovits, Theodore T., Fordham University. Department of Chemistry. John Mulcahy Hall. Room 638. Bronx, NY 10458 Hiatt, Howard H., Brigham and Women's Hospital. Department of Medicine. 75 Francis Street, Boston, MA 021 15 Highstein, Stephen M., Washington University, Department of Otolaryngology, Box 8115. 4566 Scott Avenue, St. Louis, MO 631 10 Hildebrand. John G., University of Arizona, ARL Division of Neurobiology, P.O. Box 210077. Tucson, AZ 85721-0077 Hill, Richard W., Michigan State University, Department of Zoology, East Lansing, MI 48824 Hill, Susan D., Michigan State University, Department of Zoology, East Lansing. MI 48824 Hillis, Llewellya W., Marine Biological Laboratory. Woods Hole, MA 02543 Hinchcliffe, Edward H., University of Massachusetts Medical School, Department of Cell Biology. 377 Plantation Street. Worcester, MA 01605 Hinkle, Gregory J., Bioinformatics Group. Cereon Genomics. 45 Sidney St.. Cambridge. MA 02139 Hinsch, Gertrude W., University of South Florida, Department of Biology, Tampa. FL 33620 Hinsch, Jan, Leica, Inc.. 110 Commerce Drive. Allendale. NJ 07401 Hobbie, John E.. Marine Biological Laboratory. The Ecosystems Center. Woods Hole. MA 02543 Hodge, Alan J.. 3843 Mount Blackburn Avenue. San Diego. CA 921 1 1 Hoffman, Joseph F., Yale University School of Medicine. Cellular and Molecular Physiology, 333 Cedar Street, New Haven, CT 06520-8026 Hollytield, Joe G., The Cleveland Clinic. Opthalmic Research, 9500 Euclid Avenue, Cleveland, OH 44195 Members of the Corporation R77 Holz, George G., IV, New York University Medical Center. Department of Physiology and Neuroscience, Medical Sciences Building. Room 442. 550 First Avenue. New York. NY 10016 Hopkinson, Charles S., Jr., Marine Biological Laboratory. Woods Hole. MA 02543 Houk. James C., Northwestern University Medical School, 303 East Chicago Avenue. Ward 5-315. Chicago. IL 60611-3008 Hoy. Ronald R., Cornell University. Section of Neurobiology and Behavior. 215 Mudd Hall. Ithaca. NY 14853 Huang, Alice S., California Institute of Technology, Mail Code 1-9. Pasadena, CA 91125 Hul'nagel-ZackrotT, Linda A., University of Rhode Island. Department of Microbiology, Kingston, RI 02881 I liiiiiiiniii. William D., Ohio University. Department of Biological Sciences, Athens, OH 45701 Humphreys, Susie H., Food and Drug Administration, HFS-308, 200 C Street, SW, Washington, DC 20204-0001 Humphreys, Tom, University of Hawaii. Kewalo Marine Laboratory, 41 Ahui Street. Honolulu. HI 96813 Hunt. Richard T., ICRF. Clare Hall Laboratories. South Mimms Potter's Bar. Herts EN6-3LD. England Hunter, Robert D., Oakland University. Department of Biological Sciences. Rochester. MI 48309-4401 Huxley, Hugh E., Brandeis University. Rosenstiel Center. Biology Department. Waltham. MA 02154 Ilan, Joseph, Case Western Reserve University, School of Medicine, Department of Anatomy. Cleveland, OH 44106 Ingoglia, Nicholas A., New Jersey Medical School, Department of Pharmacology/Physiology, 185 South Orange Avenue, Newark. NJ 07103 Inoue, Saduyki, McGill University, Department of Anatomy, 3640 University Street, Montreal, PQ H3A 2B2, Canada Inoue, Shinya, Marine Biological Laboratory. Woods Hole, MA 02543 Isselbacher. Kurt J., Massachusetts General Hospital Cancer Center, Charlestown. MA 02129 Issidorides, Marietta Radovic, National and Capodistrian University of Athens, Department of Psychiatry, Eginition Hospital, 74, Vas. Sophias Avenue. 1 15 28 Athens, Greece Izzard, Colin S., SUNY-Albany, Department of Biological Sciences, 1400 Washington Avenue. Albany, NY 12222 Jacobs, Neil, Hale and Dorr, 60 State Street, Boston, MA 02109 Jaffe, Laurinda A., University of Connecticut Health Center, Department of Physiology, Farmington Avenue, Farmington, CT 06032 Jaffe, Lionel, Marine Biological Laboratory, Woods Hole. MA 02543 Jeffery, William R., University of Maryland, Department of Biology, College Park, MD 20742 Johnston, Daniel, Baylor College of Medicine. Division of Neuroscience, One Baylor Plaza. Room S740. Houston, TX 77030 Josephson, Robert K., University of California. School of Biological Science. Department of Psychobioiogy, Irvine, CA 92697 Kaczmarek, Leonard K., Yale University School of Medicine. Department of Pharmacology. 333 Cedar Street, New Haven, CT 06520 Kaley, Gabor, New York Medical College, Department of Physiology. Basic Sciences Building, Valhalla, NY 10595 Kaltenbach. Jane, Mount Holyoke College, Department Biological Sciences. South Hadley, MA 01075 Kaminer, Benjamin, Boston University Medical School. Physiology Department. 80 East Concord Street. Boston, MA 02 1 1 8 Kaneshiro, Edna S., University of Cincinnati, Biological Sciences Department, JL 006. Cincinnati. OH 45221-0006 Kaplan, Ehud, Mount Sinai School of Medicine. I Gustave Levy Place. Box 1 183. New York. NY 10029 Karakashian. Stephen J., Apartment 16-F. 165 West 91st Street. New York. NY 10024 Karlin, Arthur, Columbia University, Center for Molecular Recognition, 630 West 168th Street, Room 11-401, New York, NY 10032 Karnovsky, Morris John, Harvard Medical School, Department of Pathology, 200 Longwood Avenue, Boston, MA 021 15 Keller, Hartmut Ernst, Carl Zeiss, Inc., One Zeiss Drive, Thornwood. NY 10594 Kelley, Darcy B., Columbia University, Department of Biological Sciences, 911 Fairchild. Mailcode 2432, New York, NY 10027 Kelly, Robert E., 5 Little Harbor Road, Woods Hole, MA 02543 Kemp, Norman E., University of Michigan, Department of Biology, Ann Arbor, MI 48109 Kendall, John P., Faneuil Hall Associates, 176 Federal Street, 2nd Floor, Boston, MA 02110 Kerr, Louis M., Marine Biological Laboratory, Woods Hole, MA 02543 Keynan, Alexander, Israel Academy of Science and Humanity. P.O. Box 4040, Jerusalem. Israel Khan, Shahid M. M., Albert Einstein College of Medicine, Department of Physiology and Biophysics. 1300 Morris Park Avenue, Room U273, Bronx, NY 10461 Khodakhah, Kamran, University of Colorado School of Medicine, Department of Physiology and Biophysics, 4200 East 9th Avenue, C-240. Denver, CO 80262 Kiehart, Daniel P., Duke University Medical Center. Department of Cell Biology. Box 3709, 308 Nanaline Duke Building, Durham. NC 27710 Kim ill-Ill. David, University of California, Department of Physics, 0319 9500 Gilman Drive, La Jolla, CA 92093 Klessen, Rainer, (address unknown) Klotz, Irving M., Northwestern University. Department of Chemistry, Evanston, IL 60201 Knudson, Robert A., Marine Biological Laboratory, Woods Hole, MA 02543 Koide, Samuel S., The Rockefeller University, The Population Council, 1230 York Avenue, New York, NY 10021 Kornberg, Hans, Boston University, The University Professors, 745 Commonwealth Avenue. Boston. MA 02215 Kosower, Edward M., Tel-Aviv University, Department of Chemistry, Ramat-Aviv. Tel Aviv, 69978, Israel Krahl. Maurice E., 2783 West Casas Circle, Tucson, AZ 85741 (deceased 2000) Krane, Stephen M., Massachusetts General Hospital. 55 Fruit Street. Bulf-165. Boston, MA 02114 Krauss, Robert, P.O. Box 291, Denton, MD 21629 Kravitz, Edward A., Harvard Medical School, Department of Neurobiology, 220 Longwood Avenue, Boston, MA 021 15 Kriebel, Mahlon E., SUNY Health Science Center, Department of Physiology. Syracuse, NY 13210 Kristan, William B., Jr., University of California. Department of Biology 0357, 9500 Gilman Drive. La Jolla, CA 92093-0357 Kropinski, Andrew M., Queen's University, Department of Microbiology/Immunology, Botterell Hall, Room 74, Kingston, Ontario K7L 3N6, CANADA R78 Annual Report Kuffler, Damien P., Institute of Neurobiology, 201 Boulevard del Valle. San Juan 00901. PR knlins. William J., Hospital for Sick Children, Biochemistry Research. 555 University Avenue, Toronto, Ontario M5G 1X8. Canada Kunkel, Joseph G., University of Massachusetts, Department of Biology, Amherst. MA 01003 Kuzirian, Alan M., Marine Biological Laboratory. Woods Hole, MA 02543-1015 Laderman, Aimlee D., Yale University, School of Forestry and Environmental Studies, 370 Prospect Street. New Haven, CT 06511 Landeau, Laurie J., Listowel, Inc.. 2 Park Avenue, Suite 1525, New York, NY 10016 Landis, Dennis M. D., University Hospital of Cleveland, Department Neurology, 1 1 100 Euclid Avenue. Cleveland, OH 44106 Landis, Story C., National Institutes of Health, Building 36, Room 5A05, 36 Convent Drive. Bethesda. MD 20892-4150 Landowne. David, University of Miami Medical School, Department of Physiology and Biophysics, PO Box 016430, Miami. FL 33101 Langford, George M., Dartmouth College, Department of Biological Sciences, 6044 Oilman Laboratory, Hanover. NH 03755 Laskin, Jeffrey, University of Medical and Dentistry of New Jersey. Robert Wood Johnson Medical School. 675 Hoes Lane. Piscataway, NJ 08854 Lasser-Ross, Nechama, New York Medical College. Department of Physiology. Valhalla, NY 10595 Laster, Leonard, University of Massachusetts Medical School, 55 Lake Avenue, North, Worcester, MA 01655 Laties, Alan, Scheie Eye Institute, Myrin Circle, 5 1 North 39th Street, Philadelphia, PA 19104 Laufer, Hans, University of Connecticut, Department of Molecular and Cell Biology, U-125, 75 North Eagleville Road Storrs, CT 06269- 3125 Lazarow, Paul B., Mount Sinai School of Medicine. Department of Cell Biology and Anatomy, 1190 Fifth Avenue, Box 1007, New York, NY 10029-6574 Lazarus, Maurice, Federated Department Stores. Sears Crescent, City Hall Plaza, Boston, MA 02108 Leadbetter, Edward R., University of Connecticut. Department of Molecular and Cell Biology. U-131, Beach Hall, Room 249, 354 Mansfield Road. Storrs, CT 06269-2131 Lederberg, Joshua, The Rockefeller University, Suite 400 (Founders Hall), 1230 York Avenue, New York, NY 10021 Lee, John J., City College of CUNY, Department of Biology, Convent Avenue and 138th Street, New York. NY 10031 Lehy, Donald B., 35 Willow Field Drive. North Falmouth, MA 02556 Leighton, Stephen B., Beecher Instruments, P.O. Box 8704, Silver Spring, MD 20910 Lerner, Aaron B., Yale University School of Medicine, Department of Dermatology. P.O. Box 3333, New Haven, CT 06510 Levin, Jack, Veterans Administration, Medical Center, 1 1 1 H2, 4150 Clement Street. San Francisco. CA 94121 Levine, Michael, University of California. Department MCB. 401 Barker Hall. Berkeley. CA 94720 Levine, Richard B., University of Arizona. Division of Neurobiology, Room 61 1. Gould Simpson Building. PO Box 210077. Tucson, AZ 85721-0077 Levinthal, Francoise, Columbia University, Department of Biological Sciences, Broadway and H6th Street, New York, NY 10026 Levitan, Irwin B., University of Pennsylvania. School of Medicine, 218 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 19104-6074 I link, Richard W., University of Minnesota School of Medicine. Cell Biology and Neuroanatomy Department. 4-135 Jackson Hall, 321 Church Street, Minneapolis. MN 55455 Lipicky. Raymond J., Food and Drug Administration. CDER/ODEI/ HFD-1 10. 5600 Fishers Lane. Rockville, MD 20857 Lisman. John E., Brandeis University. Molecular and Cell Biology, 415 South Street. Waltham, MA 02454-9110 Liuzzi. Anthony, 180 Beacon Street. #8G, Boston. MA 021 16 Llinas, Rodolfo R., New York University Medical Center, Department of Physiology/Biophysics, 550 First Avenue. Room 442, New York. NY 10016 Lobel, Phillip S., Boston University Marine Program, Marine Biological Laboratory. Woods Hole, MA 02543 Loew, Franklin M., Becker College. 61 Sever Street, Worcester, MA 01615-0071 Loewenstein, Birgit Rose, 102 Ransom Rd.. Falmouth, MA 02540 Loewenstein, Werner R., 102 Ransom Rd.. Falmouth. MA 02540 London, Irving M., Harvard-MIT. Division. E-25-551, Cambridge, MA 02139 Longo, Frank J., University of Iowa. Department of Anatomy. Iowa City, IA 52442 Luckenbill, Louise M., 430 Sippiwissett Road. Falmouth. MA 02540 Macagno, Eduardo R., Columbia University. 109 Low Memorial Library. Mail Code 4306, New York, NY 10027 MacNichol Edward R., Jr., Boston University School of Medicine. Department of Physiology. 80 East Concord Street. Boston. MA 02118 Maglott-Duffield, Donna R., American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852-1776 Maienschein, Jane Ann, Arizona State University. Department of Physiology. P.O. Box 872004. Tempe, AZ 85287-2004 Malbon, Craig C., SUNY, University Medical Center, Pharmacology- HSC, Stony Brook. NY 11794-8651 Malchow, Robert P., University of Illinois, Department of Biology. M/C 066, 845 West Taylor Street. Chicago. IL 60607 Manalis. Richard S., Indiana-Purdue University. Department of Biological Science, 2101 Coliseum Boulevard East, Fort Wayne, IN 46805 Manz, Robert D., P.O. Box 428, Glen Mills. PA 19342 Margulis. Lynn, University of Massachusetts, Department of Geosciences. Morrill Science Center, Box 35820, Amherst, MA 01003-5820 Marinucci, Andrew C., 102 Nancy Drive, Mercerville, NJ 08619 Martinez, Joe L., Jr., The University of Texas, Division of Life Sciences, 6900 North Loop 1604 West, San Antonio, TX 78249-0662 Martinez-Palomo, Adolfo, CINVESTAV-IPN, Sec. de Patologia Experimental. 07000 Mexico. D.F.A.P. 140740. Mexico Mastroianni, Luigi, Jr., Hospital of University of Pennsylvania, 106 Dulles, 3400 Spruce Street. Philadelphia. PA 19104-4283 Mauzerall, David, Rockefeller University. 1230 York Avenue, New York, NY 10021 McAnelly. M. Lynne, University of Texas. Section of Neurobiology, School of Life Sciences, Austin. TX 78712 McCann, Frances V., Dartmouth Medical School. Department of Physiology. Lebanon. NH 03756 McLaughlin, Jane A., Marine Biological Laboratory 1 , Woods Hole, MA 022543 McMahon, Robert F., University of Texas, Arlington, Department of Biology. Box 19498. Arlington, TX 76019 Meedel, Thomas, Rhode Island College, Biology Department. 600 Mount Pleasant Avenue, Providence, RI 02908 Members of the Corporation R79 Meinertzhagen, Ian A., Dalhousie University, Department of Psychology. Halifax, NS B3H 4J1, Canada Meiss, Dennis K., Immunodiagnostic Laboratories, 488 McCormick Street. San Leandro. CA 94577 Melillo, Jerry M., Marine Biological Laboratory, Ecosystems Center. Woods Hole. MA 02543 Mellon, DeForest, Jr., University of Virginia. Department of Biology, Gilmer Hall. Charlottesville. VA 22903 Mellon, Richard P., P.O. Box 187, Laughlintown, PA 15655-0187 Mendelsohn, Michael E., New England Medical Center. Molecular Cardiology Laboratory, NEMC Box 80, 750 Washington Street, Boston, MA 021 11 Mensinger, Allen F., University of Minnesota. Biology Department, LSCI 211. Duluth. MN 55812 Merriman, Melanie Pratt, 751 1 Beach View Drive, North Bay Village. FL 33141 Meselson, Matthew, Harvard University. Fairchild Biochemistry Building. 7 Divinity Avenue. Cambridge, MA 02138 Miledi. Ricardo, University of California, Irvine. Department of Psychobiology, 2205 Biology Science II. Irvine. CA 92697-4550 Milkman, Roger D., University of Iowa, Department of Biological Sciences. Biology Building, Room 318, Iowa City. IA 52242-1324 Miller, Andrew L., Flat 2A, Block 2, Greon Park, Razor Hill, Clearwater Bay. Kowloon. Hong Kong Miller, Thomas J., Analogic, 8 Centennial Drive. Peabody, MA 01960 Mills, Robert, 6410 2P 1 Avenue W, #311. Brandenton, FL 34210 (deceased 2001) Misevic, Gradimir, University Hospital of Basel, Department of Research, Mebelstr. 20, CH-4031 Basel, Switzerland Mitchell, Ralph, Harvard University, Division of Applied Sciences, 29 Oxford Street, Cambridge, MA 02138 Miyakawa, Hiroyoshi, Tokyo College of Pharmacy, Laboratory of Cellular Neurobiology, 1432-1 Horinouchi, Hachiouji. Tokyo 192-03, Japan Miyamoto, David M., Drew University. Department of Biology. Madison. NJ 07940 Mi/Hi. Merle, Tulane University, Department of Cell and Molecular, Biology. New Orleans. LA 70118 Moreira, Jorge E., National Institutes of Health, NICHD. Department of Cell and Molecular Biol.. Bethesda. MD 20852 Morin, James G., Cornell University, Department of Ecology and Evolutionary Biology, G14 Stimson Hall, Ithaca. NY 14853-2801 Morrell, Leyla deToledo, Rush-Presbyterian St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612 Morse, Stephen S., 275 Central Park West, New York, NY 10024 Mote, Michael L, Temple University. Department of Biology, Philadelphia, PA 19122 Muller, Kenneth J., University of Miami School of Medicine, Department of Physiology and Biophysics, 1600 NW 10th Avenue. R-430. Miami. FL 33136 Murray, Andrew W., University of California, Department of Physiology. Box 0444, 513 Parnassus Avenue, San Francisco, CA 94143-0444 Nabrit, Samuel M., 686 Beckwith Street. SW. Atlanta. GA 30314 Nadelhoffer, Knute J., Marine Biological Laboratory. 7 MBL Street, Woods Hole. MA 02543 Nagel, Ronald L., Albert Einstein College of Medicine. 1300 Morris Park Avenue. Bronx, NY 10461 Naka, Ken-ichi, 2-9-2 Tatumi Higashi, Okazaki, 444. Japan Nakajima, Yasuko, University of Illinois. College of Medicine. Anatomy and Cell Biology Department, MAT 512, Chicago, IL 60612 Narahashi, Toshio, Northwestern University Medical School. Department of Pharmacology. 303 East Chicago Avenue. Chicago. IL 60611 Nasi, Enrico, Boston University School of Medical. Department of Physiology, R-406. 80 East Concord Street, Boston, MA 02118 Neill, Christopher, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Nelson, Margaret C., Cornell University, Section of Neurobiology and Behavior, Ithaca. NY 14850 Nicholls, John G., SISSA, Via Beirut 2, 1-34014 Trieste, Italy Nickerson, Peter A., SUNY at Buffalo, Department of Pathology. Buffalo. NY 14214 Nicosia, Santo V., University of South Florida, College of Medicine, Box 1 1, Department of Pathology, Tampa. FL 33612 Noe, Bryan D., Emory University School of Medicine. Department of Anatomy and Cell Biology. Atlanta, GA 30322 Norton, Catherine N., Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Nusbaum, Michael P., University of Pennsylvania School of Medicine, Department of Neuroscience, 215 Stemmler Hall. Philadelphia. PA 19104-6074 O'Herron, Jonathan, Lazard Freres and Company, 30 Rockefeller Plaza, 59th Floor, New York, NY 10020-1900 Obaid, Ana Lia, University of Pennsylvania School of Medicine, Neuroscience Department, 234 Stemmler Hall, Philadelphia, PA 19104-6074 Ohki, Shinpei, SUNY at Buffalo, Department of Biophysical Sciences, 224 Cary Hall, Buffalo. NY 14214 Oldenbourg. Rudolf, Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Olds, James L., George Mason University, Krasnow Institute for Advanced Studies, Mail Stop 2A1, Fairfax. VA 22030-4444 Olins, Ada L., Foundation for Blood. 69 U.S. Route One, P.O. Box 190. Scarborough, ME 04070-0190 Olins, Donald E., Foundation for Blood, 69 U.S. Route One. P.O. Box 190. Scarborough. ME 04070-0190 Oschman, James L., 827 Central Avenue. Dover. NH 03820 Palazzo, Robert E., University of Kansas. Department of Physiology and Cell Biology. Lawrence, KS 66045 Palmer, John D., University of Massachusetts, Department of Zoology, 221 Morrill Science Center, Amherst, MA 01003 Pant, Harish C., National Institutes of Health, NINCDS, Laboratory of Neurochemistry, Building 36, Room 4D20, Bethesda, MD 20892 Pappas, George D., University of Illinois, Psychiatric Institute, 1601 W. Taylor Street, MC 912, Chicago, IL 60612 Pardee, Arthur B., Dana-Farber Cancer Institute, D810. 44 Binney Street. Boston. MA 02115 Pardy. Rosevelt L., University of Nebraska. School of Life Sciences, Lincoln, NE 68588 Parmentier. James L., Massachusetts General Hospital, Partners/Fenway/Shattuck Center for Aids Research, 149 13^ Street, Room 5219, Charlestown, MA 02129 Pederson, Thoru, University of Massachusetts Medical Center. Worcester Foundation Campus. 222 Maple Avenue, Shrewsbury, MA 01545 Perkins, Courtland D., 400 Hilltop Terrace, Alexandria. VA 22301 Person, Philip, 137-87 75th Road, Flushing, NY 1 1367 Peterson, Bruce J., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 R80 Annual Report Pethig, Ronald. University College of North Wales, School of Electronic Engineering, Bangor, Gwynedd. LL 57 IUT, United Kingdom Pfohl, Ronald J., Miami University, Department of Zoology, Oxford. OH 45056 Pierce, Sidney K., Jr., University of South Florida. Department of Biology. SCA 110, 4202 East Fowler Avenue, Tampa, FL 33620 Pleasure, David E., Children's Hospital, Neurology Research, 5th Floor, Ambramson Building, Philadelphia. PA 19104 Poindexter, Jeanne S., Barnard College. Columbia University. 3009 Broadway, New York. NY 10027-6598 Pollard, Harvey B., U.S.U.H.S., 4301 Jones Bridge Road, Bethesda, MD 20814 Pollard. Thomas D., Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037 Porter, Beverly H., 5542 Windysun Court. Columbia. MD 21045 Porter, Mary E., University of Minnesota, Department of Cell Biology and Neuroanatomy, 4-135 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455 Potter, David D., Harvard Medical School. Department of Neurobiology. 25 Shattuck Street. Boston, MA 021 15 Potts, William T., University of Lancaster. Department of Biology, Lancaster, England Powers, Maureen K., University of California. Department of Molecular & Cellular Biology, Life Sciences Addition, Berkeley, CA 94720 Prendergast, Robert A., 29 Pondlet Place, Falmouth. MA 02540 Prior, David J., Northern Arizona University, Arts and Sciences Dean's Office, Box 5621, Flagstaff, AZ 8601 1 Prusch, Robert D., Gonzaga University, Department of Life Sciences, Spokane, WA 99258 Purves, Dale, Duke University Medical Center, Department of Neurobiology, Box 3209, 101-1 Bryan Research Building, Durham, NC 27710 Quigley, James P., The Scripps Research Institute. Department of Vascular Biology, 10550 N. Torrey Pines Road VB-1, La Jolla, CA 92037 Rabb, Irving W., 1010 Memorial Drive, #20A. Cambridge, MA 02138 Rabin. Harvey, 1 102 Ralston Road, Rockville, MD 20852 Rabinowitz, Michael B., Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Rafferty, Nancy S., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Rakowski, Robert F., Finch University of Health Sciences, The Chicago Medical School, Department of Physiology and Biophysics, 3333 Greenbay Road, N. Chicago, IL 60064 Ramon, Fidel, Universidad Nacional Autonoma de Mexico, Division EStreet Posgrado E Invest., Facultad de Medicina. 04510, D.F., Mexico Rastetter, Edward B., Marine Biological Laboratory, The Ecosystems Center. Woods Hole. MA 02543 Rebhun, Lionel I., University of Virginia, Department of Biology, Gilmer Hall 45, Charlottesville. VA 22901 Reddan, John R., Oakland University, Department of Biological Sciences. Rochester, MI 48309-4401 Reese, Thomas S., National Institutes of Health, NINDS. Department of Neurobiology, Building 36, Room 2A-21. 36 Convent Drive, Bethesda, MD 20892 Reinisch, Carol L., Marine Biological Laboratory. 7 MBL Street, Woods Hole. MA 02543 Rickles, Frederick R., 3910 Highwood Court. N.W., Washington, DC 20007 Rieder, Conly L., Wadsworth Center, Division of Molecular Medicine, P.O. Box 509, Albany. NY 12201-0509 Riley. Monica, Marine Biological Laboratory. 7 MBL Street, Woods Hole. MA 02543 Ripps, Harris, University of Illinois at Chicago, Department of Ophthalmology/Visual Sciences, 1855 West Taylor Street, Chicago, IL 60612 Ritchie, J. Murdoch, Yale University School of Medicine, Department of Pharmacology, 333 Cedar Street, New Haven. CT 06510 Rome, Lawrence C., University of Pennsylvania, Department of Biology, Leidy Labs, Philadelphia. PA 19104 Rosenbluth, Jack, New York University School of Medical. Department of Physiology and Biophysics, RR 714, 400 East 34th Street, New York, NY 10016 Rosenbluth, Raja, Simon Fraser University, Institute of Molecular Biology and Biochemistry. Burnaby, BC V5A 1S6, Canada Rosenfield, Allan, Columbia University School of Public Health, 600 West 168th Street. New York. NY 10032-3702 Rosenkranz, Herbert S., 130 Desoto Street, Pittsburgh. PA 15213-2535 Ross, William N., New York Medical College, Department of Physiology, Valhalla, NY 10595 Rottenfusser, Rudi, Marine Biological Laboratory. 7 MBL Street, Woods Hole, MA 02543 Rowland, Lewis P.. Neurological Institute, 710 West 168th Street, New York, NY 10032 Ruderman, Joan V., Harvard Medical School, Department of Cell Biology. C2-428. 240 Longwood Avenue. Boston, MA 021 15 Rummel, John D., NASA Headquarters. Office of Space Science. Washington. DC 20546 Rushforth, Norman B., Case Western Reserve University, Department of Biology, Cleveland, OH 44106 Russell-Hunter, William D., 71 1 Howard Street, Easton, MD 21601- 3934 Saffo, Mary Beth, Harvard University, MCZ Labs 408, 26 Oxford Street, Cambridge, MA 02138 Salama, Guy, University of Pittsburgh, Department of Physiology. Pittsburgh. PA 15261 Salmon, Edward D., University of North Carolina, Department of Biology, CB 3280, Chapel Hill, NC 27514 Salyers, Abigail, University of Illinois, Department of Microbiology. B-103, 601 South Goodwin Avenue, Urbana, IL 61801 Salzberg, Brian M., University of Pennsylvania School of Medicine, Department of Neuroscience, 215 Stemmler Hall, Philadelphia, PA 19104-6074 Sanger, Jean M., University of Pennsylvania School of Medicine. Department of Anatomy, 36th and Hamilton Walk, Philadelphia, PA 19104 Sanger, Joseph W., University of Pennsylvania Medical Center, Department of Cell and Developmental Biology. 36th and Hamilton Walk. Philadelphia, PA 19104-6058 Saunders, John W., Jr., 118 Metoxit Road. P.O. Box 3381. Waquoit. MA 02536 Schachman, Howard K., University of California, Molecular and Cell Biology Department, 229 Stanley Hall, #3206, Berkeley, CA 94720- 3206 Schatten, Gerald P., Oregon Health Sciences University, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, OR 97006 Members of the Corporation R81 Schmeer, Arlene C., Mercenene Cancer Research Institute. 790 Prospect Street. New Haven. CT 06511 Schuel, Herbert. SUNY at Buffalo. Department of Anatomy/Cell Biology. Buffalo. NY 14214 Schwartz, Lawrence. University of Massachusetts. Department of Biology. Morrill Science Center. Amherst. MA 01003 Schweitzer. A. Nicola. Brigham and Women's Hospital. Immunology Division. Department of Pathology. 221 Longwood Avenue, LMRC 521, Boston, MA 02115 Segal, Sheldon J., The Population Council. One Dag Hammarskjold Plaza, New York, NY 10036 Senft, Stephen Lament, Yale University, Neuroengineering/Neuroscience Center, P.O. Box 208205, New Haven. CT 06520-8205 Shanklin. Douglas R., University of Tennessee. Department of Pathology, Room 576, 800 Madison Avenue, Memphis. TN 381 17 sli.i-.li.ii . Nadav, The Interuniversity Institute of Eilat. P.O. Box 469, Eilat 88103. Israel Shashoua, Victor E., Harvard Medical School. Ralph Lowell Labs. McLean Hospital, 115 Mill Street, Belmont, MA 02178 Shaver, Gaius R., Marine Biological Laboratory, The Ecosystems Center. Woods Hole. MA 02543 Shaver. John R., Michigan State University. Department of Zoology. East Lansing. MI 48824 Sheetz, Michael P., Duke University Medical Center. Department of Cell Biology. Bx 3709. 388 Nanaline Duke Building. Durham. NC 27710 Shepro, David, Boston University. CAS Biology, 5 Cummington Street, Boston. MA 022 1 5 Shimomura. Osamu, Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Shipley. Alan M., P.O. Box 943. Forestdale. MA 02644 Silver, Robert B., Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Siwicki, Kathleen K., Swarthmore College, Biology Department. 500 College Avenue. Swarthmore. PA 19081-1397 Skinner, Dorothy M., 24 Gray Lane, Falmouth. MA 02540 Sloboda, Roger D., Dartmouth College, Department of Biological Science, 6044 Gilman, Hanover. NH 03755-1893 Sluder, Greenfield, University of Massachusetts Medical School. Room 324. 377 Plantation Street. Worcester. MA 01605 Smith, Peter J. S., Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Smith, Stephen J., Stanford University School of Medicine, Department of Molecular and Cellular Physiology. Beckman Center. Stanford, CA 94305 Smolowitz. Roxanna S., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Sogin, Mitchell L., Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Sorenson, Martha M., Cidade Universitaria-UFRJ, Department Bioquimica Medica-ICB. 21941-590 Rio de Janerio. Brazil Speck, William T., Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Spector, Abraham, Columbia University. Department of Ophthalmology, 630 West 168th Street. New York. NY 10032 Speksnijder, Johanna E., DeMeent 12. 3984JJ Odijk. The Netherlands Spray, David C., Albert Einstein College of Medicine. Department of Neuroscience. 1300 Moms Park Avenue. Bronx. NY 10461 Spring, Kenneth R., National Institutes of Health, 10 Center Drive. MSC 1598, Building 10. Room 6N260, Bethesda, MD 20892-1603 Steele, John H., Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Steinacker, Antoinette, University of Puerto Rico. Institute of Neurobiology. 201 Boulevard Del Valle. San Juan. PR 00901 Steinberg, Malcolm. Princeton University, Department of Molecular Biology, M-18 Moffett Laboratory. Princeton. NJ 08544-1014 Stemmer, Andreas C., Institut filr Robotik. ETH-Center. 8092 Zurich, Switzerland Stenflo, Julian. University of Lund, Department of Clinical Chemistry. Malmo General Hospital. S-205 02 Malmo, Sweden Stetten, Jane Lazarow, 4701 Willard Avenue, #1413. Chevy Chase. MD 20815-4627 Steudler, Paul A., Marine Biological Laboratory, The Ecosystems Center, Woods Hole, MA 02543 Stokes, Darrell R., Emory University, Department of Biology, 1510 Clifton Road NE. Atlanta. GA 30322-1 100 Stommel, Elijah W., Dartmouth Hitchcock Medical Center. Neurology Department, 1 Medical Drive, Lebanon, NH 03756 Stracher. Alfred, SUNY Health Science Center, Department of Biochemistry. 450 Clarkson Avenue. Brooklyn. NY 11203 Strumwasser, Felix, P.O. Box 923. East Falmouth. MA 02536-2278 Stuart, Ann E., 1818 North Lakeshore Drive. Chapel Hill, NC 27514 Sugimori, Mutsuyuki. New York University Medical Center. Department of Physiology and Neuroscience. Room 442, 550 First Avenue. New York. NY 10016 Summers. William C., Western Washington University. Huxley College of Environmental Studies, Bellingham, WA 982259181 Suprenant, Kathy A., University of Kansas, Department of Physiology and Cell Biology. 4010 Haworth Hall, Lawrence. KS 66045 Sydlik, Mary Anne, Hope College. Peale Science Center. 35 East 12th St./PO Box 9000. Holland, MI 49422 Szent-Gyorgyi, Andrew G., Brandeis University. Molecular and Cell Biology, 415 South Street. Waltham, MA 02454-91 10 Tamm, Sidney L., Boston University. CAS Biology. 5 Cummington Street. Boston, MA 02215 Tanzer, Marvin L., University of Connecticut School of Dental Medicine. Department of Biostructure and Function. Farmington. CT 06030-3705 Tasaki, Ichiji, National Institutes of Health. NIMH. Laboratory of Neurobiology, Building 36, Room 2B-16. Bethesda. MD 20892 Taylor, D. Lansing, Cellomics. Inc., 635 William Pitt Way. Pittsburgh. PA 15238 Taylor, Edwin W'., University of Chicago, Department of Molecular Genetics, 920 E. 58th Street, Chicago. IL 60637 Teal, John M., 567 New Bedford Lane. Rochester, MA 02770 Telfer, William H., University of Pennsylvania. Department of Biology. Philadelphia. PA 19104 Telzer, Bruce, Pomona College, Department of Biology, Thille Building. 175 West 6th Street, Claremont, CA 91711 Terasaki, Mark, University of Connecticut Health Center, Department of Physiology, 263 Farmington Avenue, Farmington. CT 06032 Townsel, James G., Meharry Medical College. Department of Anatomy and Physiology. 1005 DB Todd Boulevard. Nashville. TN 37208 Travis. David M., 19 High Street. Woods Hole. MA 02543-1221 Treistman. Steven N., University of Massachusetts Medical Center. Department of Pharmacology, 55 Lake Avenue North. Worcester. MA 01655 Trigg, D. Thomas, One Federal Street. 9th Floor, Boston, MA 022 1 1 Troll, Walter, NYU Medical Center. Department of Environmental Medicine. 550 First Avenue. New York. NY 10016 Troxler, Robert F., Boston University School of Medicine. Department of Biochemistry. 80 East Concord Street. Boston. MA 021 18 Tucker, Edward B., Baruch College. CUNY. Department of Natural Sciences, 17 Lexington Avenue, New York, NY 10010 R82 Annual Report Turner, Ruth D., Harvard University, Museum of Comparative Zoology, Mollusk Department, Cambridge. MA 02138 (deceased 2000) Tweedell, Kenyon S., University of Notre Dame, Department of Biological Sciences, Notre Dame, IN 46556-0369 Tykocinski. Mark L., Case Western Reserve University, Institute of Pathology, 2085 Adelbert Road, Cleveland, OH 44106 Tytell, Michael, Wake Forest University, Bowman Gray School of Medicine. Department of Anatomy and Neurobiology. Winston- Salem, NC 27157 Ueno, Hiroshi, Kyoto Universily, AGR Chemistry, Faculty of Agriculture. Sakyo, Kyoto 606-8502, Japan Valiela, Ivan, Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 Vallee, Richard, University of Massachusetts Medical Center, Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury. MA 01545 Valois, John J., 420 Woods Hole Road, Woods Hole, MA 02543 Van Dover, Cindy Lee, The College of William and Mary. Biology Department. 328 Millington Hall, Williamsburg, VA 23187 Van Holde, Kensal E., Oregon State University, Biochemistry and Biophysics Department. Corvallis, OR 97331-7503 Vogl, Thomas P., Environmental Research Institute of Michigan, 1101 Wilson Boulevard, Arlington. VA 22209 Wainwright, Norman R., Marine Biological Laboratory. 7 MBL Street. Woods Hole, MA 02543 Waksman, Byron H., New York University Medical Center. Department of Pathology. 550 First Avenue, New York. NY 10016 Wall, Belly, 9 George Street. Woods Hole, MA 02543 Wangh, Lawrence J., Brandeis University, Department of Biology, 415 South Street. Waltham. MA 02254 Warner, Robert C., 1609 Temple Hills Drive, Laguna Beach, CA 9265 1 Warren, Leonard, Wistar Institute, 36th and Spruce Streets, Philadelphia. PA 19104 Waterbury, John B., Woods Hole Oceanographic Institution, Department of Biology, Woods Hole. MA 02543 Waxman, Stephen G., Yale Medical School. Neurology Department. 333 Cedar Street. P.O. Box 208018, New Haven. CT 06510 Weber, Annemarie, University of Pennsylvania School of Medicine, Department of Biochemistry and Biophysics. Philadelphia, PA 19066 Weeks, Janis C., University of Oregon. Institute of Neuroscience, Eugene, OR 97403-1254 Weidner, Earl, Louisiana State University, Department of Biological Sciences, 502 Life Sciences Building, Baton Rouge, LA 70803 Weiss, Alice Sara, 105 University Boulevard West. Silver Spring, MD 20901 Weiss, Dieter G., University of Rostock, Institute of Zoology, D- 18051 Rostock, Germany Weiss, Leon P., University of Pennsylvania School of Veterinary Medicine, Department of Animal Biology, Philadelphia. PA 19104 Weiss, Marisa C., Paoli Memorial Hospital, Department of Radiation Oncology. 255 W. Lancaster Avenue, Paoli, PA 19301 Weissmann, Gerald, New York University Medical Center. Department of Medicine/Division Rheumatology, 550 First Avenue, New York, NY 10016 Westerfield, Monte, University of Oregon, Institute of Neuroscience, Eugene. OR 97403 Whittaker, J. Richard, University of New Brunswick, Department of Biology, BS 451 1, Fredericton, NB E3B 6E1, Canada Wiesel, Torsten N., Rockefeller University, 1230 York Avenue, New York, NY 10021 Wilkens, Lon A., University of Missouri. Department of Biology. 8001 Natural Bridge Road. St. Louis. MO 63121-4499 Wilson, Darcy B., Torrey Pines Institute, 3550 General Atomics Court, Building 2. Room 138. San Diego. CA 92121 Wilson, T. Hastings, Harvard Medical School. Department of Physiology, 25 Shattuck Street, Boston, MA 02 1 1 5 Witkovsky, Paul, NYU Medical Center. Department of Ophthalmology. 550 First Avenue, New York, NY 10016 Wittenberg, Beatrice, Albert Einstein College of Medicine. Department nt Physiology and Biophysics. Bronx, NY 10461 Wittenberg, Jonathan B., Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, NY 10461 Wonderlin, William F., West Virginia University, Pharmacology and Toxicology Department, Morgantown, WV 26506 Worden, Mary Kate, University of Virginia, Department of Neuroscience, McKim Hall Box 230. Charlottesville, VA 22908 Worgul, Basil V., Columbia University, Department of Ophthalmology, 630 West 168 Street. New York. NY 10032 Wu, Chau Hsiung, Northwestern University Medical School, Department of Pharmacology (S215). 303 East Chicago Avenue, Chicago. IL 60611-3008 Wyttenbach, Charles R., University of Kansas. Biological Sciences Department. 2045 Haworth Hall, Lawrence, KS 66045-2106 Zakon, Harold H., University of Texas, Section of Neurobiology, School of Life Science, Austin. TX 787 1 2 Zigman, Seymour, Marine Park Condominiums, 1 74 Queen Street. Unit 10-F, Falmouth, MA 02540 Zigmond, Michael J., University of Pittsburgh, S-526 Biomedical Science Tower, 3500 Terrace Street. Pittsburgh, PA 15213 Ziinmerberg, Joshua J., National Institutes of Health, LCMB. NICHD, Building 10, Room KID 14, 10 Center Drive. Bethesda, MD 20892 Zottoli, Steven J., Williams College. Department of Biology, Williamstown. MA 01267 Zucker, Robert S., University of California, Neurobiology Division, Molecular and Cellular Biology Department, Berkeley, CA 94720 Members of the Corporation R83 MBL Associates Executive Board Ruth Ann taster. President Jack Pearce. Vice President Kitty Brown. Treasurer Molly N. Cornell, Secretary Duncan Aspinwall. Membership Chair Tammy Smith Amon Barbara At wood Julie Child Seymour Cohen Elizabeth Farnham Michael Fenlon Pat Ferguson Sallie A. Giffen Alice Knowles Rebecca Lash Cornelia Hanna McMurtrie Joan Pearlman Virginia Reynolds Volker Ulbnch Associates Liaison/Gift Shop Coordinator Kendall B. Bohr Patron Judge and Mrs. John S. Langford Sustaining Associate Mr. and Mrs. G. Nathan Calkins, Jr. Mrs. Janet F. Gillette Dr. and Mrs. Edward F. MacNichol, Jr. Supporting Associate Mr. and Mrs. William O. Burwell Mr. and Mrs. Thomas Claflin Mrs. George H. A. Clowes Dr. and Mrs. James D. Ebert Mr. and Mrs. David Fausch Mr. Mike Fenlon and Ms. Linda Sallop Dr. and Mrs. James J. Ferguson. Jr. Mrs. Janet F. Gillette Mrs. Mary L. Goldman Mr. and Mrs. Lon Hocker Mr. and Mrs. Arthur King Dr. and Mrs Leonard Laster Drs. Luigi and Elaine Mastroianni Mr. and Mrs. Walter J. Salmon Mrs. Anne W. Sawyer Dr. John Tochko and Mrs. Christina Myles- Tochko Mr. and Mrs. John J. Valois Mr. and Mrs. Leslie J. Wilson Familv Membership Dr. and Mrs. Edward A. Adelberg Mr. and Mrs. David C. Ahearn Dr. and Mrs. Dean C. Allard. Jr. Drs. James and Helene Anderson Dr. and Mrs. Samuel C. Armstrong Mr. and Mrs. Duncan P. Aspinwall Mr. and Mrs. Donald R. Aukamp Mr. and Mrs. John M. Baitsell Mr. and Mrs. David Bakalar Dr. and Mrs. Robert B. Barlow, Jr. Mr. and Mrs. John E. Barnes Dr. and Mrs. Robert M. Berne Drs. Harriet and Alan Bemheimer Mr. and Mrs. Robert O. Bigelow Dr. and Mrs. Edward G. Boettiger Mr. and Mrs. Kendall B. Bohr Dr. and Mrs. Thomas A. Borgese Dr. and Mrs. Francis P. Bowles Dr. and Mrs. John B. Buck Dr. and Mrs. John E. Bums Mr. and Mrs. D. Bret Carlson Dr. and Mrs. Richard L. Chappell Dr. and Mrs. Frank M. Child Dr. and Mrs. Arnold M. Clark Mr. and Mrs. James M. Cleary Dr. and Mrs. Laurence P. Cloud Drs. Harry Conner and Carol Scott-Conner Mrs. Neal Cornell Mr. and Mrs. Norman C. Cross Dr. and Mrs. John M. Cummings Mr. and Mrs. Joel P. Davis Mr. and Mrs. F. Gerald Douglass Dr. and Mrs. John E. Dowling Dr. and Mrs. Arthur Brooks DuBois Dr. and Mrs. Michael J. Fishbein Mr. and Mrs. Harold Frank Mr. and Mrs. Howard G. Freeman Dr. and Mrs. Robert A. Frosch Dr. and Mrs. John J. Funkhouser Dr. and Mrs. Mordecai L. Gabriel Dr. and Mrs. Sydney Gellis Dr. and Mrs. James L. German, III Dr. and Mrs. Harold S. Ginsberg Dr. and Mrs. Murray Glusman Drs. Alfred and Joan Goldberg Mr. and Mrs. Charles Goodwin, III Mr. and Mrs. Anthony D. Green Dr. and Mrs. Thomas C. Gregg Dr. and Mrs. Newton H. Gresser Mr. and Mrs. Peter A. Hall Dr. and Mrs. Harlyn O. Halvorson Dr. and Mrs. Richard Bennet Harvey Dr. and Mrs. J. Woodland Hastings Dr. Robert R. Haubrich Mr. and Mrs. Gary G. Hayward Dr. and Mrs. Howard H. Hiatt Mr. and Mrs. David Hibbitt Dr. and Mrs. John E. Hobble Mr. and Mrs. Gerald J. Holtz Drs. Francis Hoskin and Elizabeth Farnham Dr. and Mrs. Robert J. Huettner Dr. and Mrs. Shinya Inoue Dr. and Mrs. Kurt J. Isselbacher Mrs. Mary D. Janney Dr. and Mrs. Benjamin Kaminer Mr. and Mrs. Paul W. Knaplund Mr. and Mrs. A. Sraney Knowles, Jr. Mr. and Mrs. Walter E. Knox Sir and Lady Hans Kornberg Dr. and Mrs. S. Andrew Kulin Mr. Ezra and Dr. Aimlee Laderman Mr. and Mrs. Trevor Lambert Dr. and Mrs. George M. Langford Dr. and Mrs. Hans Laufer Dr. and Mrs. Berton J. Leach Dr. and Mrs. John J. Lee Mr. and Mrs. Stephen R. Levy Mr. and Mrs. Robert Livingstone, Jr. Dr. and Mrs. Laszlo Lorand Mr. and Mrs. Francis C. Lowell, Jr. Dr. Isabelle and Mr. Bernard Manuel Mr. and Mrs. Joseph C. Martyna Mr. and Mrs. Frank J. Mather, UJ Dr. and Mrs. Robert T. McCluskey Dr. and Mrs. William M. McDermott Dr. and Mrs. Jerry M. Melillo Mr. and Mrs. Wesley J. Merrill 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 Dr. and Mrs. John E. Naugle Dr. Pamela Nelson and Mr. Christopher Olmsted Mr. and Mrs. Frank L. Nickerson Dr. and Mrs. Clifford T. O'Connell Mr. and Mrs. James J. O'Connor Mr. and Mrs. David R. Palmer Mr. and Mrs. Robert Parkinson Mr. and Mrs. Richard M. Paulson, Jr. Dr. and Mrs. John B. Pearce Mr. and Mrs. William J. Pechilis Mrs. Nancy Pendleton Mr. and Mrs. John B. Peri Dr. and Mrs. Courtland D. Perkins Dr. and Mrs. Philip Person Mr. and Mrs. Frederick S. Peters Mr. and Mrs. E. Joel Peterson Mr. and Mrs. Harold Pilskaln Mr. and Mrs. George H. Plough Dr. and Mrs. Aubrey Pothier, Jr. Mr. and Mrs. Allan Putnam Dr. and Mrs. Lionel I. Rebhun Dr. and Mrs. George T. Reynolds Dr. and Mrs. Harris Ripps Dr. Paul B. Rizzoli Ms. Jean Roberts Drs. Priscilla and John Roslansky Mr. and Mrs. John D. Ross Dr. and Mrs. John W. Saunders, Jr. Dr. and Mrs. R. Walter Schlesinger R84 Annual Report Mr. and Mrs. Harold H. Sears Dr. and Mrs. Sheldon J. Segal Mr. and Mrs. Daniel Shearer Dr. and Mrs. David Shepro Mr. and Mrs. Bertram R. Silver Mr. and Mrs. Jonathan O. Simonds Drs. Frederick and Marguerite Smith Dr. and Mrs. Alan B. Stembach Dr. and Mrs. William K. Stephenson Mr. and Mrs. E. Kent Swift, Jr. Mr. and Mrs. Gerard L. Swope. Ill Mr. Norman N. Tolkan Dr. and Mrs. Walter Troll Prof, and Mrs. Michael Tytell Mr. and Mrs. Volker Ulbrich Dr. and Mrs. Gerald Weissmann Dr. and Mrs. Paul S. Wheeler Dr. and Mrs. Martin Keister White Mr. and Mrs. Geoffrey G. Whitney, Jr. Mr. and Mrs. Lynn H. Wilke Dr. and Mrs. T. Hastings Wilson Mrs. Sumner Zacks Dr. Linda and Mr. Erik Zettler Dr. and Mrs. Seymour Zigman Individual Membership Drs. Fred and Peggy Alsup Mrs. Tammy Smith Amon Mr. Dean N. Arden Mrs. Ellen Prosser Armstrong Mrs. Kimball C. Atwood, III Mr. Everett E. Bagley Dr. Millicent Bell Mr. C. John Berg Dr. Thomas P. Bleck Ms. Avis Blomberg Mr. Theodore A. Bonn Mr. James V. Bracchitta Mrs. Jennie P. Brown Mrs. M. Kathryn S. Brown Dr. Robert H. Broyles Mrs. Barbara Gates Burwell Dr. Graciela C. Candelas Mr. Frank C. Carotenuto Dr. Robert H. Carrier Mrs. Patricia A. Case Ms. Mia D. Champion Dr. Sallie Chisholm Mrs. Octavia C. Clement Mr. Allen W. Clowes Dr. Jewel Plummer Cobb Mrs. Margaret H. Coburn Dr. Seymour S. Cohen Dr. Alan Robert Cole Ms. Anne S. Concannon Prof. D. Eugene Copeland Dr. Vincent Cowling Mrs. Marilyn E. Crandall Ms. Dorothy Crossley Ms. Helen M. Crossley Mrs. Villa B, Crowell Mrs. Alexander T. Daignault Dr. Morton Davidson Mrs. Elizabeth M. Davis Ms. Maureen Davis Ms. Carol Reimann DeYoung Ms. Shirley Dierolf Mrs. Juliette G. Dively Mr. David L. Donovan Ms. Suzanne Droban Mr. Roy A. Duffus Ms. Maureen J. Dugan Mrs. Charles Eastman Dr. Frank Egloff Ms. Judy Ernst Dr. Stephen L. Estabrooks Mrs. Eleanor B. Faithorn Mrs. Ruth Alice Fitz Ms. Sylvia M. Flanagan Mr. John W. Folino. Jr. Mrs. Kathryn W. Foster Dr. Krystyna Frenkel Mr. Paul J. Freyheit Mrs. Ruth E. Fye Mrs. Lois E. Galvin Miss Eleanor Garneld Mrs. Ruth H. Garland Mr. John Garnett Ms. Sallie A. Giffen Mrs. James R. Glazebrook Mr. Michael P. Goldring Mrs. Phyllis Goldstein Mrs. DeWitt S. Goodman Ms. Muriel Gould Mrs. Rose Grant Ms. Janet M. Gregg Mrs. Jeanne B. Griffith Mrs. Barbara Grossman Mrs. Valerie A. Hall Ms. Mary Elizabeth Hamstrom Dr. Carol W. Hannenberg Ms. Elizabeth E. Hathaway Mrs. Elizabeth Heald Mrs. Jane G. Heald Mrs. Betty G. Hubbell Miss Elizabeth B. Jackson Mr. Raymond L. Jewett Mrs. Barbara W. Jones Mrs. Joan T. Kanwisher Mrs. Sally Karush Ms. Patricia E. Keoughan Dr. Peter N. Kivy Dr. Annlee D. Laderman Mrs. Janet W. Larcom 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 Mr. Richard C. Lovenng Mrs. Margaret M. MacLeish Ms. Anne Camille Maher Mrs. Nancy R. Malkiel Ms. Diane Maranchie Dr. Miriam Jacob Mauzerall Mrs. Mary Hartwell Mavor Mr. Paul McGonigle Dr. Susan Gerbi Mcllwam Ms. Mary W. McKoan Ms. Jane A. McLaughlm Ms. Louise McManus Ms. Cornelia Hanna McMurtne Mrs. Anne L. Meigs-Brown Mr. Ted Melillo Dr. Martin Mendelson Ms. Carmen Merryman Mrs. Grace S. Metz Mrs. Mary G. Miles Mrs. Florence E. Mixer Mr. Lawrence A. Monte Mrs. Mary E. Montgomery Ms. Cynthia Moor Mr. James V. Moynihan Mrs. Eleanor M. Nace Mrs. Anne Nelson Ms. C. Marie Newman Dr. Eliot H. Nierman Mr. Edmund F. Nolan Ms. Catherine N. Norton Dr. Renee Bennett O' Sullivan Dr Arthur B. Pardee Ms. Carolyn L. Parmenter Ms. Joan Pearlman Mr. Raymond W. Peterson Ms. Elizabeth T. Price Ms. Dianne Purves Mrs. Julia S. Rankin Dr. Margaret M. Rappaport Mr. Fred J. Ravens, Jr. Ms. Mary W. Rianhard Dr. Mary Elizabeth Rice Dr. Monica Riley Mrs. Lola E. Robertson Mrs. Arlene Rogers Ms. Jean Rogers Mrs. Wendy E. Rose Mrs. Atholie K. Rosett Dr. Virginia F. Ross Dr. John D. Rummel Mr. Raymond A. Sanbom Mr. Claude Schoepf Ms. Elaine Schott Ms. Emily Schwartz-Clark Mrs. Elsie M. Scott Dr. Cecily C. Selby Mrs. Deborah G. Senft Ms. Dorothy Sgarzi Mrs. Charlotte Shemin Ms. Enid K. Sichel Dr. Jeffrey D. Silberman Mrs. Cynthia C. Smith Mr. Sean W. Smith Mrs. Louise M. Specht Dr. Guy L. Steele, Sr. Dr. Robert E. Steele Mrs. Eleanor Steinbach Mrs. Judith G. Stetson Mrs. Jane Lazarow Stetten Mrs. Elizabeth Stommel Members of the Corporation R85 Mr. Albert H. Swain Elisabeth Buck Barbara Thomson Mrs. Belle K. Taylor Jewel Cobb Alice Todd Mr. James K. Taylor Janet Daniels Elaine Troll Mr. Emil D. Tietje, Jr. Carol DeYoung Natalie Trousof Mrs. Alice Todd Fran Eastman Barbara Van Holde Mr. Arthur D. Traub Alma Ebert Doris Van Keuren Mr. D. Thomas Trigg Jane Foster Susan Veeder Ms. Natalie Trousof Becky Glazebrook Carol Ann Wagner Ms. Ciona Ulbrich Muriel Gould Mabel Whelpley Ms. Sylvia Vatuk Barbara Grossman Clare Wilber Ms. Susan Veeder Jean Halvorson Betty Wilson Mr. Lee D. Vincent Hanna Hastings Grace Witzell Mr. Arthur D. Voorhis Sally Karush Bunnie Rose Zigman Mrs. Eve Warren Marcella Katz Mr. John T. Weeks Alice Knowles Mr. Michael S. Weinstein Donna Kornberg MBL Summer Tour Guides Ms. Lillian Wendorff Evelyn Laufer Ms. Mabel Y. Whelpley Barbara Little Gloria Borgese Mrs. Barbara Whitehead Winnie Mackey Nancy Campana Mrs. Ava Whittemore Diane Maranchie Frank Child Mrs. Joan R. Wickersham Miriam Mauzerall Julie Child Mrs. Clare M. Wilber Mary Mavor Nancy Fraser Mrs. Helen Wilson Jane McCormack Sallie Giffen Ms. Nancy Woitkoski Louise McManus Nichole Graham Ms. Marion K. Wright Mary Miles Lois Harvey Mrs. Dorothy M. York Florence Mixer Lincoln Kraeuter Mrs. Margery P. Zinn Lorraine Mizell Barbara Little Helen Murphy Jennifer Machado Bertha Person Charles Mahoney MBL Gift Shop Volunteers Margareta Pothier Francis X. Mahoney Liz Price Julie Rankin Marion Adelberg Julie Rankin Howard Redpath Barbara Atwood Arlene Rogers Arlene Rogers Beth Berne Lil Saunders Pucky Roslansky Harriet Bernheimer Cynthia Smith Suzanne Thomas Avis Blomberg Peggy Smith Mary Ulbnch Gloria Borgese Louise Specht John Valois Kitty Brown Jane Stetten Margery Zinn Certificate of Organization Articles of Amendment Bylaws Certificate of Organization Articles of Amendment (On File in the Office of the Secretary of the Commonwealth) No. 3170 We, Alpheus Hyatt. President. William Stanford Stevens, Treasurer, and William T. Sedgwick, Edward G. Gardiner. Susan Mims and Charles Sedgwick Minot being a majority of the Trustees of the Marine Biological Laboratory in compliance with the requirements of the fourth section of chapter one hundred and fifteen of the Public Statutes do hereby certify that the following is a true copy of the agreement of association to constitute said Corporation, with the names of the subscribers thereto: We, whose names are hereto subscribed, do, by this agreement, associate ourselves with the intention to constitute a Corporation according to the provisions of the one hundred and fifteenth chapter of the Public Statutes of the Commonwealth of Mas- sachusetts, and the Acts in amendment thereof and in addition thereto. The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LABORATORY The purpose for which the Corporation is constituted is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruc- tion in biology and natural history. The place within which the Corporation is established or located is the city of Boston within said Commonwealth. The amount of its capital stock is none. In Witness Whereof, we have hereunto set our hands, this twenty seventh day of February in the year eighteen hundred and eighty-eight, Alpheus Hyatt, Samuel Mills, William T. Sedgwick. Edward G. Gardiner, Charles Sedgwick Minot, William G. Farlow, William Stanford Stevens. Anna D. Phillips, Susan Minis, B. H. Van Vleck. That the first meeting of the subscribers to said agreement was held on the thirteenth day of March in the year eighteen hundred and eighty-eight. In Witness Whereof, we have hereunto signed our names, this thirteenth day ot March in the year eighteen hundred and eighty-eight. Alpheus Hyatt. President. William Stanford Stevens. Treasurer. Edward G. Gardiner. William T. Sedgwick. Susan Mims, Charles Sedgwick Minot. (Approved on March 20. 1888 as follows: I hereby certify that it appears upon an examination of the within written certificate and the records of the corporation duly submitted to my inspection, that the require- ments of sections one. two and three of chapter one hundred and fifteen, and sections eighteen, twenty and twenty-one of chapter one hundred and six, of the Public Statutes, have been complied with and I hereby approve said certificate this twentieth day of March A.D. eighteen hundred and eighty-eight. Charles Endicott Commissioner of Corporations) (On File in the Office of the Secretary of the Commonwealth) We, James D. Ebert, President, and David Shepro. Clerk of the Marine Biological Labor.iior>. located at Woods Hole. Massachusetts 02543. do hereby certify that the following amendment to the Articles of Organization of the Corporation was duly adopted at a meeting held on August 15. 1975. as adjourned to August 29. 1975. by vote of 444 members, being at least two-thirds of its members legally qualified to vote in the meeting of the corporation: Voted: That the Certificate of Organization of this corporation be and it hereby is amended by the addition of the following provisions: "No Officer. Trustee or Corporate Member of the corporation shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of. or otherwise in connection with, any commitments, agreements, activities or affairs of the corporation. "Except as otherwise specifically provided by the Bylaws of the corporation, meet- ings of the Corporate Members of the corporation may be held anywhere in the United States. "The Trustees of the corporation may make, amend or repeal the Bylaws of the corporation in whole or in part, except with respect to any provisions thereof which shall by law. this Certificate or the bylaws of the corporation, require action by the Corporate Members." The foregoing amendment will become effective when these articles of amendment are filed in accordance with Chapter 180. Section 7 of the General Laws unless these articles specify, in accordance with the vote adopting the amendment, a later effective date not more than thirty days after such filing, in which event the amendment will become effective on such later date. In Witness whereof and Under the Penalties of Perjury, we have hereto signed our names this 2nd day of September, in the year 1975. James D. Ebert. President; David Shepro, Clerk. (Approved on October 24, 1975, as follows: I hereby approve the within articles of amendment and, the filing fee in the amount of $10 having been paid, said articles are deemed to have been filed with me this 24th day of October. l c )75 Paul Guzzi Secretary of the Commonwealth ) Bylaws (Revised August 7. 1992 and December 10. 1992) ARTICLE I THE CORPORATION A. Name an,/ fiirpan: The name of the Corporation shall be The Marine Biolog- ical Laboratory. The Corporation's purpose shall be to establish and maintain ,i R86 Bylaws of the Corporation R87 laboratory or station tor scientific study and investigation and a school lor instruction in biology and natural history. B. Nondiscrimination, The Corporation shall not discriminate on the basis of age, religion, color, race, national or ethnic origin, sex or sexual preference in its policies on employment and administration or in its educational and other programs. ARTICLE n MEMBERSHIP A. Memhcr\. The Members of the Corporation ("Members") shall consist of persons elected by the Board of Trustees (the "Board"), upon such terms and conditions and in accordance with such procedures, not inconsistent with law or these Bylaws, as may be determined by the Board. At any regular or special meeting of the Board, the Board may elect new Members. Members shall have no voting or other rights with respect to the Corporation or its activities except as specified in these Bylaws, and any Member may vote at any meeting of ihe Members in person only and not by proxy. Members shall serve until their death or resignation unless earlier removed with or without cause by the affirmative vote of two-thirds of the Trustees then in office. Any Member who has retired from his or her home institution may, upon written request to the Corporation, be designated a Life Member. Life Members shall not have the right to vote and shall not be assessed for dues. B. Meetings. The annual meeting of the Members shall be held on the Friday following the first Tuesday in August of each year, at the Laboratory of the Corpo- ration in Woods Hole, Massachusetts, at 9:30 a.m. The Chairperson of the Board shall piusulL- at meetings of the Corporation. If no annual meeting is held in accordance with the foregoing provision, a special meeting may be held in lieu thereof with the same effect as the annual meeting, and in such case all references in these Bylaws, except in this Article II. B., to the annual meeting of the Members shall be deemed to refer to such special meeting. Members shall transact business as may properly come before the meeting. Special meetings of the Members may be called by the Chair- person or the Trustees, and shall be called by the Clerk, or in the case of the death, absence, incapacity or refusal by the Clerk, by any other officer, upon written application of Members representing at least ten percent of the smallest quorum of Members required for a vote upon any matter at the annual meeting of the Members, to be held at such time and place as may be designated. C. Quorum. One hundred ( 100) Members shall constitute a quorum at any meeting. Except as otherwise required by law or these Bylaws, the affirmative vote of a majority of the Members voting in person at a meeting attended by a quorum shall constitute action on behalf of the Members. D. Notice of Meetings. Notice of any annual meeting or special meeting of Members, if necessary, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting at least 15 days before such meeting to each Member at his or her address as shown on the records of the Corporation. E. Waiver of Notice. Whenever notice of a meeting is required to be given a Member, under any provision of the Articles or Organization or Bylaws of the Corporation, a written waiver thereof, executed before or after the Meeting by such Member, or his or her duly authorized attorney, shall be deemed equivalent to such notice. F. Adjournments. Any meeting of the Members may be adjourned to any other time and place by the vote of a majority of those Members present at the meeting, whether or not such Members conslitute a quorum, or by any officer entitled to preside at or to act as Clerk of such meeting, if no Member is present or represented. It shall not be necessary to notify any Members of any adjournment unless no Member is present or represented at the meeting which is adjourned, in which case, notice of the adjournment shall be given in accordance with Article II. D. Any business which could have been transacted at any meeting of the Members as originally called may he transacted at an adjournment thereof. ARTICLE III ASSOCIATES OF THE CORPORATION Associates of the Corporation. The Associates of the Marine Biological Laboratory shall be an unincorporated group of persons (including associations and corporations) interested in the Laboratory and shall be organized and operated under the general supervision and authority of the Trustees. The Associates of the Marine Biological Laboratory shall have no voting rights. ARTICLE IV BOARD OF TRUSTEES A. Powers. The Board of Trustees shall have the control and management of the affairs of the Corporation. The Trustees shall elect a Chairperson of the Board who shall serve until his or her successor is elected and qualified. They shall annually elect a President of the Corporation. They shall annually elect a Vice Chairperson of the Board who shall be Vice Chairperson of the meetings of the Corporation. They shall annually elect a Treasurer. They shall annually elect a Clerk, who shall be a resident of Massachusetts. They shall elect Trustees-at-Large as specified in this Article IV. They shall appoint a Director of the Laboratory for a term not to exceed rive years, provided the term shall not exceed one year if the candidate has attained the age of 65 years prior to the date of the appointment. They shall choose such other officers and agents as they shall think best. They may fix the compensation of all officers and agents of the Corporation and may remove them at any time. They may till vacancies occurring in any of the offices. The Board shall have the power to choose an Executive Committee from their own number as provided in Article V, and to delegate to such Committee such of their own powers as they may deem expedient in addition to those powers conferred by Article V. They shall, from time to time, elect Members to the Corporation upon such terms and conditions as they shall have determined, not inconsistent with law or these Bylaws. B. Composition and Election. i 1 ) The Board shall include 24 Trustees elected by the Board as provided below; (a) At least six Trustees {"Corporate Trustees") shall be Members who are scientists, and the other Trustees ("Trustees-at-Large") shall be individuals who need not be Members or otherwise affiliated with the Corporation. (b) The 24 elected Trustees shall be divided into four classes of six Trustees each, with one class to be elected each year to serve for a term of four years, and with each such class to include at least one Corporate Trustee. Such classes of Trustees shall be designated by the year of expiration of their respective terms. (2) The Board shall also include the Chief Executive Officer, Treasurer and the Chairperson of the Science Council, who shall be ex officio voting members of the Board. (3) Although Members or Trustees may recommend individuals for nomination as Trustees, nominations for Trustee elections shall be made by the Nominating Committee in its sole discretion. The Board may also elect Trustees who have not been nominated by the Nominating Committee. C. Eligibility. A Corporate Trustee or a Trustee-at-Large who has been elected to an initial four-year term or remaining portion thereof, of which he/she has served at least two years, shall be eligible for re-election to a second four-year term, but shall be ineligible for re-election to any subsequent term until one year has elapsed after he/she has last served as a Trustee. D. Removal. Any Trustee may be removed from office at any time with or without cause, by vote of a majority of the Members entitled to vote in the election of Trustees; or for cause, by vote of two-thirds of the Trustees then in office. A Trustee may be removed for cause only if notice of such action shall have been given to all of the Trustees or Members entitled to vote, as the case may be, prior to the meeting at which such action is to be taken and if the Trustee to be so removed shall have been given reasonable notice and opportunity to be heard before the body proposing to remove him or her. E. Vacancies. Any vacancy in the Board may be filled by vote of a majority of the remaining Trustees present at a meeting of Trustees at which a quorum is present. Any vacancy in the Board resulting from the resignation or removal of a Corporate Trustee shall be filled by a Member who is a scientist. F. Meetings. Meetings of the Board shall be held from time to time, not less frequently than twice annually, as determined by the Board. Special meetings of Trustees may be called by the Chairperson, or by any seven Trustees, to be held at such time and place as may be designated. The Chairperson of the Board, when present, shall preside over all meetings of the Trustees. Written notice shall be sent to a Trustee's usual or last known place of residence at least two weeks before the meeting. Notice of a meeting need not be given to any Trustee if a written waiver of notice executed by such Trustee before or after the meeting is filed with the records of the meeting, or if such Trustee shall attend the meeting without protesting prior thereto or at its commencement the lack of notice given to him or her. G. Quorum and Action by Trustees. A majority of all Trustees then in office shall constitute a quorum. Any meeting of Trustees may be adjourned by vote of a majority of Trustees present, whether or not a quorum is present, and the meeting may be held as adjourned without further notice. When a quorum is present at any meeting of the Trustees, a majority of the Trustees present and voting (excluding abstentions) shall decide any question, including the election of officers, unless otherwise required by law, the Articles of Organization or these Bylaws. H. Transfers of Interests in Land. There shall be no transfer of title nor long-term lease of real property held by the Corporation without prior approval of not less than two-thirds of the Trustees. Such real property transactions shall be finally acted upon at a meeting of the Board only if presented and discussed at a prior meeting of the Board. Either meeting may be a special meeting and no less than four weeks shall elapse between the two meetings. Any property acquired by the Corporation after December 1. 1989 may be sold, any mortgage or pledge of real property (regardless R8S Annual Report of when acquired) to secure borrowings by the Corporation may be granted, and any transfer of title or interest in real property pursuant to the foreclosure or endorsement of any such mortgage or pledge of real property may be effected by any holder of a mortgage or pledge of real property of the Corporation, with the prior approval of not less than two-thirds of the Trustees (other than any Trustee or Trustees with a direct or indirect financial interest in the transaction being considered for approval) who are present at a regular or special meeting of the Board at which there is a quorum. ARTICLE V COMMITTEES A. Executive Committee. There shall be an Executive Committee of the Board of Trustees which shall consist of not more than eleven (11) Trustees, including c\ officio Trustees, elected by the Board. The Chairperson of the Board shall act as Chairperson of the Executive Committee and the Vice Chairperson as Vice Chairperson. The Executive Committee shall meet at such times and places and upon such notice and appoint such subcommittees as the Committee shall determine. The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meetings of the Board except those powers specifically withheld, from time to time, by vote of the Board or by law. The Executive Committee may also appoint such committees, including persons who are not Trust- ees, as it may, from time to time, approve to make recommendations with respect to matters to be acted upon by the Executive Committee or the Board. The Executive Committee shall keep appropriate minutes of its meetings, which shall be reported to the Board. Any actions taken by the Executive Committee shall also be reported to the Board. B. Nominating Committee. There shall be a Nominating Committee which shall consist of not fewer than four nor more than six Trustees appointed by the Board in a manner which shall reflect the balance between Corporate Trustees and Trustees- at-Large on the Board. The Nominating Committee shall nominate persons for election as Corporate Trustees and Trustees -at -Large, Chairperson of the Board. Vice Chairperson of the Board. President, Treasurer, Clerk, Director of the Laboratory and such other officers, if any, as needed, in accordance with the requirements of these Bylaws. The Nominating Committee shall also be responsible for overseeing the training of new Trustees. The Chairperson of the Board of Trustees shall appoint the Chairperson of the Nominating Committee. The Chairperson of the Science Council shall be an e\ officio voting member of the Nominating Committee. C. Science Council. There shall he a Science Council (the "Council") which shall consist of Members of the Corporation elected to the Council by vote of the Members of the Corporation, and which shall advise the Board with respect to matters con- cerning the Corporation's mission, its scientific and instructional endeavors, and the appomtmenl and promotions of persons or committees with responsibility for mailers requiring scientific expertise. Unless otherwise approved by a majority of the mem- bers of the Council, the Chairperson of the Council shall be elected annually by the Council. The chief executive officer of the Corporation shall be an ex officio voting member of the Council. D. Board of Overseers. There shall be a Board of Overseers which shall consist of not fewer than five nor more than eight scientists who have expertise concerning matters with which the Corporation is involved. Members of the Board of Overseers may or may not be Members of the Corporation and may be appointed by the Board of Trustees on the basis of recommendations submitted from scientists and scientific organizations or societies. The Board of Overseers shall be available to review and offer recommendations to the officers. Trustees and Science Council regarding scientific activities conducted or proposed by the Corporation and shall meet from time to time, not less frequently than annually, as determined by the Board of Trustees. E. Board Committees Generally. The Trustees may elect or appoint one or more other committees (including, but not limited to, an Investment Committee, a Devel- opment Committee, an Audit Committee, a Facilities and Capital Equipment Com- mittee and a Long-Range Planning Committee) and may delegate to any such committee or committees any or all of their powers, except those which by law, the Articles of Organization or these Bylaws the Trustees are prohibited from delegating; provided that any committee to which the powers of the Trustees are delegated shall consist solely of Trustees. The members of any such committee shall have such tenure and duties as the Trustees shall determine. The Investment Committee, which shall oversee the management of the Corporation's endowment funds and marketable securities shall include as e\ officio members, the Chairperson of the Board, the Treasurer and the Chairperson of the Audit Committee, together with such Trustees as may be required for not less than two-thirds of the Investment Committee to consist of Trustees. Except as otherwise provided by these Bylaws or determined by the Trustees, any such committee may make rules for the conduct of its business, but. unless otherwise provided by the Trustees or in such rules, its business shall be conducted as nearly as possible in the same manner as is provided by these Bylaws for the Trustees. F. Actions Without a Meeting. Any action required or permitted to be taken at any meeting of the Executive Committee or any other committee elected by the Trustees may be taken without a meeting if all members of such committees consent to the action in writing and such written consents are filed with the records of meetings. Members of the Executive Committee or any other committee elected by the Trustees may also participate in any meeting by means of a telephone conference call, or otherwise lake action in such a manner as may. from time to time, be permitted by law. G. Manual oj Procedures. The Board of Trustees, on the recommendation of the Executive Committee, shall establish guidelines and modifications thereof to be recorded in a Manual of Procedures. Guidelines shall establish procedures for: (1) Nomination and election of members of the Corporation, Board of Trustees and Executive Committee; (2) Election of Officers; (3) Formation and Function of Standing Committees. ARTICLE VI OFFICERS A. Enumeration. The officers of the Corporation shall consist of a President, a Treasurer and a Clerk, and such other officers having the powers of President. Treasurer and Clerk as the Board may determine, and a Director of the Laboratory. The Corporation may have such other officers and assistant officers as the Board may determine, including (without limitation) a Chairperson of the Board, Vice Chairper- son and one or more Vice Presidents, Assistant Treasurers or Assistant Clerks. Any two or more offices may be held by the same person. The Chairperson and Vice Chairperson of the Board shall be elected by and from the Trustees, but other officers of the Corporation need nol be Trustees or Members. If required by the Trustees, any officer shall give the Corporation a bond for the faithful performance of his or her duties in such amount and with such surety or sureties as shall be satisfactory to the Trustees. B. Tenure. Except as otherwise provided by law, by the Articles of Organization or by these Bylaws, the President. Treasurer, and all other officers shall hold office until the first meeting of the Board following the annual meeting of Members and thereafter, until his or her successor is chosen and qualified. C. Resignation. Any officer may resign by delivering his or her written resignation to the Corporation al its principal office or to the President or Clerk and such resignation shall be effective upon receipt unless it is specified to be effective at some other lime or upon the happening of some other event. D. Removal. The Board may remove any officer with or without cause by a vote of a majority of the entire number of Trustees then in office, at a meeting of the Board called for lhat purpose and for which notice of the purpose thereof has been given, provided that an officer may be removed for cause only after having an opportunity to be heard by the Board at a meeting of the Board at which a quorum is personally present and voting. E. Vacancy. A vacancy in any office may be filled for the unexpired balance of the term by vote of a majority of the Trustees present at any meeting of Trustees at which a quorum is present or by written consent of all of the Trustees, if less than a quorum of Trustees shall remain in office. F. Chairperson. The Chairperson shall have such powers and duties as may be determined by the Board and. unless otherwise determined by the Board, shall serve in that capacity for a term coterminous with his or her term as Trustee. G. Vice Chairperson. The Vice Chairperson shall perform the duties and exercise the powers of the Chairperson in the absence or disability of the Chairperson, and shall perform such other duties and possess such other powers as may be determined by the Board. Unless otherwise determined by the Board, the Vice Chairperson shall serve for a one-year term. H. Director. The Director shall be the chief operating officer and, unless otherwise voted by the Trustees, the chief executive officer of the Corporation. The Director shall, subject to the direction of the Trustees, have general supervision of the Laboratory and control of the business of the Corporation. Al the annual meeting, the Director shall submit a report of the operations of the Corporation for such year and a statement of its affairs, and shall, from lime to time, report to the Board all matters within his or her knowledge which the interests of the Corporation may require to be brought lo its notice. I. Deputy Director. The Deputy Director, if any, or if there shall be more than one, the Deputy Directors in the order determined by (he Trustees, shall, in the absence or disability of the Director, perform ihe duties and exercise the powers of the Director and shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. Bylaws of the Corporation R89 J. President. The President shall have the powers and duties as may be vested in him or her by the Board. K. Treasurer and Assistant Treasurer. The Treasurer shall, subject to the direction of the Trustees, have general charge of the financial affairs of the Corporation, including its long-range financial planning, and shall cause to be kept accurate books of account. The Treasurer shall prepare a yearly report on the financial status of the Corporation to be delivered at the annual meeting. The Treasurer shall also prepare or oversee all filings required by the Commonwealth of Massachusetts, the Internal Revenue Service, or other Federal and State Agencies. The account of the Treasurer shall be audited annually by a certified public accountant. The Assistant Treasurer, if any, or if there shall be more than one, the Assistant Treasurers in the order determined by the Trustees, shall, in the absence or disability of the Treasurer, perform the duties and exercise the powers of the Treasurer, shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. L. Clerk and Assistant Clerk. The Clerk shall be a resident of the Commonwealth of Massachusetts, unless the Corporation has designated a resident agent in the manner provided by law. The minutes or records of all meetings of the Trustees and Members shall be kept by the Clerk who shall record, upon the record books of the Corporation, minutes of the proceedings at such meetings. He or she shall have custody of the record books of the Corporation and shall have such other powers and shall perform such other duties as the Trustees may, from time to time, prescribe. The Assistant Clerk, if any, or if there shall be more than one. the Assistant Clerks in the order determined by the Trustees, shall, in the absence or disability of the Clerk, perform the duties and exercise the powers of the Clerk and shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. In the absence of the Clerk and an Assistant Clerk from any meeting, a temporary Clerk shall be appointed at the meeting. M. Other Powers and Duties. Each officer shall have in addition to the duties and powers specifically set forth in these Bylaws, such duties and powers as are custom- arily incident to his or her office, and such duties and powers as the Trustees may, from time to time, designate. ARTICLE VII AMENDMENTS These Bylaws may be amended by the affirmative vote of the Members at any meeting, provided that notice of the substance of the proposed amendment is stated in the notice of such meeting. As authorized by the Articles of Organization, the Trustees, by a majority of their number then in office, may also make, amend or repeal these Bylaws, in whole or in part, except with respect to (a I the provisions of these Bylaws governing (i) the removal of Trustees and (n) the amendment of these Bylaws and (b) any provisions of these Bylaws which by law, the Articles of Organization or these Bylaws, requires action by the Members. No later than the time of giving notice of meeting of Members next following the making, amending or repealing by the Trustees of any Bylaw, notice thereof stating the substance of such change shall be given to all Members entitled to vote on amending the Bylaws. Any Bylaw adopted by the Trustees may be amended or repealed by the Members entitled to vote on amending the Bylaws. ARTICLE Vm INDEMNITY Except as otherwise provided below, the Corporation shall, to the extent legally permissible, indemnify each person who is, or shall have been, a Trustee, director or officer of the Corporation or who is serving, or shall have served at the request of the Corporation as a Trustee, director or officer of another organization in which the Corporation directly or indirectly has any interest as a shareholder, creditor or otherwise, against all liabilities and expenses (including judgments, fines, penalties, and reasonable attorneys' fees and all amounts paid, other than to the Corporation or such other organization, in compromise or settlement) imposed upon or incurred by any such person in connection with, or arising out of. the defense or disposition of any action, suit or other proceeding, whether civil or criminal, in which he or she may be a defendant or with which he or she may be threatened or otherwise involved, directly or indirectly, by reason of his or her being or having been such a Trustee, director or officer. The Corporation shall provide no indemnification with respect to any matter as to which any such Trustee, director or officer shall be finally adjudicated in such action, suit or proceeding not to have acted in good faith in the reasonable belief that his or her action was in the best interests of the Corporation. The Corporation shall provide no indemnification with respect to any matter settled or comprised unless such matter shall have been approved as in the best interests of the Corporation, after notice thai indemnification is involved, by (i) a disinterested majonty of the Board of the Executive Committee, or (ii) a majority of the Members. Indemnification may include payment by the Corporation of expenses in defending a civil or cnminal action or proceeding in advance of the final disposition of such action or proceeding upon receipt of an undertaking by the person indemnified to repay such payment if it is ultimately determined that such person is not entitled to indemnification under the provisions of this Article VIII, or under any applicable law . As used in the Article VIII, the terms "Trustee." "director," and "officer" include their respective heirs, executors, administrators and legal representatives, and an "interested" Trustee, director or officer is one against whom in such capacity the proceeding in question or another proceeding on the same or similar grounds is then pending. To assure indemnification under this Article VIII of all persons who are determined by the Corporation or otherwise to be or to have been "fiduciaries" of any employee benefits plan of the Corporation which may exist, from time to time, this Article VIII shall be interpreted as follows: (i) "another organization" shall be deemed to include such an employee benefit plan, including without limitation, any plan of the Corpo- ration which is governed by the Act of Congress entitled "Employee Retirement Income Secunty Act of 1974," as amended, from time to time, ("ERISA"); (ii) "Trustee" shall be deemed to include any person requested by the Corporation to serve as such for an employee benefit plan where the performance by such person of his or her duties to the Corporation also imposes duties on. or otherwise involves services by, such person to the plan or participants or beneficiaries of the plan; (iii) "fines" shall be deemed to include any excise tax plan pursuant to ERISA; and (iv) actions taken or omitted by a person with respect to an employee benefit plan in the performance of such person's duties for a purpose reasonably believed by such person to be in the interest of the participants and beneficiaries of the plan shall be deemed to be for a purpose which is in the best interests of the Corporation. The right of indemnification provided in this Article VIII shall not be exclusive of or affect any other rights to which any Trustee, director or officer may be entitled under any agreement, statute, vote of Members or otherwise. The Corporation's obligation to provide indemnification under this Article VIII shall be offset to the extent of any other source of indemnification of any otherwise applicable insurance coverage under a policy maintained by the Corporation or any other person. Nothing contained in the Article shall affect any rights to which employees and corporate personnel other than Trustees, directors or officers may be entitled by contract, by vote of the Board or of the Executive Committee or otherwise. ARTICLE IX DISSOLUTION The consent of every Trustee shall be necessary to effect a dissolution of the Marine Biological Laboratory. In case of dissolution, the property shall be disposed of in such a manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Trustees then in office in accordance with the laws of the Com- monwealth of Massachusetts. ARTICLE X MISCELLANEOUS PROVISIONS A. Fiscal Year. Except as otherwise determined by the Trustees, the fiscal year of the Corporation shall end on December 31st of each year. B. Seal. Unless otherwise determined by the Trustees, the Corporation may have a seal in such form as the Trustees may determine, from time to time. C. Execution of Instruments. All checks, deeds, leases, transfers, contracts, bonds, notes and other obligations authorized to be executed by an officer of the Corporation in its behalf shall be signed by the Director or the Treasurer except as the Trustees may generally or in particular cases otherwise determine. A certificate by the Clerk or an Assistant Clerk, or a temporary Clerk, as to any action taken by the Members. Board of Trustees or any officer or representative of the Corporation shall as to all persons who rely thereon in good faith be conclusive evidence of such action. D. Corporate Records. The original, or attested copies, of the Articles of Organi- zation, Bylaws and records of all meetings of the Members shall be kept in Massa- chusetts at the principal office of the Corporation, or at an office of the Corporation's Clerk or resident agent. Said copies and records need not all be kept in the same office. They shall be available at all reasonable times for inspection by any Member for any proper purpose, but not to secure a list of Members for a purpose other than in the interest of the applicant, as a Member, relative to the affairs of the Corporation. E. Articles of Organization. All references in these Bylaws to the Articles of Organization shall be deemed to refer to the Articles of Organization of the Corpo- ration, as amended and in effect, from time to time. F. Transactions with Interested Parties. In the absence of fraud, no contract or other R90 Annual Report transaction between this Corporation and any other corporation or any firm, association, partnership or person shall be affected or invalidated by the fact that any Trustee or officer of this Corporation is pecuniarily or otherwise interested in or is a director, member or officer of such other corporation or of such firm, association or partnership or in a party to or is pecuniarily or otherwise interested in such contract or other transaction or is in any way connected with any person or person, firm, association, partnership, or corporation pecuniarily or otherwise interested therein; provided that the fact that he or she individ- ually or as a director, member or officer of such corporation, firm, association or partnership in such a party or is so interesied shall he disclosed to or shall have been known by the Board of Trustees or a majority of such Members thereof as shall be present at a meeting of the Board of Trustees at which action upon any such contract or transaction shall be taken; any Trustee may be counted in determining the existence of a quorum and may vote at any meeting of the Board of Trustees for the purpose of authon/ing any such contract or transaction with like force and effect as if he/she were not so interested, or were not a director, member or officer of such other corporation, firm, association or partnership, provided that any vote with respect to such contract or transaction must be adopted by a majority of the Trustees then in office who have no interest in such contract or transaction. 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F. Solter, J. Nixon, J. E. Beever, H. R. Gaskins, G. Olsen, S. Subramaniam. M. L. Sogin, and H. A. Lewin. 2000. Phylogenomic analysis of a protea- some gene family from early-diverging eukaryotes. J. Mol. Evol. 51: 532-543. Breton, S., N. N. Nsumu, T. Galli, I. Sabolic, P. J. S. Smith, and D. Brown. 2000. Tetanus toxin-mediated cleavage of cellubrevin inhib- its proton secretion in the male reproductive tract. Am. J. Phvsiol. Renal Physinl. 278: F7 17-725. Brothers, C., E. Marks, and R. Smolowitz. 2000. Conditions affecting growth and zoosporulation of protistan parasite QPX in culture. Biol. Bull. 199: 200-201. Burgos, M. H., M. Goda, and S. Inoue. 2000. Fertilization-induced changes in the fine structure of stratified Arbacia eggs. II. Observations with electron microscopy. Biol. Bull. 199: 213-214. Bush, M. B., M. C. Miller, P. E. De Oliveira, and P. A. Colinvaux. 2000. Two histories of environmental change and human disturbance in eastern lowland Amazonia. 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