THE Number I GT/^i A ih ICAL, We walk in the footsteps of visionaries, ^nd see with the eyes of disciples. T^ Galileo up close: astronomer, physicist, tf mathematician, dethroner of Aristotle, defender ( of Copernicus and, yes, credited with making Double convex lenses and the first biological with a microscope. A compound microscope. Hooke: 43 years later, the compound microscope eveals living things are composed of cells. Leeuwenhoek: invents the first practical microscope and discovers bacteria. Armed with the knowledge and inspiration of these jnd other Olympians, secure in the history of our own iccomplishments and aware that the mind's eye should ilways focus on challenging the enigmatic as well as he undeniable, Olympus has created the BX2 series of :linical and research microscopes, noteworthy for their nhancements in optics, imaging and ergonomics. And each so individual, it deserves the nomenclature Ay Microscope. From possibility to actuality, it is wonderful to behold. \ posse ad esse est mirabile visu. TheBX4l The new standard in laboratory microscopy performance, ver- satility, operation. Uncanny digitized imaging: enhanced with the DP11 digital camera, delivering high-resolution image recording beyond the norm. Unusually insightful optics: observe magnifications from 1.25x to lOOx without changing the condenser. Choice of condensers includes Abbe, swing- out, phase-c and darkllol Unerringly precise- motion stage: selectable for right/ left hand. I)i speed, ease, comfort, an. accuracy. Uncommonly easy-to-operate controls: extended fine-focus knob; ed illumination jtile X-Y control. croscope Senes The BX45 The new standard in ergonomic microscopy. A dramatic metamor- phosis: innova- &Q" live, intelligent, insightful design improvements specifically addressing the needs of those spending hours looking through a microscope. The lowest low-position stage: three inches lower than standard microscopes, offering the promise of minimum effort and hand movement when changing specimens. The tilting, telescoping observation tube: a marvel of adjustability, it can be changed to accommodate any . 80mm higher and 70mm to the operator than a convention- al tilting tube. And the eye- i iurward by 45mm from 0-25 degrees. The new stan- -m,.^« dard in research microscopy. . ,( The words "new" and "standard" really don't do it ' justice. How about "ingenious" or "original," "paragon" or "prototypical." Prepare yourself for a higher power. The 6-position filter cube turret: Not 4, not 5, but 6, so single and multi- band imaging of new fluorochromes and fluorescent proteins is faster, simpler and exclusively tailored The rectangul ours alone. By cleverly matcnms the camera's field of view, only the area requiring fluorescence excita- tion is exposed. Which means that the surrounding areas are protected — - from photo bleaching. The fluorescence excitation bal- ancers: another Olympus exclusive. A continuously variable excitation bandwidth. Visualization is enhanced, differentiation of multi-labels is immeasurably improved, capturing of images is at an unheard-of level. Trinle Labeled Specimen (1 to r): . with prevalent FITC ; • with prevalent DAPI and Cy3 • with balanced fluorescence emission wider mirror aspherical lens The aspherical collector lens: sui generis, in a class by itself. Specimens appear brighter and more evenly illuminated; and achromatic performance now incorporates wavelengths from UV to IR. The universal ^^ condenser: with 8 positions, it can accept optical inserts for DIC, phase, brightfield and dark- field illumination. And the advanced Nomarski DIC system has been expanded and optimized to encompass prisms for enhanced contrast and image resolution. OLYMPUS FOCUS ON LIFE Visit us at www.olympusamerica.com or call 1-800-455-8236 © Olympus America Inc. THE BIOLOGICAL BULLETIN f *VOOnc w~i . ^ ua 'CSl i ah • AUGUST 2000 Editor Associate Editors Section Editor Online Editors Editorial Board Editorial Office "-uc,,^;,;. MICHAEL J. GREENBERG Louis E. BURNETT R. ANDREW CAMERON CHARLES D. DERBY MICHAEL LABARBERA SHINYA INDUE, Imaging and Microscopy JAMES A. BLAKE, Keys to Marine Invertebrates of the Woods Hole Region WILLIAM D. COHEN, Marine Models Electronic Record and Compendia PETER B. ARMSTRONG ERNEST S. CHANG THOMAS H. DIETZ RICHARD B. EMLET DAVID EPEL GREGORY HINKLE MAKOTO KOBAYASHI ESTHER M. LEISE DONAL T. MANAHAN MARGARET MCFALL-NGAI MARK W. MILLER TATSUO MOTOKAWA YOSHITAKA NAGAHAMA SHERRY D. PAINTER J. HERBERT WAITE RICHARD K. ZIMMER PAMELA CLAPP HINKLE VICTORIA R. GIBSON CAROL SCHACHINGER CAROL MARRAMA "' The Whitney Laboratory, University Grice Marine Biological Laboratory, College of Charleston California Institute of Technology Georgia State University University of Chicago Marine Biological Laboratory ENSR Marine & Coastal Center, Woods Hole Hunter College, City University of New York University of California, Davis Bodega Marine Lab., University of California, Davis Louisiana State University Oregon Institute of Marine Biology, Univ. of Oregon Hopkins Marine Station, Stanford University Cereon Genomics, Cambridge, Massachusetts Hiroshima University of Economics, Japan University of North Carolina Greensboro University of Southern California Kewalo Marine Laboratory, University of Hawaii Institute of Neurobiology, University of Puerto Rico Tokyo Institute of Technology, Japan National Institute for Basic Biology, Japan Marine Biomed. Inst., Univ. of Texas Medical Branch University of California, Santa Barbara University of California, Los Angeles Managing Editor Staff Editor Editorial Associate Subscription & Advertising Secretary Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS Cover Among the "primitive" chitons of the suborder Lepidopleurina, the eggs have a smooth jelly coat, and the sperm — equipped with a typical, prominent acrosome — probably can enter an egg at any point on its surface. All other chitons have eggs with more elaborate spinous or cupulous hulls that focus sperm to specific regions on the surface. Moreover, these sperm have evolved a long nuclear filament tipped by a minute acrosome which interacts with the egg in specific ways. Differences in the form of the egg hull and in the mechanism of fertilization among chitons are providing insights into the evo- lution of this ancient molluscan taxon. In this issue (pp. 59-67), John Buckland-Nicks and Alan Hodgson describe fertilization in Cullocliiton castaneus from South Africa. This chiton retains a mixture of primitive and derived characters that together produce a novel mechanism of fertiliza- tion, which is represented on the cover. In the background of the cover is an unfertilized egg of C. castaneus, from which the jelly coat and part of the vitelline layer have been stripped to reveal a honeycomb of egg membrane cups. In the intact egg, these cups coincide with the bases of regularly spaced pores in the jelly coat. Fertile sperm seeking the egg locate one of these external pores and swim down it to the vitelline layer. The minute acrosome digests a pore, and the needle-like nuclear filament bridges the distance to the egg membrane cup. The micrograph superimposed on the background shows a fertilizing sperm in the process of injecting chromatin into the egg cortex. The unusual aspect of fertilization in these chitons is that the sperm organelles are apparently abandoned in a membrane bag on the egg surface. If this is indeed the case, then inheritance, not only of mitochondria, but also of centrioles and other cytoplasmic components, would be maternal. CONTENTS VOLUME 199. No. 1: AUGUST 2000 RESEARCH NOTE Bolton, Toby F., Florence I. M. Thomas, and Celere N. Leonard Maternal energy investment in eggs and jelly coats sur- rounding eggs of the echinoid Aitariti punctulata .... PHYSIOLOGY Johnson, M. L., P. M. J. Shelton, E. Gaten, and P. J. Herring Relationship of dorsoventral eyeshine distributions to habitat depth and animal size in mesopelagic de- capods Dietz, T. H., A. S. Udoetok, J. S. Cherry, H. Silverman, and R. A. Byrne Kidney function and sulfate uptake and loss in the freshwater bivalve T»xt>ln\nia texasensis Vollmer, Steven V., and Peter J. Edmunds Allometric scaling in small colonies of the scleractin- ian coral Siderastrea sidimi (Ellis and Solander) .... DEVELOPMENT AND REPRODUCTION Torrado, Mario, and Alexander T. Mikhailov Frog Lim-1-like protein is expressed predominantly in the nervous tissue, gonads, and early embryos of the bivalve mollusc Afyfr'/f/.s gattopravinciatis Gibson, Glenys D., and Jennifer M. L. Harvey Morphogenesis during asexual reproduction in Py- gospio eh'gmis Claparede (Annelida, Polychaeta) .... 14 21 50 59 68 Watson, G. J., F. M. Langford, S. M. Gaudron, and M. G. Bentley Factors influencing spawning and pairing in the scale worm Harmothoe imbricata (Annelida: Polychaeta). . . Buckland-Nicks, John, and Alan N. Hodgson Fertilization in C.nll/>< Intuit untnni'in (Mollusca) Hirose, M., R. A. Kinzie III, and M. Hidaka Early development of zooxanthella-containing eggs of the corals Pocillo/xn-a vemimsfi and P. ryilouxi with special reference to the distribution of zooxanthellae CELL BIOLOGY Wakefield, Timothy S., Mark A. Fanner, and Stephen C. Kempf Revised description of the fine structure of in \ilii "Zooxanthellae" genus .S'v'"'"'"/'''""" ECOLOGY AND EVOLUTION Barnes, David K. A., and Mathew H. Dick Overgrowth competition between clades: implica- tions for interpretation of the fossil record and over- growth indices 85 76 4 1 Annual Report of the Marine Biological Laboratory Rl THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to Subscription Manager, THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. 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Aulhors (or delegates lor foreign ABSTRACTS listings). authors) will receive page proofs of articles shortly before publi- H. A few well-known international journals in their preferred cation. They will be charged the current cost of printers' time for forms rather than WORLD LIST or USASI usage (e.g.. Nature. corrections to these (other than corrections of printers' or editors' Science Evolution NOT Nature, Lond., Science. N.Y.: Evolution. errors). Other than these charges for authors' alterations. The La caster Pa ) Biological Bulletin does not have page charges. Reference: Biol. Bull. 199: 1-5. (August 20(10) Maternal Energy Investment in Eggs and Jelly Coats Surrounding Eggs of the Echinoid Arbacia punctulata TOBY F. BOLTON'-*, FLORENCE I. M. THOMAS1, AND CELERE N. LEONARD2 1 Department of Biology, University of South Florida, Tampa, 4202 East Fowler Avenue, Tampa, Florida 33620-5150: and ~ Department qf Biology, Tuskeegee University, Tuskeegee, Alabama 36088 In free-spawning marine invertebrates, the amount of maternal energv that is invested in each egg has profound implications for all life-history stages of the offspring. The eggs ofechinoids are freely spawned into the water and are surrounded by several structurally complex extracellular layers. These extracellular layers, or jelly coats, do not contribute energy to embryonic development but must im- pose an energy cost on the production of each egg. The investment of maternal energy reserves in the jelly coats of echinoid eggs may have important implications for the number of eggs that can be produced (i.e.. fecundity) and the amount of energy that can be invested in each egg. We estimated the degree to which maternal energy is invested in the jelly coats surrounding eggs of the echinoid Arbacia punctulata. Estimates were derived from measurements of the amount of energy contained in the combined eggs and jelly coats, and in the eggs alone. The amount of energy contained in A. punctulata eggs ranged from 2.70 to 5.53 X 10~4 J egg~l. The amount of energy contained in the jelly coats ranged from 0.13 to 0.48 X 10~4 J jelly coat ~ '. The mean concentration of energy in the eggs was 2.15 mm '' and 0.29 J mm~~ in the jelly coats. These results indicate that bet\veen 3% and 11% (mean = 7%) of the total energy invested in each A. punctulata egg is partitioned to the jelly coat alone. A significant positive relationship was found between the volumes of the jelly coats and the amount of energy they contained. Based on this relationship and an analysis of differences in the size of jelly coats between echinoid species, we suggest that the degree to which en- ergy is invested in jelly coats may vary among echinoid species and is therefore likely to be an important life-history characteristic of these organisms. Received 2 February 1999; accepted 18 April 2000. * To whom correspondence should be addressed. In free-spawning marine invertebrates, the egg contains all the maternal energy provisioned for the development of each offspring. The amount of maternal energy invested in individual eggs is central to many theories on the evolution of life-history patterns in marine invertebrates and is widely considered to have profound implications for all stages of marine invertebrate life cycles (1-5). The eggs ofechinoids are freely spawned into the water column where fertilization and development take place. Several extracellular layers surround the eggs of echinoids. These extracellular layers (commonly, and from here on, referred to as "jelly coats") are structurally complex, consisting of several concentric layers of polysaccharide fiber networks embedded in a glycoprotein matrix (6, 7). The jelly coats surrounding the eggs of echinoids are thought to play important roles in fertilization processes (8-10) and may also protect eggs from physical forces that they are exposed to during and after spawning (11, 12). The jelly coats of some echinoid species disintegrate soon after contact with seawater ( 13) or following fertilization, and do not contribute energy to embryonic development. Although the jelly coats surrounding the eggs of echi- noids do not contribute energy to embryonic development, they must impose an energy cost on the production of each egg. Assuming that the amount of maternal energy available for reproduction is finite, the investment of energy in jelly coats may have important life-history implications. These potential implications include a reduction in the number of eggs that can be produced (i.e., fecundity), a reduction in the amount of energy that can be invested in each egg, or both. Although previous studies have shown that there is substan- tial extra-embryonic investment in the gelatinous matrices of egg masses that are deposited on the benthos by some marine invertebrates (14, 15), the energy invested in jelly T. F. BOLTON ET AL coats surrounding the eggs of a free-spawning species has not been considered specifically. We estimated the amount of maternal energy that is invested in the jelly coats surrounding eggs of the echinoid Arbacia punctulata. These estimates were derived from wet oxidation (16. 17) measurements of the amount of energy contained in the combined eggs and jelly coats, and in the eggs alone. The wet oxidation method yields an estimate of the amount of organic carbon contained in a sample, which can be directly interpreted as a measure of the amount of energy that it contains. This method has been used in previous studies of maternal energy investment in marine invertebrate eggs ( 18-21 ), so our data can be directly com- pared with earlier results. The amount of energy contained in the combined egg and jelly coat (mean ± SD = 3.97 ± 0.79 X 1(T4 J egg~' ) was significantly higher (paired sample t test: / = 8.33, df = 9, P < 0.0001 ) than the amount of energy contained in the egg alone (mean ± SD = 3.69 ± 0.57 X 10 ~4 J egg"'; Table 1 ). The average ( ±SD) amount of energy contained in the jelly coat was 0.28 ± 0.10 X 10~4 J jelly coat"1, and constituted 7.4% of the total amount of energy contained in the combined egg and jelly coat (Table 1 ). The concentrations of energy (mean joules per cubic millimeter) in the eggs and jelly coats were calculated from the amount of energy each contained (Table 1 ) and their respective volumes (Table 2). The concentration of energy in eggs was 2.15 J mm'-1 (i.e., 3.69 X 10~4 X 5847 = 2.15 J mm"3, where the combined volumes of 5847 eggs are equivalent to 1 mm3). The concentration of energy in the jelly coats was 0.29 J mm"-' (i.e.. 0.28 X 10~4 X 10,416 = 0.29 J mm"3, where the combined volumes of 10.416 jelly coats are equivalent to 1 mm3). The concentration of energy in the egg was 7.4 times greater than the concentration of energy in the jelly coats (i.e., concentration of energy in the eggs [2.15 J mm 3] divided by the concentration of energy in the jelly coat [0.29 J mm"3] = 7.4). Before exposure to seawater, the volume (mean ± SD) of the combined egg and jelly coat was 2.67 ± 0.30 X 10~4 mm3, and the volume of the egg alone was 1.71 ±0.19 X 1()~4 mm3 (Table 2). The volume of the jelly coat alone was 0.96 ± 0.48 X 1() 4 mm3 (Table 2); thus, the jelly coat constituted 36% of the volume of the combined egg and jelly coat prior to exposure to seawater (i.e., 0.96/2.67 X 100 = 35.9%). After exposure to seawater, the volume of the jelly coats increased substantially to 9.27 ± 2.42 • 10~4 mm3 (Table 2) and constituted 84%- of the combined volume of the egg and jelly coat. Linear regression analyses on data contained in Tables 1 and 2 showed no significant relationship between the vol- umes of the jelly coats and the volumes of the eggs. Simi- larly, no significant relationship was found between the amount of energy contained in the jelly coats and the amount of energy contained in the eggs. A significant pos- Table 1 The iiiinnini of energy contained (mean ± SD x IQ~4 J, n = 10) in the combine J egg and jelly coat, and in the egg and jelly coat alone for each female Arbacia punctulata; the proportion of total energy in the combined egg and jelly coal that is partitioned to jelly coat is also given Combined egg Female and jelly coat Energy partitioned Jelly coat to jelly coat (<£) 1 4.06 (0.20) 3.93(0.18) 0.13 3.3 T 3.50(0.22) 3.15 (0.13) 0.35 100 3 5.53(0.17) 5.32(0.31) 0.21 3.X 4 4.45(0.15) 4.12(0.34) 0.32 7.2 5 4.77(0.69) 4.29(0.43) 0.48 10.1 6 3.86(0.30) 3.59(0.25) 0.26 6.7 7 3.42(0.41) 3.05 (0.44) 0.37 10. N g 2.70(01)2) 2.47 (0.46) 0.23 8.5 i) 3.46(0.30) 3.11 (0.07) 0.34 9.8 ID 4.04(0.57) 3.89(0.85) 0.14 3.5 Overall 3.97 (0.79) 3.69(0.57) 0.28(0.10) 7.4(2.8) Specimens of Arbacia punctulata were collected subtidally between July and August 1998 from marina walls at Panama City. Florida. Eggs were obtained from 10 of these specimens by intra-coelomic injection of 0.5-1 ml 0.5 M KC1. The amount of energy contained in the combined egg and jelly coat and in the egg alone was determined using a modification of the wet oxidation method given by Parsons ct al. ( 16). Energy determinations were made from large samples of eggs that were estimated to yield at least 7.8 joules (J). The jelly coats were removed from half of the eggs obtained from each female by pouring them through a 100-/u,m Nytex screen. Thus, samples of eggs with and without jelly coats were obtained from each female for analysis. The concentration of eggs in each sample was determined by replicate counts (n = 7-20) of 10-/M1 aliquots of well-suspended eggs from each sample. To ensure that the eggs were not damaged by the removal of the jelly coats, eggs were examined microscopically (400x magnification) for any signs of injury to the egg membrane or leakage of yolk from the egg. The viability of eggs from rive females was assessed from fertilization assays in which samples of eggs with and without jelly coats were incubated in dilute sperm suspensions (dry sperm diluted hy 10 J in seawater). Embryos were allowed to divide to the four-cell stage before being recorded as viable. The proportion of eggs with jelly coats that were fertili/.ed was compared to the proportion of eggs without jelly coats that were fertilised from each female (paired sample / test, a = 5%, on arcsine transformed proportions). Three subsamples of eggs with jelly coats and without jelly coats were taken from samples of eggs from each female and placed in separate containers. The jelly coat material was eliminated from subsamples hy removing the supernatant above the eggs and refilling the container with seawater that had been filtered through a 0.22-jiuii membrane. This process was repeated several times with all subsamples of eggs. To ensure that all of the jelly coat material had been removed from the subsamples, a vital stain (Janus green) was added to the final supernatant solutions, which were then examined microscopically. The amount of energy contained in each egg (mean ± SD joules egg" ' ) was calculated from the total amount of energy in each subsample and the number of eggs that each subsample contained. The concentrations of energy (joules per cubic millimeter) in the eggs and jelly coats were calculated from the amount of energy each contained (Table I ) and their respective volumes (Table 2). A paired sample t test (a = 5%) was used to determine whether there were differences in the amount of energy contained in the combined eggs with jelly coats compared to the amount of energy contained in the egg alone. Relationships between the volumes and the amounts of energy contained in eggs and jelly coats were examined using linear regression analyses. The significance of these relationships were tested by one-way ANOVA (a = 5%). PARTITIONING OF MATERNAL ENERGY TO EGGS AND JELLY COATS SURROUNDING EGGS Table 2 Volumes (mean ± SD X 10 "J mm', n = 10) i>f llic cimihiitcd CKK <»'rc and after contact with seawater, fur Arbacia punctulata Before contact with seawater After contact with seawater Female Egg Egg and jelly coat Jelly coal Egg and jelly coat Jelly coat 1 .43(0.22) 3.32(0.77) 1.89 10.61 (3.08) 9.18 2 L14(0.44) 3.49(0.74) 1.35 11.78(1.63) 9.64 3 .73 (0.79) 2.61 (0.35) 0.84 7.45(1.761 5.75 4 .16(0.31) 2.78(0.73) 1.62 9.69(4.04) 8.53 5 .67(0.23) 2.56(0.56) 0.89 15.23(2.84) 13.56 6 .73 (0.79) 2.42(0.31) 0.64 12.77(1.92) 1 1.04 7 .73 (0.79) 2.40(0.17) 0.67 11.65(1.60) 9.92 8 .74(0.18) 2.29(0.32) 0.55 12.22(1.98) 10.42 9 .70(0.14) 2.22(0.25) 0.52 11.15(1.81) 9.45 II) .49 (0.52) 2.56(061 ) 0.57 7.20(1.19) 5.21 Overall .71 (0.14) 2.67 (0.30) 0.96(0.48) 11.02(2.51) 9.27 (2.42) The volumes of the combined eggs and jelly coats and of the eggs alone were calculated from their respective diameters (D) and the equation for the volume of a sphere (4/3ir[D/2]3). The volumes of the jelly coats were calculated by subtracting the volumes of the eggs alone from the volumes of the combined eggs and jelly coats. Before the eggs of Arbacia punctulata contact seawater (i.e., prior to spawning), the jelly coats lie in close proximity to the eggs. After contact with seawater, the jelly coats hydrate and increase substantially in volume. The volumes of jelly coats before hydration were used in calculations of the amount of energy they contain. To determine the pre-hydration volume, the thickness of the coat was measured, using an ocular micrometer in a compound microscope (200X magnification), from the distance between adjacent eggs and added to the mean diameter of the eggs. The edges of jelly coats after exposure to seawater were visualized by adding india ink to the egg suspension, and diameters were measured in the manner described above. itive relationship was apparent between the amount of en- ergy contained in the jelly coats and their volumes (r~ 0.482, F == 7.44. P = 0.025). However, no significant relationship was found between the amount of energy con- tained in the eggs and the volumes of the eggs. Microscopic examination of eggs from which the jelly coats had been removed did not reveal any damage to the integrity of the membrane surrounding the eggs. Eggs from which jelly coats had been removed were fertilized at the same rate as eggs with jelly coats at a standard sperm concentration. Thus we assume that the removal of the jelly coats did not result in any leakage of yolk from the eggs or any reduction in their viability. Our results indicate that approximately 7% (range = 3%-l 1%) of the maternal energy invested in the combined eggs and egg jelly coats of A. punctulata is partitioned to the jelly coats alone (Table 1 ). The amount of energy contained in the eggs of the A. punctulata tested in this study was about half of that reported for this species in a previous study (18). Similarly, the concentration of energy in the eggs of the A. punctulata measured here is about half of the average concentration of energy contained in eggs of free- spawning marine invertebrates with planktotrophic larval development (22). Large differences in the amount of en- ergy contained in eggs from different populations of marine invertebrate species have been reported previously ( 19. 20). Differences in the quality of the yolk content of eggs be- tween populations of the echinoid Arbacia lixula have also been reported (23, 24). These population differences in the energy content of the egg and of the quality of the yolk may be the result of variation in the quality and quantity of food available to the adult (22) or of differences in the produc- tivity of the waters in which larvae develop (25). The degree to which maternal energy is partitioned to the jelly coats of A. punctulata eggs (mean = 7.490 is small relative to the amount of extra-embryonic energy parti- tioned to the gelatinous matrices of benthic egg masses of some other marine invertebrates. Although these gelatinous matrices contain less energy per unit weight than the eggs they encompass, they constitute a large proportion of the total maternal energy investment in the mass. For example, in species of the prosobranch gastropod genus Conns, up to 50% of the maternal energy invested in egg masses is partitioned to the gelatinous matrix (14). Similarly, in spe- cies of opisthobranch gastropods, up to 589r of the total energy investment in egg masses is partitioned to the gelat- inous matrix (15). While the amount of energy invested in the jelly coats of ,4. punctulata eggs is small relative to that of the gelatinous matrices of benthic egg masses, it may nonetheless have important life-history implications. Although the jelly coats of echinoid eggs do not contribute energy to embryonic or larval development, they do impose energy costs on the production of each egg. Within the context of current life- history theory (1-5), the investment of energy in the pro- duction of jelly coats may influence the number of eggs T. F. BOLTON ET AL. Table 3 ize indices of the jelly coats surrounding the eggs of six echinoid species Diameter of Source of Diameter of egg combined egg and Relative size index Echinoids data* n (fini) jelly coat (^m) (±SD) Strongylocentrotus purpuraius 1 NA 80 120 1 .50 Strongylocentrotus franciscanus 1 NA 130 196 1.51 Strongylocentrotus droebachiensis 2 50 160 260 1.61 (0.16) A rhticia punctulata 3 100 69 126 1.83(0.15) Lytechinus variegalus 2 12? 143 298 2.09(0.27) DenJra.iter excentricns 4 NA 125 205 1.64 The size indices are the ratio of the diameter of the combined egg and jelly coat (after contact with seawater) to the diameter of the egg alone. A larger index indicates larger jelly coat relative to the size of the egg. SD = standard deviation; NA = not available. * 1— Lessios, 1990 (25); 2— Bolton and Thomas, unpubl. data; 3— this study; 4— Timko. 1479 (26). produced, the degree to which energy is invested in indi- vidual eggs, or both. Assuming that the maternal energy available for reproduction is finite, and that the amount of energy in each egg is constant, the investment of energy in jelly coats may compromise the number of eggs that could be produced (i.e., may reduce fecundity). This study indi- cates that approximately 7% of total energy investment in the combined egg and jelly coats is partitioned to the jelly coats alone. Accepting the assumptions given above, the investment of energy in jelly coats may reduce the potential fecundity of A. punctulata by about 7%. Alternatively, assuming that the amount of energy available for reproduc- tion is constant, and that the number of eggs produced is also constant, the partitioning of energy to jelly coats may reduce the amount of energy that could be invested in each egg. If this is the case, the investment of energy in jelly coats may compromise offspring growth, survivorship, and reproductive output. No significant relationships were apparent between the volumes of jelly coats and eggs or the amount of energy contained in jelly coats and eggs. This indicates that the amount of maternal energy invested in jelly coats is inde- pendent of the amount of energy invested in eggs. Similarly, no relationship was found between the amount of energy invested in eggs and the volume of the eggs. A significant relationship was apparent, however, between the amount of energy invested in jelly coats and the volume of jelly coats. This suggests that it may be possible to infer the relative degree to which maternal energy is invested in the egg jelly coats of different species from the volumes of these coats. The proportion of maternal energy invested in jelly coats relative to that invested in eggs is likely to vary among echinoid species. For example, an index of relative size of the jelly coats surrounding eggs of a particular species can be obtained by taking the ratio of the diameter of the egg plus jelly coat to the diameter of the egg alone. Thus, the relative size of the jelly coats to the size of the egg can be compared among species independently of actual differ- ences in egg size. When this index is calculated for the few echinoid species for which data are available (26. 27). differences are apparent (Table 3). Since the amount of energy contained in the jelly coats of A. punctulata is positively related to the volume of the jelly coats, it is possible that the proportion of energy invested in the jelly coat relative to that invested in the egg could be inferred from this index. If this is the case, ecologically important differences in the degree to which energy is invested in jelly coats may exist among echinoid species. The jelly coats surrounding the eggs of echinoids are not unique: the eggs of many free-spawning marine inverte- brates are surrounded by extracellular structures and exhibit enormous diversity in size, structure, and form (28-30). Therefore, the investment of energy in the extracellular structures surrounding their eggs may impose substantial reproductive costs on many of these species and should be considered in theories of their life-history evolution. Further measurements of the degree to which maternal energy is invested in the extracellular structures surrounding the eggs of free-spawning marine invertebrates are clearly needed. Acknowledgments This research was supported by NSF grants IBN-9723779 and OCE-9701434 awarded to Dr. Florence I. M. Thomas. We thank Julien Lartigue. Chris Cornelisen. Gregory De- Lozier. Kristen Edwards, and two anonymous reviewers for helpful comments on this manuscript. Literature Cited 1. Vance, R. R. 1973a. 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De\: 35: 181-188. 7 Bonnell, B. S., S. H. Keller, V. D. Vacquier, and D. E. Chandler. 1994. The sea urchin jelly coat consists of globular glycoproteins bound to a fibrous fucan superstructure. Dev. Biol. 162: 313-324. 8. Vaquier, M., and G. W. Moy. 1977. Isolation of binding: the protein responsible for the adhesion of sperm to sea urchin eggs. Proc. Natl. Acad. Sci. USA 74: 2456-2460. 9. Nomura, K., and S. Isaka. 1985. Synthetic study of the structure- activity relationship of sperm activating peptides from the jelly coat of sea urchin eggs. Biochem. Biophys. Res. Commun. 126: 974-982. 10. Levitan, D. R. 1996. Effects of gamete traits on fertilization in the sea and the evolution of sexual dimorphism. Nature 382: 153-155. 1 1 Thomas, F. I. M., K. A. Edwards, T. F. Bolton, M. A. Sewell, and J. A. Zande. 1999. Mechanical resistance to shear stress: the role of echinoderm egg extracellular layers. Biol. Bull. 197: 7-10. 1 2. Thomas, F. I. M., and T. F. Bolton. 1999. Shear stress experienced by echinoderm eggs in the oviduct during spawning: potential role in the evolution of egg properties. J. Exp. Biol. 202: 3111-3119. 13. Kidd, P. 1978. The jelly and vitelline coats of the sea urchin egg: new ultrastructural features. J. Ultrastruct. Res. 64: 204-215. 14. Perron, F. E. 1981. The partitioning of reproductive energy between ova and protective capsules in marine gastropods of the genus Conus. Am. Nat. 118: 110-118. 15. Lee, C. E., and R. R. Strathmann. 1998. Scaling of gelatinous clutches: effects of siblings' competition for oxygen on clutch size and parental investment per offspring. Am. Nat. 151: 293-310. 16. Johnson, M. J. 1949. A rapid micromethod for estimation of non- volatile organic matter. J. Biol. Client. 181: 707-711. 17 Parsons, T. R., Y. Malta, and C. M. Lalli. 1984. A Manual of Chemical and Biological Methods for Sea Water Analysis. Pergamon Press, New York. 173 pp. 18. Strathmann, R. R., and K. Vedder. 1978. Size and organic content of eggs of echinoderms and other invertebrates as related to develop- mental strategies and egg eating. Mar. Biol. 39: 305-309. 19. McEdward, L. R., and L. K. Coulter. 1987. Egg volume and energetic content are not correlated among sibling offspring of starfish: implications tor life-history theory. Evolution 41: 914-917. 20 McEdward, L. R., and F. Chia. 1991. Size and energy content of eggs from echinoderms with pelagic lecithotrophic development. J. Exp. Mar. Bio/. Ecol. 147: 95-102. 21. McEdward, L. R., and S. F. Carson. 1987. Variation in egg or- ganic content and its relationship with egg size in the starfish Solaster stimpsoni. Mar. Ecol. Prog. Ser. 37: 159-169. 22. Jaeckle, W. B. 1995. Variation in the size, energy content, and biochemical composition of invertebrate eggs: correlates to the mode of larval development. Pp. 49-77 in Ecology of Marine Invertebrate Lan'ae. L. R. McEdward, ed. CRC Press, Boca Raton, FL. 23. George, S. B. 1990. Population and seasonal differences in egg quality of Arbacia lixula (Echinodermata: Echinoidea). Invertebr. Re- prod. Dev. 17: 111-121. 24. George, S. B., C. Cellario, and L. Fenaux. 1990. Population dif- ferences in egg quality of Arbacia lixula (Echinodermata: Echinoideal: proximate composition of eggs and larval development. J. Exp. Mar. Biol. Ecol. 141: 107-118. 25. Lessios, H. A. 1990. Adaptation and phylogeny as determinants of egg size in echinoderms from two sides of the Isthmus of Panama. Am. Nat. 135: 1-13. 26. Timko, P. 1979. Larviphagy and oophagy in benthic invertebrates: a demonstration for Dendraster excentricus. Pp. 91-98 in Reproductive Ecology of Marine Invertebrates, S. E. Stancyk. ed. University of South Carolina Press, Columbia, SC. 27. Strathmann, M. F. 1987. Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast. University of Washington Press, Seattle. 28. Berrill, N. J. 1975. Chordata: Tunicata. Pp. 252-255 in Reproduc- tion of Marine Invertebrates. Vol. II. A. L. Giese and J. S. Pearse, eds. Academic Press, New York. 29. Pearse, J. S. 1975. Polyplacophora. Pp. 39-43 in Reproduction of Marine Invertebrates. Vol. V. A. L. Giese and J. S. Pearse, eds. Academic Press, New York. 30. Mozingo, N. M., V. D. Vaquier, and D. E. Chandler. 1995. Struc- tural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization. Mol. Reprod. Dev. 41: 493- 502. Reference: Binl. Bull. 199: 6-13. (AucuM 2000) Relationship of Dorsoventral Eyeshine Distributions to Habitat Depth and Animal Size in Mesopelagic Decapods M. L. JOHNSON1'*, P. M. J. SHELTON1. E. GATEN4, AND P. J. HERRING: Department of Biology, University of Leicester, University Road, Leicester LEI 7RH, UK; and Southampton Oceanography Centre, Empress Dock, Southampton SO14 3ZH, UK Abstract. Eyeshine distribution patterns recorded from the eyes of 19 mesopelagic decapod species were examined and related to the depths at which the species are found. For most species examined, eyeshine was found to be brighter ventrally than dorsally. Deep-water decapod species that do not undergo diel vertical migrations had brighter dorsal eyeshine than migratory species. Eyeshine intensity in- creased with body size in five of the species examined and decreased in two. These changes in eyeshine intensity may be an adaptation to variations in depth distributions that occur with increasing body size. It is suggested that the depth and size-related changes reflect the importance of remaining camouflaged in the mesopelagic realm and are an example of ecologically functional development. Introduction Many species active at low light levels possess a well- developed reflective tapetum behind the retina that effec- tively doubles the path length of light through the photore- ceptor cells (Lythgoe, 1979). This doubling increases the photon-capturing efficiency of the eye without requiring an increase in eye size (Land, 1981 ). In most arthropod species that have superposition compound eyes, light reflected by the tapetum and not absorbed by the rhabdoms is visible as eyeshine (Kunze, 1979). Eyeshine consists of a circular patch of light that tills about half of the eye and represents the effective aperture (Land, 1981). Since approximately 80%-90% of the blue-green light entering the eye is ab- Received 18 February 2000; accepted 12 June 2000. * Present address: Biology Department. University of Hull, (Scarbor- ough Campus), Filey Road, Scarborough. YO1 1 3ZZ. UK t To whom correspondence should he addressed. E-mail: gat@le.ac.uk sorbed by the rhabdoms (Johnson, 1998), eyeshine is or- ange-red when the eye is illuminated with white light. The ecology of the mesopelagic realm is dominated by the vertical distribution of its inhabitants (Herring and Roe, 1988). Few expeditions have comprehensively studied the way in which species are distributed in the water column in a particular area over any significant length of time. As a result, we have little knowledge of how ecological factors affect vertical distributions and daily vertical migrations. In addition, the picture is complicated by community variation and differences in migratory behavior that may be associ- ated with factors such as the intensity and angular distribu- tion of light, hydrography, season, reproduction, ontogeny, feeding, interspecific interactions, and capture methods (Foxton, 1970; Jerlov, 1974; Longhurst. 1976; Marshall, 1979; Roe. 1984; Domanski. 1985; Herring and Roe, 1988; Gonzalez el 0.001, n = 128). The relative eyeshine patch diameter resulting from green incident light (0.33 ± 0.09 mm) was 27.5% smaller than that resulting from white light (0.46 ± 0.10 mm). Because there was a significant difference in the diameter of the eyeshine patch under white and green light, this study used only the measurements of diameter and intensity made under green illumination, which more closely resembles the light that mesopelagic species would experience normally (Kirk, 1983). Eyeshine distribution along the ilorsoventn/l av/'.v For the 19 species examined, the general trend is for eyeshine to be brightest ventrally (Table 1 ). The degree to which eyeshine intensity varies around the eye differs be- tween species. In nonmigratory and deeper-living species, such as Acanthephyra pelagica and Acanthephyra styloro- stratis, eyeshine is usually of similar intensity dorsally and ventrally (Fig. 2a). Migratory species that come close to the surface during the night, such as Parapandalus richardi, Plesionika mania (Fig. 2b), Opiophorus spinosus, and Sys- tellaspis debilis (Fig. 2 c, d), have eyeshine that is brighter ventrally than dorsally. In Sergestes cornicttlum, because it hangs vertically during normal swimming, the eyeshine pattern is offset by 90° so that the more reflective posterior region of the eye is directed downwards (Shelton et 5 mm range) were obtained for nine species. For five species (Acanthephyra pelagica, Oplopho- rus spinosus, Sergia grandis, Sergia robustus, and Systel- laspis cristata) both dorsal and ventral eyeshine increased with carapace length (Table 1 ). For two species (Plesionika martia and Sergestes corniculum) there was no clear trend with regard to dorsal eyeshine, but ventral eyeshine was greater in larger specimens. In Notostomus auriculatus and Systellaspis debilis, both dorsal and ventral eyeshine inten- sity decreased with increasing carapace length. A typical example of eyeshine distribution in large and small speci- mens is shown by O. spinosus (Fig. 2c). S. debilis is unusual in that eyeshine intensity is markedly greater in small spec- imens than large (Fig. 2d). For four species where size ranges and numbers captured permitted, dorsal eyeshine was plotted against carapace length (Fig. 2 e, f). The two 10 M. L. JOHNSON ET AL a) Non-migrants b) Migrants 125- S 100- cfl o ? 75 M c u = 50- ~* Acanthephyra pelagica "* Acanthephyra stvloroslratis 50 100 150 200 Degrees from dorsal • Parapandalus richardi Z) O) 125 - lOOn 75- * Plesionika martia «> c 50- £ TTJ u 25 - *^i^_r*^±J- -L U D - 50 100 150 200 Degrees from dorsal 100- 50-1 c ) Oplophorus spinosits Cl = 4 mm Cl = 16mm 0 50 100 150 200 Degrees from dorsal e )Systellaspis debilis & S. cristata • ^'yi/t'//«5/?(5 debilis ° Systellaspis cristata § 75 H 5 10 15 20 Carapace length (mm) D on o 60- d) Systellaspis debilis 150- 100- 5(1 - I 50 100 150 Degrees from dorsal f) Oplophorus spinosus & Acanthephyra pelagica • Oplophorus spinosus ° Acanthephyra pelagica 5 1(1 15 20 25 Carapace length (mm) Figure 2. Size- and depth-related variations in eyeshine distributions, (a. b) Dorsoventral eyeshine intensity distributions for four species of decapod. In the two migratory species Parapandalus richardi and Plesionika ihi cveshine intensity is markedly brighter ventrally than dorsally. In the deep migratory (Acanthephyra ] and nonmigratory (A. stylorostratis) species, eyeshine does not vary significantly from dorsal to ventral (c. d) Dorsoventral eyeshine distributions for two species of migratory decapod showing how eyeshine intensity differs between large and small specimens. In Oplophorus spinosus. eyeshine is brightest in large specimens. In S\-\iflla.\r>is debilis, eyeshine is brightest in smaller specimens, (e, f) Changes in dorsal eyeshine intensity with increasing carapace length for four species of mesopelagic decapod. For two species (Syslellaspis cristata and Oplophorus \y>m«.v»,v). positive and significant correlations were found (/; = 3, r = 0.896, P < 0.05 and n = 16. /• = 0.459, P < 0.10 respectively). Although a positive trend is also found for Acantheph\ra pelagica (it = 8, i = 0.614). it is not significant. In the case of Systellaspis Jehilis. dorsal eyeshine intensity decreases with increasing carapace length (n = 15, r = 0.560. P < 0.05). EYESHINE DISTRIBUTION IN DECAPODS II deep-water species (Acanthephyra pelagica and Systelluspis cristata) showed a significant increase in dorsal eyeshine intensity with increasing carapace length. In Oplophorus spinosus, the increase in dorsal eyeshine intensity was less pronounced, and in Systellaspis debilis, as is also demon- strated by Figure 2b, eyeshine actually decreased with in- creasing carapace length. Depth distribution and eyeshine intensity In the present study it was found that for the largest size classes of each of the 19 species examined (Table 1), there were significant correlations between loglo depth and loglo dorsal eyeshine intensity (Fig. 3 a. b). In the case of the relationship between eyeshine intensity and daytime depth, the correlation was markedly improved when Oplophorus spinosus was excluded from the analysis. This species has much higher dorsal eyeshine intensity for its daytime depth distribution than would normally be expected. It is possible that this anomaly is related to the unusually small amplitude of its vertical migration pattern (Foxton, 1970), which sug- gests that this species may be able to light adapt (thereby reducing eyeshine) to some degree. Ventral eyeshine ap- pears to vary independently of depth (Fig. 3 c, d). Analysis of variance showed that migratory species have significantly lower (F = 3.12, P = 0.095) log,,, dorsal eyeshine intensity (1.69 ± 0.21, n = 13) than nonmigrants (1.85 ± 0.15, n = 6). A comparison of ventral eyeshine between the two groups showed that there was no significant difference (F = 2.29, P = 0.15) between migratory (1.81 ± 0.14) and nonmigratory species (1.92 ± 0.15). Discussion Eyeshine intensity varies as a result of the efficiency and quantity of reflecting and absorbing pigments within the eye (Gaten et «/., unpubl.). Our examination of mesopelagic decapods has demonstrated that the distribution patterns of their dorsoventral eyeshine intensity vary with the species' estimated habitat depths. With increasing habitat depth. tu a) Day depth v. Dorsal eyeshine intensity 2.1 -I 0 l.q- •5 1.8- c .1 1.7- 1 1.6- 1.4 O.s b) Night depth v. Dorsal eyeshine intensity 1.9- 1.x - 1.7- 1 .6 - 1.5- 1.4 Depth (m) Depth (m) 1.9- 1.8- 1.7- 1.6- c) Day depth v. Ventral eyeshine intensity 1.5 2.5 3 3.5 Depth (m) 3 C/5 O d ) Night depth v. Ventral eyeshine intensity I 9- 1.8- 1.7- 1.6- 1.5- 1.5 2.5 Deplh (m) Figure 3. Eyeshine intensity in relation to depth distributions for adult mesopelagic species (n = I9| with least-squares lines fitted. Significant positive correlations were found for dorsal eyeshine intensity and day depth [(a) r = 0.68, P < 0.001] and night depth [(b) r = 0.81. P < 0.001]. When Optophoms spinosus (O.s in Fig. 3a) — which exhibits higher dorsal eyeshine intensity than expected for its estimated daytime depth distribu- tion— is excluded, r = O.X2. Ventral eyeshine intensity was poorly correlated with both day [(c) ;• = 0.34, n.s.] and night |(d) r = 0.17, n.s.] depths. 3.5 12 M. L. JOHNSON ET AL. dorsal eyeshine was brighter, and the difference in intensity between dorsal and ventral regions of the eye decreased. In all species examined except Systellaspis debilis and Noto- stomus aitriculatus. eyeshine intensity in at least one region of the eye increased with carapace length. Ventral eyeshine showed no significant depth-related trend. The variation in eyeshine distribution suggests that dorsal eyeshine intensity is related to the degree to which each shrimp species is exposed to downwelling light. This is supported by theo- retical evidence which suggests that for any given eye, there are ideal distributions of reflecting and shielding pigments that optimize both sensitivity and resolution (Warrant and Mclntyre, 1991). For most superposition compound eyes, the tapetum should, ideally, be formed of reflecting pigment enclosing the proximal third of each rhabdom. This has the effect of doubling the path length of light (by reflecting unabsorbed photons back through the target rhabdom) and restricting the bleed of light between adjacent rhabdoms. Despite the prevailing low levels of ambient light normally experienced by mesopelagic species, the distributions of these pigments generally deviate from the theoretical ideal (for maximum sensitivity) in the dorsal part of the eye (Gaten el al, 1992; Johnson, 1998). This suggests that there is a requirement to remain camouflaged that outweighs the need for highly sensitive vision dorsally. Generally we have found that with increasing carapace length, eyeshine intensity increases, and that the increase is more pronounced dorsally than ventrally. If the supposition that dorsal eyeshine intensity is determined by habitat depth is true, then it follows that where eyeshine increases with carapace length, juvenile mesopelagic decapods should be found closer to the surface than adults. Size-related differ- ences in vertical distribution have been observed for some mesopelagic decapods, euphausiids, and copepods (Foxton. 1970; Baker, 1970; Hays, 1996). The study of the ontogeny of eye anatomy of mesopelagic decapods has shown that juveniles often have apposition eyes, that superposition optics develop with age, and that the ventral portion devel- ops first (Gaten and Herring, 1995). Our description of the way in which eyeshine distribution patterns develop with size agrees with this finding. The current results are consistent with the view that gradients of reflectivity along the dorsoventral axis and dorsal holes in the tapetum reduce the visibility of the eye to predators (Shelton et al.. 1992, 2000). The gradients are necessary because of the characteristic distribution of irra- diance in the ocean. Here the brightness of upwelling light is two orders of magnitude lower than that of the down- welling light, and the light field is symmetrical about the vertical axis (Kirk, 1983). Low reflectivity in upwardly looking parts of the eye reduces the contrast between the light reflected from the tapetum and that arising from the dim background. In downwardly looking parts of the eye, a highly reflective ventral tapetum is unlikely to increase visibility, because the levels of upwelling light are low. The variations in eyeshine distribution shown here are an example of how the development of sense organs can be linked, in a functional manner, to variations in depth distri- bution. Small decapods can only have small eyes and are limited in the degree to which they can vertically migrate by the inverse relationship between body size and energy re- quirements for swimming (Longhurst, 1976). A small ap- position eye is sufficient in the relatively well-lit upper regions of the pelagic realm, where juvenile and adolescent pelagic decapods and euphausiids are to be found (Baker, 1970; Foxton, 1970; Marshall, 1979). but as shrimps in- crease in size and daily movement to the ecological refuge provided by depth (King and Butler, 1985) becomes a viable strategy, their eyes develop to suit a more oligophotic en- vironment. Acknowledgments We gratefully acknowledge the assistance given by the officers and crew of RRS Discoveiy and Tony Rice of Southampton Oceanography Centre for allowing MLJ to participate in cruise 204. Many thanks also to Jeremy Brooks for technical assistance with eyeshine measurements and to Matt Sheehy for his comments on the manuscript. MLJ was supported by NERC grant GT4/92/5/A. PMJS and PJH acknowledge support of NERC grants GR9/0019A and GR3/11212. Literature Cited Baker, A. de C. 1970. The vertical distribution of euphausiids near Fuerteventura. Canary Islands ('Discovery' SOND Cruise. 1965). J. Mar. Bin/. A.isoc. UK 50: 301-342. Burkenroad, M. D. 19-43. A possible function of bioluminescence. J. Mar. Ret,. 5: 161-164. Cartes, J. E., J. C. Sorbe, and F. Sarda. 1994. Spatial distribution of deep-sea decapods and euphausiids near the bottom in the northwestern Mediterranean. J. £171. Mar. Biol. Ecol. 179: 131-144. Crosnier, A., and J. Forest. 1973. Les Crevettes ProfonJes de L'AtlantU/iu' Oriental Tropical. Volume XIX. ORSTOM, Paris. Domanski, P. A. 1985. The Azores front: a zoogeographic boundary'.' UNESCO Tech. ft//). Mar. Sri. No. 49: 73-83. Foxton, P. 1970. The vertical distribution of pelagic decapods (Crusta- cea: Natantia) collected on the Sond Cruise 1965. J. Mar. Biol. Assoc. UK 50: 939-960. Gaten, E., and P. J. Herring. 1995. Morphology of the reflecting superposition eyes of larval oplophorid shrimps. J. Mnrphol. 225: 19-29. Gaten, E., P. M. J. Shelton, and P. J. Herring. 1992. Regional mor- phological variations in the compound eyes of certain mesopelagic shrimps in relation to their habitat. J. Mar. Biol. Assoc. UK 72: 61-75. Gonzalez, J. A., M. V. Tuset, I. J. Lozano, and J. I. Santana. 1997. Biology of Plesionika nan-al (Crustacea. Decapoda. Pandahdae) around the Canary Islands (Eastern Central Atlantic). Est. Coast Shelf Sci. 44: 339-350. Hays, G. C. 1996. Large-scale patterns of diel vertical migration in the North Atlantic. Deep-Sea Res. 43: 1601-1615. EYESHINE DISTRIBUTION IN DECAPODS 13 Heffernan, J. J., and T. L. Hopkins. 1981. Vertical distribution and feeding of the shrimp genera Gennadax and Bentheogennema (Deca- poda: Penaeidea) in the eastern Gulf of Mexico. J. Crustac. Biol. 1: 461-473. Herring, P. ,|. 1990. Bioluminescent communication in the sea. Pp. 245-278 in Light and Life in the Sea. P. J. Herring. A. K. Campbell. M. Whitfield, and L. Maddock. eds. Cambridge University Press. Cam- bridge. Herring. P. J., and H. S. J. Roe. 1988. The photoecology of pelagic oceanic decapods. Symp. Zoo/. Soc. Loud. 59: 263-290. Hiller-Adams, P., and J. F. Case. 1988. Eye size of pelagic crustaceans as a function of habitat depth and possession of photophores. Vision. Res. 28: 667-680. Jerlov, N. G. 1974. Significant relationships between optical properties of the sea. Pp. 77-94 in Optical Aspects of Oceanography, N. G. Jerlov, ed. Academic Press, London. Johnson, M. L. 1998. Aspects of visual function and adaptation of deep-sea decapods. Ph.D. Thesis, University of Leicester. Leicester, England. 262 pp. Johnson, M. L., P. M. J. Shelton, and E. Gaten. 2(100. Temporal responses in the eyes of marine decapods from coastal and deep sea habitats. Mar. Biol. 136: 243-24S. King, M. G., and A. J. Butler. 1985. Relationship of life history patterns to depth in deep-water candean shrimps (Crustacea: Natantia). Mar. Biol. 86: 129-138. Kirk, J. T. O. 1983. Light and Photosynthesis in Aquatic Ecosystems. Cambridge University Press. Cambridge. Kunze, P. 1979. Apposition and superposition eyes. Pp. 441-502 in Handbook of Sensory Physiologv. H Autrum, ed. Springer- Verlag, Berlin. Land, M. F. 1981. Optics and vision in invertebrates. Pp. 471-492 in Handbook of Sensory Physiology, H. Autrum. ed. Spnnger-Verlag, Berlin. Laughlin. S. B., and M. Weckstrom. 1993. Fast and slow photorecep- tors — a comparative study of the functional diversity of coding and conductances in the Diptera. J. Comp. Physiol. A 172: 593-609. Longhurst, A. R. 1976. Vertical migration. Pp. 1 16-137 in The Ecology of the Seas. D. H. Cushmg and J. J. Walsh, eds. Blackwell Scientific, Oxford. Lvthgoe. J. N. 1979. The Ecology of Vision. Oxford University Press. Oxford. Marshall. N. B. 1979. Development!, in Deep-Sea Biology (Blandford Press. Poole, England. Morin, J. G., and A. C. C'ohen. 1991. Bioluminescent displays, court- ship and reproduction in ostracodes. Pp. 1-16 in Crustacean Sexual Biology. T. B. J. Martin, ed. Columbia University Press, New York. Nilsson, H. L. 1982. Rhabdom breakdown in the eye of Cirolana no- reulis (Crustacea) caused by exposure to daylight. Cell Tissue Res 227: 633-639. Nilsson, H. L., and M. Lindstriim. 1983. Retinal damage and sensitivity loss of a light-sensitive crustacean compound eye (Cirolana borealis). J. Exp. Biol. 107: 277-292. Roe, H. S. J. 1984. The diel migrations and distributions within a mesopelagic community in the north east Atlantic. 2. Vertical migra- tions and feeding of mysids and decapod Crustacea. Prog. Oceanogr. 13: 269 -3 IS. Roe, H. S. J., and D. M. Shale. 1979. A new multiple rectangular mid-water trawl (RMT 1 + 8 M) and some modifications to the Institute of Oceanographic Sciences' RMT 1 + 8. Mar. Biol. 50: 283-288. Shelton, P. M. J., E. Gaten, and C. J. Chapman. 1986. Accessory pigment distribution and migration in the compound eye of Nephrops norvegicus (L.) (Crustacea: Decapoda). J. Exp. Mar. Biol. Ecol. 98: 185-198. Shelton, P. M. J., E. Gaten, and P. J. Herring. 1992. Adaptations of tapeta in the eyes of mesopelagic shrimps to match the oceanic irradi- ance distribution. J. Mar. Biol. Assoc. UK 72: 77-88. Shelton, P. M. J.. E. Gaten, M. L. Johnson, and P. J. Herring. 2000. The 'eye-blink' response of mesopelagic Natantia; eyeshine patterns and the escape reaction. Crustacean Issues 12: 253-260. Warrant, E. J., and P. D. Mclnt.vre. 1991. Strategies for retinal design in arthropod eyes of low F-number. J. Comp. Physiol. A 168: 499- 512. Wild, R. A., E. Darlington, and P. J. Herring. 1985. An acoustically controlled cod-end system for the recovery of deep-sea animals at in situ temperatures. Deep-Sen Res. 32: 1583-1589. Reference: Biol. Bull. 199: 14-20. (August 2000) Kidney Function and Sulfate Uptake and Loss in the Freshwater Bivalve Toxolasma texasensis T. H. DIETZ1 •*, A. S. UDOETOK1. J. S. CHERRY1, H. SILVERMAN1, AND R. A. BYRNE2 ^Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803; and ~ Department of Biology, State University of New York College at Fredonia, Fredonia, New York 14063 Abstract. Toxolasma texasensis acclimated to an artificial pondwater (PW) maintained a concentration of SO4 in the blood of about 1-2 mmol I"1. The anion transport inhibitor DIDS (5, 5'-diisothiocyanatostilbene 2, 2'-disulfonic acid) reduced the uptake of 35SO4 from the bathing medium by 54%. The clearance of polyethylene glycol (PEG) injected into the blood of T. texasensis ranged between 0.8 and 1.3 ml g ' dry tissue h~', and provided an estimate of renal filtration in PW-acclimated animals. The clearance of ra- dioactive ^SO., simultaneously injected into the same ani- mal was about 16% of the PEG clearance, suggesting that sulfate was being reabsorbed by the kidney. Para-aminohip- puric acid was cleared about 4.6 times faster than PEG. indicating that this organic acid was subjected to secretion in addition to filtration. When the normal osmotic gradient was abolished by acclimating T. texasensis to 10% seawater (SW), the PEG clearance decreased to 0.17 ml g ' dry tissue h~ '. Sulfate clearance in animals acclimated to PW or 10% SW was the same. However, in mussels acclimated to 10% SW, the calculated amount of SO4 reabsorbed was significantly reduced relative to mussels acclimated to PW. T. texasensis conserved SO4 when acclimated to PW, and reduced reabsorption when acclimated to the sulfate-rich 10% SW. When mussels acclimated to 10% SW were returned to PW, there was a transient increase in sulfate clearance during the first 8 h because filtration exceeded reabsorption. Received 2 February 201 .0. accepted 16 May 2000. * To whom correspondence should he addressed. E-mail: zothom@lsu.edu. Abbreviations: DIDS. 5, S'-diisothiocyanatostilbene 2, 2'-disultomc acid; PAH. para-aminohippuric acid: PEG. polyethylene glycol; PW. arti- licial pondwater: SW. artificial seawater. Introduction The characteristics of solute (K, La, Na, sucrose, manni- tol) penetration through the epithelia of the unionid Toxo- lasma texasensis are intermediate to those of other fresh- water bivalves (Dietz and Byrne, 1990; Dietz el ai, 1995; Wilcox and Dietz, 1995; Byrne and Dietz, 1997; Zheng and Dietz, 1998b; Dietz and Byrne, 1999). Previous studies indicated that the passive movements of solutes and water across the epithelia of T. texasensis were relatively slower that those of the dreissenid Dreissena polymorpha (Scheide and Dietz, 1986; Dietz and Byrne, 1997. 1999). To maintain ionic homeostasis, an animal must be able to accumulate and retain solutes; then to preserve water balance, it must excrete a volume of water equivalent to that taken up osmotically. Kidney filtration can be estimated by measur- ing the clearance of marker solutes from the blood, and it is a useful index from which kidney function and osmotic water movement can be monitored (Potts, 1954b; Murphy and Dietz, 1976; Hevert, 1984; Kirschner, 1991; Dietz and Byrne, 1997, 1999). Unionid bivalves accumulate sulfate at the rather slow rate of 0.04 jumol g"1 dry tissue h"1 (Dietz, 1978). Thus, sulfate is a relatively nonpenetrating anion, and therefore it has been used for short-term studies of independent ion transport (Krogh, 1939; Scheide and Dietz, 1982: Byrne and Dietz, 1997; Zheng and Dietz, 1998a; Dietz and Byrne, 1999). However, sulfate is present in millimolar concentra- tions in the blood of freshwater mussels (Potts, 1954a; Dietz and Byrne, 1999) and is a component of various organic molecules (e.g., amino acids, mucopolysaccharides) found in molluscs (Eriksson ct ai. 1984; Kornprobst et ai, 1998). Sulfate balance was studied recently in D. polymorpha. which has the highest epithelial solute permeability of any freshwater bivalve tested (Dietz and Byrne, 1999). In the present study, kidney function was examined in a unionid 14 RENAL FUNCTION IN A FRESHWATER CLAM 15 bivalve, Toxolasma texasensis. The unionids have a longer freshwater ancestry (Triassic) than the dreissenids, which invaded freshwater in the Pleistocene (Haas, 1969). Sulfate uptake and the characteristics of the renal clearance and conservation of SO4 that was injected into the body fluids were studied. Materials and Methods Animal acclimation Toxolasma (=Carunculina) texasensis was collected from ponds near Baton Rouge, Louisiana. The animals were stored, unfed for at least 1 week before use, in aerated artificial pondwater (PW) at 22° ± 2°C. The pondwater composition (in millimoles per liter) was 0.5 NaCl. 0.4 CaCl2, 0.2 NaHCO,. 0.2 MgSO4, 0.05 KC1 (Dietz et PEG]. Sulfate clearance was calculated by the method de- scribed above for PEG. The sulfate concentration in the blood of each specimen was measured and converted into the amount of sulfate filtered (micromoles of sulfate per gram of dry tissue per hour) into the kidney by multiplying the PEG clearance (milliliters of blood cleared of PEG per gram of dry tissue per hour) by the blood sulfate concen- tration (micromoles of sulfate per milliliter of blood). Knowing the specific activity (dpm jamo!"1) of 35SO4 in the blood, the quantity of 35SO4 excreted (dpm per gram of dry tissue per hour) was converted into the total quantity of sulfate eliminated (micromoles of sulfate per gram of dry tissue per hour) for each specimen, and this value repre- sented sulfate excretion. The sulfate reabsorption was cal- culated as the difference between the filtered and excreted sulfate values for each animal. Clearance studies were performed on mussels acclimated either to PW or to 10% SW: animals in PW were hyperos- motic to the bathing medium; those in 107r SW were isosmotic. Animals acclimated to 10% SW were transferred to PW for 1, 4, 8. 24, 48. or 72 h to observe the changes in renal clearance when they experienced an increased osmotic gradient. Clearance was measured for I h, ending at each time interval specified, and the amounts of sulfate filtered, excreted, and reabsorbed were calculated. Para-aminohippuric acid (PAH, 194 Da) was injected into mussels together with PEG ( 15 jul. 1 jaCi) to compare clearance values. The method used was similar to that described above tor the double-label 'H-PEG and 35SO4 studies. Both 'H- and "C-label for both PEG and PAH, and identical results were obtained. The clearance of PAH was Table 1 Blh 0.1) for both "SO4 and ^H-PEG. The SO4 clearance in 10% SW-acclimated animals ap- peared to be unchanged relative to the PW-acclimated mus- sels. However, because the clearances of SO4 and PEG were similar, these data suggest that 10% SW-acclimated animals had reduced their reabsorption of SO4 (Table 2). The SO4 concentration in the blood of T. texasensis acclimated to 10% SW was significantly higher (P < 0.05) than in PW- acclimated controls (Table 3), but was the same as in the 10% SW bathing medium (-2.7 mmol 1~' SO4). When T. texasensis was transferred from 10% SW into Table 3 Concentration of solutes in the blood o/Toxolasma texasensis acclimated to pondwater {PW), 10% seawater (SW), or returned to PW for various periods Treatment mosm kg ' Ion concentration, mmol I ' Total solute Na K Ca Mg Cl S04 10% SW 110 ± If 45.1 ± 0.7e 1.6 ± O.le 2.1 ± 0.2a 3.3 ± 0.3e 41.1 ± 1.7e 2.8 ± 0.2c 1 h PW 102 ± 2e 41.0 ± 0.7d 1.1 ± O.Od 2.8 ± 0.2b 3.6 ± 0.2e 36.2 ± I. Id 2.1 ± 0.4ab 4hPW 79 ± Id 29.6 ± 0.7c 0.9 ± O.Ocd 2.1 ± O.la 2.7 ±0.2d 24.9 ± 0.8c 2.4 ± 0.1 he 8 h PW 67 ± Ic 27.7 ± 0.5c 0.7 ± O.lbc 2.6 ± O.lb 1.8 ± 0.2c 21.0 ± 0.3b 1.4 ± 0.2a 24 h PW 55 ± Ib 20.1 ± 0.7b 0.6 ± O.Oab 3.0 ± 0.2bc 1.3 ± O.lbc 15.5 ± 0.6a 1.4 ± 0.2a 48 h PW 45 ± 3a 16.4 ± l.la 0.4 ± O.Oa 2.6 ± O.lb 1.0 ± O.lb 12.0 ± 0.8a 1.4 ± 0.2a 72 h PW 46 ± la 16.6 ± 0.2a 0.6 ± O.lab 2.9 ± 0.2b 0.8 ± O.lab 12.3 ± 0.5a 2.1 ± O.labc PW 44 ± 2a 19.1 ±0.9b 0.5 ± O.Oa 3.5 ±0.2c 0.4 ± O.Oa 12.7 ±0.7a 1.7 ±0.2a Data are expressed as mean ± 1 standard error, with 5-1 1 animals for each treatment. Values within a column that have different letters are significantly different using Fisher's protected least significant difference method (P < 0.05). 18 T. H. DIETZ ET AL. PW. PEG and "SO4 clearance increased (Table 2). Al- though there was an immediate rise in osmotic uptake of water, only PEG clearance increased significantly during the first hour relative to 10% SW animals, but clearance of both solutes was elevated by 4 h. The clearance of PEG was restored to the same level as that found in PW mussels by 72 h. In contrast, the 35SO4 clearance remained elevated for 8 h and then returned to PW control levels by 24 h. The elevation in SO4 excretion was due to a significant increase in filtration. During the first hour after transfer to PW, the SO4 clearance remained statistically the same as the PEG clearance. By 4 h, the SO4 clearance was significantly less (P < 0.05) than the corresponding PEG clearance. The reduction in SO4 clearance was due to the rapid restoration of sulfate reabsorption (Table 2). During the first hour of re-acclimation to PW, the sulfate concentration in the blood returned to the same level as in the PW-acclimated controls; recovery was due to dilution caused by the osmotic uptake of water combined with increased levels of filtration (Table 3). However, 48 h were required for the total solute and most of the other measured ions to return to PW levels (Table 3). To determine whether the renal tissue of T. texasensis could secrete organic acids, radioactive PEG and PAH were both injected into PW-acclimated animals. The clearance was 1.26 ± 0.08 mlg~' dry tissue h"1 for PEG, and 5.75 ± 0.65 ml g~' dry tissue h ' for PAH (/; = 10). PAH is a smaller molecule than PEG, but the volume of blood cleared of PAH by filtration was likely to be the same as for PEG. The additional PAH clearance was due to secretory mech- anisms and amounted to 4.49 ml g~' dry tissue h"1; this value was 3.5 times the amount of PAH cleared by filtration. Discussion Toxolasma texasensis has a tubular kidney with func- tional characteristics similar to those found in other inver- tebrates and vertebrates. The kidney forms urine by ultra- filtration, for which PEG serves as a useful marker (Hevert, 1984). Some solutes can be added to the urine by the process of secretion, as well as filtration, and PAH is an organic acid that is subject to secretory activity. Most of the PAH eliminated by the kidney of T. texasensis was through secretory mechanisms. The importance of secretion in the elimination of PAH has also been documented in the snail Achatina fulica (Martin et ul.. 1965). At low concentrations of PAH in the blood, most of this solute is secreted by the snail kidney rather than filtered. The third major process responsible for urine formation is solute reabsorption. In this study, we have focused on the characteristics of sulfate reabsorption by the kidney of T. texasensis. Toxolasiua texasensis was able to maintain a sulfate concentration in the blood of about 1-2 mmol 1" ' while acclimated to an artificial PW containing 0.2 mmol 1 ' SO4. Sulfate balance was maintained by transport systems in the epithelia, including the kidney. Sulfate concentrations in the blood and pericardial fluid are the same, which suggests that the anion is freely filtered in molluscs (Potts and Todd, 1965) as it is in vertebrates (Mudge et «/.. 1973). Thus, filtration in bivalves was assumed to be the same for sulfate as for PEG, but the renal reabsorption of SO4 reduced its clearance from the blood by more than 80% relative to PEG clearance. Renal reabsorption of sulfate was reduced in animals that were acclimated to 10% SW for almost 2 weeks. During acclimation to 10% SW, the concentration of SO4 in the blood increased to about 2.8 mmol 1~ ' (equal to the bathing medium). These data contrast with the some- what more rapid SO4 transport rates observed in Dreissena polymorphtt, and with the apparent cessation of SO4 reab- sorption in that species when acclimated to 10% SW (Dietz and Byrne. 1999). The low blood SO4 concentration of 0.7 mmol 1~' reported for the unionid Anodonla cygnea (Potts, 1954a) is similar to concentrations we observed in T. tex- asensis, but is less than half the concentration found in D. pol\morpha (Dietz and Byrne, 1999). In previous studies, the clearance values for PEG, inulin. and high-molecular weight dextran from the blood of D. pol\morpha were similar, and we concluded that these three solutes were probably measuring the renal filtration rate (Dietz and Byrne, 1997, 1999). PEG clearance values in PW-acclimated T. texasensis were about I ml g~ ' dry tissue h~', and were similar to inulin clearances reported for the freshwater snail L\mmiea stagnalis (de With and van der Schors, 1984). When T. texasensis was acclimated to 10% SW. the PEG clearance decreased to about 0.2 ml g~' dry tissue h '. Although pondwater-acclimated D. polymorpha clear the blood of PEG at about double the rate observed for T. texasensis, the response by the kidney was the same in both species when the osmotic gradient was abolished by acclimation to 10% SW (Dietz and Byrne, 1997. 1999; this study). In the sulfate-rich 10% SW environment. T. texasensis became isosmotic and isoionic for SO4 and reduced its renal reabsorption. All freshwater bivalves studied become isos- motic when exposed to dilute seawater (Wilcox and Dietz, 1998; Jordan and Deaton, 1999). They have limited toler- ance, but may survive in an environment in which total solutes approach 400 mosm kg"1. Freshwater bivalves can maintain cellular volume regulation under moderate os- motic challenges; their ability to mobilize free amino acids is restricted, however, and this restriction may be responsi- ble for the limit to their survival (Dietz et al., 1998; Jordan and Deaton. 1999). Transferring mussels from 10% SW to PW would in- crease the osmotic uptake of water and the subsequent excretion of water by the kidney. After the transfer, PEG clearance was rapidly elevated to values exceeding those observed for PW-acclimated T. texasensis. Because of ele- RENAL FUNCTION IN A FRESHWATER CLAM 19 vated filtration, it was 24 h before SO4 clearance returned to PW control levels, even though SO4 reabsorption was im- mediately reestablished. D. polymorpha also required about 24 h to reestablish SO4 reabsorption to PW control levels (Dietz and Byrne, 1999). However. D. polymorpha did not elevate PEG clearance above that found in PW-acclimated animals. Unlike the unionids, D. pol\nwrpha has maximum renal filtration (PEG clearance) when acclimated to PW, and filtration cannot be increased even when the mussel is subjected to higher osmotic uptake of water (Dietz and Byrne. 1999). Recent studies have examined mechanisms of sulfate transport in a variety of preparations (Larsen and Simonsen, 1988; Cole and Rastogi, 1991; Tenenhouse and Martel. 1993; Grassl, 1996; Dietz and Byrne, 1999). Toad skin is capable of active sulfate influx from a Ringer's solution containing 1 mmol 1~' SO4, using an anion exchange mech- anism (Larsen and Simonsen, 1988). The anion transport inhibitor. DIDS. significantly decreased the amount of SO4 label that accumulated in the blood of freshwater bivalves, suggesting that sulfate uptake was linked to an anion exchange mechanism (Dietz and Byrne. 1999; this study). Sulfate reabsorption is subject to regulatory mechanisms in freshwater bivalves. 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EDMUNDS Department of Biology, California State University, 18111 Nordhoff Street, Northridge. California 91330 Abstract. Although most physiological traits scale allo- metrically in unitary organisms, it has been hypothesized that modularity allows for isometric scaling in colonial modular taxa. Isometry would allow increases in size with- out functional constraints, and is thought to be of central importance to the success of a modular design. Yet, despite its potential importance, scaling in these organisms has received little attention. To determine whether scleractinian corals are free of allometric constraints, we quantified met- abolic scaling, measured as aerobic respiration, in small colonies (S40 mm in diam.) of the scleractinian Siderastrea siderea. We also quantified the scaling of colony surface area with biomass, since the proposed isometry is contin- gent upon maintaining a constant ratio of surface area to biomass (or volume) with size. Contrary to the predicted isometry, aerobic respiration scaled allometrically on bio- mass with a slope (b) of 0.176, and colony surface area scaled allometrically on biomass with a slope of 0.730. These findings indicate that small colonies of S. siderea have disproportionately high metabolic rates and SA:B ra- tios compared to their larger counterparts. The most prob- able explanations for the allometric scaling of aerobic res- piration are ( 1 ) a decline in the SA:B ratio with size such that more surface area is available per unit of biomass for mass transfer in the smallest colonies, and (2) the small size, young age, and disproportionately high growth rates of the corals examined. This allometric scaling also demonstrates that modularity, alone, does not allow small colonies of S. Received 23 September 1999; accepted 30 May 2000. * To whom correspondence should be addressed. Current address: De- partment of Organismic and Evolutionary Biology. 16 Divinity Ave.. Box 90. Harvard University. Cambridge. MA 02138. E-mail: svollmer@oeb. harvard.edu siderea to overcome allometric constraints. Further studies are required to determine whether allometric scaling is characteristic of the full size range of colonies of S. siderea. Introduction Body size affects diverse biological variables ranging from physiological to life-history traits (Schmidt-Nielson, 1984). In unitary organisms, most processes scale allometri- cally (Schmidt-Nielson, 1974) — that is, they change dispro- portionately with size — as a result of physical and geomet- ric constraints on body size, structure, and function (Gould, 1966; Schmidt-Nielson, 1974). Classic examples of these constraints include the limits that the skeleton places on the size of terrestrial mammals (Schmidt-Nielson, 1974; Econo- mos, 1981) and the limits that flight muscles place on the size of flying birds (Pennycuick, 1972). Constraints often are inherent to the design of organisms, yet they can be minimized, in theory, by minor changes in geometry or shape (Brody, 1945; Gould, 1966). Profound changes in size, however, require design modifications, including elab- orate structural changes and the development of complex internal systems (Gould, 1966; Schmidt-Nielson, 1984). Such changes probably evolve over relatively long time scales (Gould, 1966, 1977). The relationship between surface area and volume is fundamentally important to scaling arguments, especially for surface-area-related phenomena such as metabolism and thermal regulation (Gould, 1966; Schmidt-Nielson, 1984). because most processes scale allometrically as a result of decreasing ratios of surface area to volume (SA:V) that are associated with volumetric increases in body size (Gould. 1966). In cases where geometric similarity is maintained with increasing size (that is. where there is geometric isom- 21 22 S. V. VOLLMER AND P. J. EDMUNDS etry, or constancy of shape), surface area (v) scales to the two-third power of volume (.v) according to the allometric equation: v = a.\J'. This equation describes a case of func- tional allometry arising from geometric isometry, where a is a constant and b is the scaling exponent with a predicted value of .067 (Schmidt-Nielson, 1984; Peters, 1983). Thus, all things being equal and with geometric isometry, all surface-area-related processes (heat loss, gas exchange, etc.) also should scale to the two-thirds power of mass, i.e., the surface rule (Rubner, 1883). However, most organisms do not maintain geometric similarity as they grow (McMahon, 1973), and therefore the scaling exponents of many surface- area-related processes deviate from the predicted value of 0.67 (Kleiber, 1932, 1961). Metabolism, for example, often scales to the three-quarter power in interspecific analyses (b = 0.75) (Zeuthen, 1953; Hemmingsen, 1960). whereas intraspecific exponents vary widely depending on the or- ganism (Altman and Dittmer, 1968; Peters, 1983). Such deviations can be explained, in part, by geometric allometry involving changes in the shape of exchange surfaces; these changes maintain high SA;V ratios and minimize surface- area-related constraints (Gould, 1966). In an extreme case, organisms might overcome geometric constraints entirely by maintaining a constant SA:V ratio with increasing body size (Gould, 1966). However, the maintenance of a constant SA:V ratio is likely only in organisms with relatively un- usual body plans — for example, the dorsoventral flattening in flatworms (Gould, 1966), and the incorporation of non- respiring biomass in corals, bryozoans, hydroids, and other colonial modular organisms (Gould, 1966; Sebens. 1987a). It has been hypothesized that colonial modular organisms overcome the allometric constraints typically associated with volumetric increases in body size by maintaining a constant SA:V ratio as colony size increases (Jackson, 1979; Hughes and Hughes, 1986). Purportedly, this is achieved by subdividing the biomass of the colony into individual units (i.e., modules) of similar size (Hughes and Hughes, 1986) and growing through modular iteration (Jackson, 1979; Hughes and Cancino, 1985). As a result, physiological processes should not be functionally constrained by declin- ing SA:V ratios, but instead should scale proportionally (i.e., isometrically, b = I ) to both the number of modules and the total colony biomass (Jackson, 1979; Sebens, 1979, 1987a), thereby allowing indeterminate colony growth (Se- bens, 1987a). In turn, this proposed isometry is thought to be critical to the success of colonial modular organisms (Hughes and Hughes. 1986). because it should provide access to the beneficial fitness consequences of increased size (Jackson. 1977; Sebens. 1982; Hughes and Jackson, 1985; Karlson, 1988) without the constraints of allometry. However, despite the theoretical importance of isometry in colonial modular organisms, few studies have tested this prediction, and the available data are contradictory. Aerobic respiration, for example, scales isometrically with mass in the bryozoan Elect ra pilnsa (b = 0.97; Hughes and Hughes, 1986) but allometrically in the soft coral Alcyoiiiiini side- riiiin (h = 0.88; Sebens, 1987b). Moreover, chemical engi- neering and mass transfer theory predict that many colonial modular organisms with simple geometries should display allometric scaling (Patterson. 1992a). In this study, we revisit scaling in colonial modular organisms to determine whether their body plans do, indeed, provide a comprehen- sive escape from allometric constraints. More specifically, we test the null hypotheses that aerobic respiration (hereaf- ter referred to as respiration) and the surface-area-to-bio- mass (SA:B) ratio scale isometrically (i.e., proportionately) in the scleractinian Siderastreu siderea. Respiration was selected to examine the scaling of physiological traits be- cause of its importance in generating ATP for synthetic and muscular work. The SA:B ratio was selected as a proxy for the SA:V ratio because biomass (B) can be determined easily with a gravimetric approach, and it is proportional to volume with a constant tissue density. Siderastrea siderea was used as a model system for a colonial modular taxon because, as a scleractinian, it pro- vides a consummate example of this structural clade. Ad- ditionally, S. siderea is ecologically important on Caribbean reefs (Goreau, 1959) and can be identified readily to species (Foster, 1979, 1980). The study was restricted to small (<40 mm diam.), juvenile colonies (Soong, 1993) because they are tractable to investigation within the constraints of lab- oratory chambers designed to measure metabolism. Juvenile corals also have a strong effect on the population biology of reef corals (Bak and Meesters, 1999), and thus studies of their biology are likely to result in a better understanding of the processes driving coral demography. The full size range of S. side rea (to = 1 m diam. and > 100 y old. Foster, 1979) was not included because large colonies were rare at the study site (the north coast of Jamaica) and cannot be ac- commodated easily in laboratory apparatus. Thus, although the results of this study provide a valid test of scaling in an important life-history stage of a colonial modular taxon, the findings cannot be extrapolated beyond the size range of the colonies investigated. Materials and Methods Respiration Small colonies of Siderastrea siderea were collected from 8.5 m depth on the forereef at Dairy Bull, about 2 km east of Discovery Bay. Jamaica, in January 1997. They were transported to the Discovery Bay Marine Laboratory (DBML) where they were epoxied (Z-Spar A-788) to tiles made of acrylic plastic. The epoxy was applied to the exposed skeleton so that only living coral tissue was left uncovered. Within 24 h. the tiles were secured to racks and returned to the collection site to recover. After more than 1 week of recovery, corals were selected haphazardly from ALLOMETRIC SCALING IN SMALL CORALS 23 the racks, returned to the laboratory, and placed in a dark- ened container supplied with flowing seawater. The corals were kept in darkness overnight, prior to respiration mea- surements, to avoid the confounding effect of light history on the respiration of symbiotic corals (Edmunds and Da- vies. 1988). Respiration rates were measured as oxygen flux using polarographic oxygen electrodes that were connected to an oxygen meter (Cameron OM400) and inserted into the top of clear acrylic chambers. The chambers were designed to expose the corals to unidirectional flow while retaining the minimal volumes necessary for respirometry with small organisms (Fig. 1). A small chamber was used for corals roughly 20 mm in diameter, and a large chamber for corals 21 to 40 mm in diameter. Both chambers consisted of a circular working area with volumes of 332 and 680 ml, respectively, and were regulated at ambient seawater tem- perature (26°C) using a water jacket and bath (Haake Dl). Water flow inside the chambers was created by a stirbar rotating at a constant rate. Flow rates at the periphery of the chambers, where the corals were located, were quantified using brine shrimp cysts (Johnson and Sebens, 1993), and were not significantly different between chambers (Mann Water Jacket \ Working Volume Stirbar Electrode 6 cm ' Figure 1. Plan view of the respiration chamber (drawn to scale). The chamber consisted of a cylindrical working volume (5 cm high x 14 cm in diam.. 680 ml in volume) surrounded by a water jacket. A centrally located stirbar created a unidirectional flow (5.8 ± 0.1 cm s~'( over the coral colony located on the periphery of the chamber; arrows indicate direction of flow. Whitney U test, £/, = 683.5, n]2 = 40, P = 0.26). The pooled flow rate for both chambers was 5.8 ± 0.1 cm s ' (mean ± SE. ;; = 80). Two oxygen electrodes were used (Strathkelvin E5046 and YSI Model 5739), and both were calibrated using a zero solution (0.01 M sodium tetraborate and sodium sultite) and air-saturated seawater. Salinities were determined using a refractometer, barometric pressure was recorded, and oxy- gen solubilities were determined from Weiss (1970). Corals were placed into the chambers filled with filtered seawater (0.45 n-m. FSW). and respiration rates were measured in darkness following 15-min acclimation to the chamber. All measurements were completed at an oxygen saturation above 80% (Edmunds and Davies, 1986), and data were recorded using a data acquisition system (Datacan, Sable Systems). Controls were run daily in the same manner using FSW alone. The rates of change in pO-, in the experimental and control trials were calculated using simple linear regres- sion (r2 > 0.94). After accounting for controls, the respira- tion rate per coral (micromoles of oxygen per coral per hour) was calculated to examine metabolic scaling. Surface area and biomass After respiration measurements were completed, surface areas were estimated using the aluminum foil method (Marsh, 1970). In this technique, aluminum foil was molded over the surface of the coral; the foil was then removed, dried, and weighed; and the surface area was estimated using a previously derived relationship between area and weight. Dry tissue biomass was quantified by preserving the corals in 5% formalin in seawater, decalcifying in 5% HNO3. and drying the resulting tissue tunic at 60°C for 7 days (Edmunds and Davies, 1986). Preliminary experiments using tissue from the anemone Anthopleura xanthogram- niica demonstrated that the formalin and acid treatment resulted in a loss of 2.7% ± 0.7% (mean ± SE, n = 10) of the dry tissue. Therefore, the values of dry tissue biomass in the present study are likely to be slightly conservative. Statistical analyses Logarithmic linear regression was used to examine the scaling relationships. The slope of the regression provides the scaling exponent (/?), and all analyses were completed using natural logarithms (In). The scaling of metabolism was estimated by a regression analysis with the log of respiration (per coral) as the dependent variable and the log of dry tissue biomass as the independent variable. Changes in the ratio of surface area to volume were estimated by regression analysis with the log of surface area as the dependent variable and the log of dry tissue biomass as the independent variable, assuming that biomass and volume are related linearly. Model II (reduced major axis) regres- sion analyses were used because the independent variables 24 S. V. VOLLMER AND P. J EDMUNDS O £ o 5- 3- 1- a, c« -1- -3' A -20246 In Dry Tissue (mg) O "o 0 £ c -2 ^o *4— I 03 i_ 'a, -4 •a -6- -20246 In Dry Tissue (mg) Figure 2. Respiration plotted against biomass in small colonies of Siderastrea siderea. (A) Regression of the log of the respiration rate per coral on the log of dry tissue biomass: regression equation: y = 0.176 x + 1.717, ,• = 0.494. The slope of 0.176 ± 0.031 (±SE. » = 26 corals) deviates significantly from 1 (/ = 26.59. df = 24, P < 0.0001 ). indicating allometric scaling. (B) Regression of the log of the mass-specific respiration rate on the log of dry tissue biomass (recalculated from the data in Fig. 2A); regression equation: v = -0.924 .v - 1.9752. r = 0.986. were subject to measurement error (Ricker, 1973; Sokal and Rohlf, 1995). In this technique, the slope (or scaling expo- nent) is obtained by dividing the standard error of the dependent variable by the standard error of the independent variable, which results in a slope greater than that generated by least-squares linear regression (Sokal and Rohlf, 1995). The null hypothesis of isometry was tested using a t test (//0: b =: 1), where a significant deviation (P < 0.05) indicates an allometric relationship. Results Respiration Respiration rates were estimated in 26 corals ranging in diameter between 3 and 37 mm. The regression of the log of respiration rate (micromoles of oxygen per coral per hour) on the log of colony dry tissue biomass (Fig. 2a) was significant (F( , 24l = 7.952, P < 0.01 ). and produced a slope of 0.176 ± 0.031 (±SE, n = 26 corals), which deviated significantly from 1 (t = 26.59. df = 24. P < 0.0001 ). This significant departure from a slope of 1 indicates that respi- ration scaled allometrically on biomass such that respiration increased disproportionately more slowly than colony size (biomass). As a result, a doubling of biomass corresponds to only a 13% increase in the respiration rate per colony (Fig. 2a), and a 47% decline in mass-specific respiration (micro- moles of oxygen per milligram of tissue per hour) (Fig. 2b). Snrfiice area on hiomtiss The scaling of colony surface area with biomass was quantified in 25 of the 26 corals; 1 coral was excluded as an outlier due to high leverage (Sokal and Rohlf, 1995). The regression of the log of surface area on the log of the dry tissue biomass of the colonies (Fig. 3) also was significant (F(\ 23, = 294.973, P < 0.0001 ), and the slope of 0.730 ± 0.041 (±SE, n = 25 corals) deviated significantly from 1 (t = 6.60, df = 23, P < 0.0001 ). This indicates that colony surface area scaled allometrically with biomass such that surface area increases disproportionately more slowly than 4- o u I 0- -4- In Dry Tissue (mg) Figure 3. Colony surface area plotted against biomass in small colo- nies of SUIcrastrea siderea. Regression of the log of colony surface area on the log of dry tissue biomass; regression equation: v = 0.730 .v - 1.356. /• = 0.963. The slope of 0.730 ± 0.041 (±SE. n = 25 corals) deviates significantly from 1 (I = 6.60, df = 23. P < 0.0001 ), indicating allometric scaling, ALLOMETRIC SCALING IN SMALL CORALS 25 biomass, and the ratio of surface area to biomass declines with increasing colony size. As a result, a doubling of biomass corresponds to only a 66% increase in surface area. Moreover, the slope of 0.730 for surface area on biomass does not deviate significantly (: = 1.453, df = 23, P = 0.1597) from the expectation of geometric isometry (b = 0.67). Thus, the modular design of these small corals does not confer significantly higher ratios of surface area to biomass than would be expected if geometric similarity was maintained. Discussion Contrary to the isometric scaling predicted for colonial modular organisms (sensu Hughes and Hughes, 1986), res- piration and surface area scaled allometrically with biomass in small colonies of Siderastrea siderea. As a result, both mass-specific respiration and the surface-area-to-biomass (SA:B) ratio declined with colony size. Thus, although respiration scales isometrically in at least one colonial mod- ular organism — the encrusting bryozoan Electro pilosa (Hughes and Hughes, 1986) — the present results show that isometric scaling is not axiomatic with a colonial modular design. Instead, allometry describes the size-dependency of two traits in S. siderea, and has been demonstrated previ- ously for respiration in the octocoral Alcyoiiiiini siderium (Sebens, 1987b) and predicted on the basis of chemical and mass-transfer theory (Patterson, 1992a). Although compar- isons across taxa are difficult due to the wide variation in intraspecific metabolic scaling exponents, the exponent of 0.176 calculated for small colonies of S. siderea falls within the observed range (/? == 0.15 -• 1.28) of intraspecific exponents for metazoans (see Peters, 1983; Patterson, 1992a). Metabolic scaling exponents in unitary anthozoans range from 0.54 to 0.94 (Patterson, 1992a) and include the solitary scleractinian Fun^ia scutaria (b = 0.79; Krupp, 1982; exponent calculated in Patterson, I992a). As for metabolic scaling in small colonies of S. siderea, we posit that the unusually small scaling exponent (b = 0.176) is a result of the changes in the SA:B ratio and the developmen- tal stage of the small colonies investigated. Maintaining a constant surface-area-to-volume (SA:V) ratio (and, with invariate biomass density, a constant SA:B ratio) is the theoretical basis for isometry in colonial mod- ular organisms (Jackson, 1979). However, isometry can occur only where colony biomass is restricted to a single layer of modules with conserved dimensions; this design is typical of hydroids, scleractinians, and cheilostome bryozo- ans (Jackson, 1979). However, where there is metabolically active biomass outside the modules, the SA:B ratio de- creases as extra-modular biomass increases volumetrically. Thus, the extra-modular biomass in octocorals (i.e.. the coenenchyme) and compound ascidians (i.e., the gelatinous matrix) should favor allometric scaling (Jackson, 1979; Sebens, 1987a). These predictions are supported by exper- imental data from the encrusting bryozoan Electra pilosa and the fleshy octocoral Alcyonium siderium (cited above). However, although the single layer of uniformly sized pol- yps in scleractinians also should allow for a constant SA:B ratio, this is not the case for S. siderea, where colony surface area scales allometrically on biomass (b = 0.730). Thus, in small colonies of S. siderea, the SA:B ratio declines with increasing colony size, such that larger colonies have dis- proportionately more biomass than their smaller counter- parts (Fig. 3). The functional basis for the allometric scaling of the SA:B ratio is unknown, but it is probably related to calci- fication (Barnes, 1973) and the selective pressure for rapid growth in small corals (Jackson, 1977). Thus, the smallest corals may sustain high rates of linear growth (i.e.. calcifi- cation) at the expense of tissue growth, so that the existing tissues are "stretched" thinly over the increasing surface area. Then, as the colonies become larger, they may con- centrate resources on tissue growth, thereby increasing bio- mass and tissue thickness. Support for this hypothesis comes from two studies. First, reanalysis of the data of Jokiel and Morrisey ( 1986) for the coral Pocilloponi dami- cornis demonstrates allometric scaling of surface area with biomass (b = 0.700 ± 0.057, mean ± SE, n = 6) as well as respiration (b = 0.840 ± 0.041, mean ± SE, n = 6) (Jokiel and Morrisey, 1986). Thus, biomass is added more rapidly than surface area and. as in S. siderea. the resulting allo- metric scaling of the SA:B ratio provides a possible expla- nation for the allometric scaling of respiration in P. dami- cornis. Second, trade-offs in growth between skeleton and tissue, similar to those proposed for S. siderea (described above), have been reported for Porites from the Great Barrier Reef (Barnes and Lough, 1993), as have systematic differences in tissue thickness for the same species (Barnes and Lough, 1992). Indeed, the positive relationship between tissue thickness and colony height in Porites (Barnes and Lough. 1992), together with the large amount of extra- modular biomass ( ==90% by thickness, Barnes and Lough, 1992), might be prominent in this genus. Thus, variation in tissue biomass. thickness, or both with colony size may be a general feature of scleractinian corals. However, in addi- tion to putative changes in tissue thickness driving the observed changes in the SA:B ratio, it is possible that the SA:B ratio was biased by the use of the aluminum foil method (Marsh, 1970) to measure the surface area. This technique is widely used for determining the surface area of corals with relatively smooth and unconvoluted surfaces like those in 5. siderea (see Hoegh-Guldberg. 1988. for an alternative approach), but it is unable to quantify the area of the expanded polyps. Quantifying the area of expanded polyps is made difficult by their highly variable morphology and degree of expansion and, as a result, previous studies have relied on geometric approximations to obtain polyp or 26 S. V. VOLLMER AND P. J. EDMUNDS tentacle area (Sebens, 1981). Regardless of the methodolog- ical difficulties, currently there is no evidence of systematic variation in polyp dimensions with colony size (i.e.. allom- etry): moreover, polyp dimensions may be highly conserved for mass-transfer purposes (Patterson, 1992a). Thus, given that the thickness of coral tissues is known to vary (e.g., Barnes and Lough. 1992), we believe that changes in the SA:B ratio are more likely to be driven by tissue thickness than by the area of expanded polyps. Still, a definitive test of the hypothesized mechanism of variation in the SA:B ratio is required, and this will necessitate an analysis of tissue thickness and skeletal extension as a function of colony size. The allometric scaling of the SA:B ratio in S. siderea could drive the scaling of respiration through indirect ef- fects on mass transfer of metabolites to the coral tissue. Mass transfer with the surrounding seawater is determined, in part, by surface area, which decreases relative to biomass as S. siderea increases from 3 to 37 mm in diameter. Thus, all things being equal (i.e., excluding the boundary layer arguments described below) and within the size range stud- ied here, small corals should maintain relatively higher fluxes of metabolites than large corals, which could support the higher respiration rates observed in the small corals (Fig. 2). Additionally, increases in biomass will be accompanied by increases in biovolume that probably lengthen diffusion pathways (i.e., the tissue thickness) and reduce the rates of solute transport (Patterson. 1992b). For the colony size range studied, the respiration of large S. siderea therefore may be depressed by limitations on the delivery of oxygen to metabolically active tissue. This hypothesis could be tested by measuring the magnitude of the flow dependency of respiration (sensu Patterson and Sebens, 1989), with the expectation of a greater effect in larger colonies than in smaller ones. Although the scaling of the SA:B ratio provides a testable hypothesis to explain the scaling of respiration in S. siderea. it does not exclude the possibility that other factors might also be important. Of these, variation in energy expenditure among developmental phases (i.e., colony sizes) has the greatest potential to explain, in part (or entirely), the allo- metric scaling of respiration. In benthic marine inverte- brates, scaling exponents typically are affected by the size range and developmental phase of the organisms investi- gated (Zeuthen, 1953). Lower exponents are characteristic of early and late developmental phases and of the extremes of the natural size range. For example, metabolic scaling exponents (/?) for the mussel Mytilus edulis change from 0.80 in recruits «0.1 mg) to 0.95 in sub-adults (0.1 to I mg) and to 0.65 in adults (> 1 mg) (Zeuthen, 1953). The low scaling exponents in the smallest (i.e., youngest) size classes demonstrate that their metabolic rates are relatively high compared to those of the larger sub-adults, and are thought to be a consequence of the elevated energy expenditure necessary to sustain accelerated growth (Zeuthen, 1953). Size and age are poorly related in scleractinians (Hughes and Jackson. 1980), but the colonies of S. siderea used in the present study (^37 mm diam.) are young relative to the largest colonies of this species ( = 1 m diameter and > 1 00 y old; Foster. 1979). and the smallest corals (3 mm diam.) may be only a few months old (Van Moorsel, 1988). Re- gardless of age, small corals are probably exposed to selec- tive pressure for rapid growth (Jackson, 1977), as occurs in other colonial modular organisms (Jackson, 1977; Sebens, 1982; Karlson, 1988). because of the mortality risks of being small (Jackson, 1977). Thus, in addition to the SA:B explanation for allometric scaling in small colonies of S. siderea (described above), it is possible that the respiration rate (per coral) in the smallest colonies is elevated by the high metabolic rate of young tissues or by the costs of responding to the selective pressure for rapid growth. Two other hypotheses could account for allometric scal- ing of respiration in small colonies of S. siderea — namely. mass transfer effects (sensu Patterson, 1992a) and the pop- ulations of endosymbiotic zooxanthellae — but these are not supported by the available data. The "mass transfer hypoth- esis" focuses on the importance of mass transfer in moving metabolites between the coral tissue and seawater and driv- ing coral respiration (Patterson and Sebens, 1989; Patterson el ul., 1991 ). The boundary layers next to the coral have a critical role in determining rates of mass transfer (Denny, 1988; Patterson. 1992b) and are a function of the interaction of the flow regime with the size and shape of the coral colony. Based on these relationships. Patterson (1992a) predicted that metabolic scaling in aquatic organisms could be explained with a mass transfer argument. In short, changes in organism size and shape can be sufficient to alter mass transfer and support the allometric scaling of metab- olism, with exponents similar to published values (Patter- son. 1992a). For hemispherical objects like the small colo- nies of S. siderea used in the present study, the mass transfer explanation for metabolic scaling (sensu Patterson, 1992a) would predict an exponent (b) of =0.47 (Helmuth et ul.. 1997). This is 2.7-fold higher than the allometric scaling exponent we calculated for respiration in small colonies of S. siderea that have hemispherical colonies (h = 0.176). One reason for this discrepancy is that the colonies used (3-37 mm diam.) were probably too small to establish their own equilibrium boundary layers (Denny, 1988) and were, instead, affected by upstream roughness elements in the respiration chamber (Gardella and Edmunds, unpubl. data). In other words, small colonies of S. siderea may be an exception to the mass transfer argument for allometric scal- ing (sensu Patterson. I992a). because they all are too small (i.e., £37 mm diam.) to affect their own boundary layers directly. The "zooxanthellae hypotheses" focus on the role of the /ooxanthellae in contributing to the respiration of the col- ALLOMETRIC SCALING IN SMALL CORALS 27 ony (i.e., the cnidarian host plus algal symbionts) (Musca- tine ct til.. 1981; Edmunds and Davies, 1986) to account for allometric scaling of coral respiration. Thus, changes in the density or metabolic activity of zooxanthellae should affect the respiration of the colony and, if these changes are correlated with size, could affect metabolic scaling. In S. sidereu, zooxanthellae densities cannot account for allomet- ric scaling of respiration, because zooxanthellae densities scaled isometrically with biomass (Vollmer, 1999). It is possible, however, that the respiration rate per zooxanthella varied with colony size, but this possibility cannot be ex- amined experimentally at the current time because zooxan- thellar respiration can only be measured /;; vitro, and these rates may be different from those attained in hospite (Gates ct til.. 1999). In the absence of in hospite determinations of zooxanthellar respiration, and given that zooxanthellae den- sities scale isometrically, we conclude tentatively that the zooxanthellae are unlikely to be a proximal cause of the allometric scaling of respiration in small S. sidereu. This study demonstrates allometric scaling in small col- onies of Siderastrea siderea and underscores two mecha- nisms that probably drive this scaling — i.e.. disproportion- ate changes in the SA:B ratio and the developmental stage of the colonies examined. Because both mechanisms may be associated with the rapid growth necessary to escape the risky life-history stage of being small, allometric scaling probably has strong fitness consequences. While it remains to be seen whether the present findings are applicable to other species, or to a larger size range of colonies, further studies of allometric scaling in scleractinians will be valu- able. Acknowledgments This research was submitted in partial fulfillment of the Master of Science degree to S. V. Vollmer at California State University, Northridge (CSUN). We thank R. C. Car- penter, D. Carlon, and three anonymous reviewers for their comments on earlier drafts of this paper. Field work was made possible through the generosity of M. Haley and the staff of the Discovery Bay Marine Laboratory (DBML), and numerous dive buddies who assisted in the field, including G. F. River and S. Tallack. 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In a few well-known cases, the biological con- sequences of the disruption of liin-1 homeodomain (HD) genes have demonstrated the important roles of these genes in vertebrate development, especially in the nervous tissue, kidney, and gonads. Functional assay approaches require information not only about lim-l gene organization, but also about properties and tissue localization of Lim-l proteins. Although lim-l genes have been identified in certain phyla of invertebrates, no information is available on Lim-l pro- teins and genes in bivalve molluscs. Our study represents the beginning stage of identification of the Lim-l -related proteins in marine bivalves. Using antibodies against the C-terminal region of the Xenopus laevis Lim-l protein, we describe cross-reactive antigen patterns in adults and early embryos of the mussel Mytilus galloprovincialis, as well as in sea urchin and chick embryos. In adult mussels, nervous ganglia and gonads display the most prominent Lim-l im- munoreactivity. Further, the antibodies verified the predic- tion that mussel Lim-l antigens, like Lim-l HD proteins in general, can be localized in the nucleus. Moreover, antibody detection allowed us to identify the Lim-1-like antigens in unfertilized mature eggs, as well as in very early embryos of bivalve molluscs and sea urchins (Strongylocentrotus pur- pnratns). In mussel eggs and embryos, Lim-l antigens are expressed in multiple forms (40, 45, and 65 kDa), as de- tected by SDS-PAGE followed by Western blot. Taken together, the observations emphasize the conservation of the Lim-l protein expression pattern in the nervous tissue and Received 13 December 1999; accepted 8 May 2000. * To whom correspondence should be addressed: E-mail: margot @udc.es Abbreviations: LIM. an abbreviation derived from the names of three homeodomain transcription factors, containing a cysteine-rich zinc-finger domain (i.e.. LIM-domain): Lin- 11 of Caenorhabditis elegans, Isl-1 of the rat, and Mec-3 of C. elegans', MW, molecular weight; SDS-PAGE. sodium dodecylsulfate polyacrylamide gel electrophoresis; HD. homeodomain. gonads of different animal groups, and demonstrate that Lim-1-like polypeptides can be maternally accumulated in eggs and, therefore, are present in very early embryos before zygotic expression of the genes begins. Introduction A cysteine-rich zinc finger domain, named LIM, was first identified in the Caenorhabditis elegans homeobox genes lin-1 1 and mec-3, and in the rat DNA binding factor Isl-1. Then the LIM domain was found in a variety of proteins including transcription factors, cytoskeletal proteins, and LIM kinases (Dawid et ai, 1998; Bach. 2000). LIM do- mains appear to play a primary role in protein-protein interactions, through the formation of dimers with identical or different LIM domains or by binding distinct proteins (Breen et ai. 1997; Dawid et ai. 1998; Curtis and Heiling, 1998; Hobert and Ruvkun. 1998; Hobert and Westphal, 2000). Phenotypic analysis of patterns of lim gene expression reveals that the genes can participate in a number of impor- tant events in early embryonic development, as well as in cell fate determination and cell differentiation at advanced stages of organogenesis. Moreover, some lim genes are constitutively expressed in adult tissues, where they may contribute to certain tissue-specific functions (Dawid et ai. 1998; Hobert and Westphal, 2000). The early and late ontogenetic expression phases of lim genes suggest that they have multiple and distinct functions at different stages of the animal life cycle. In the latter context, the LIM containing homeodomain (HD) lim-l genes have been most exten- sively studied in a range of animals. Lim-l encodes a protein with a pair of LIM domains located N-terminal to the HD. In vertebrates, //';;/ / was originally identified in the frog, Xenopus laevis. as Xlim 1 (Taira et ai, 1992). In X. laevis, the Xlim-l is expressed in 29 30 M. TORRADO AND A. T. MIKHAILOV the Spemann's organizer during gastrula stages, and in late embryos primarily in the nervous system and kidney (Taira etui.. 1992. 1994. 1997: Wallingford et til.. 1998; Carroll et a!.. 1999a; Carroll and Vize. 1999). Similar patterns of lim-l expression have been observed in fishes (Toyama et ul.. 1995; Carroll et ai, 1999b). chickens (Tsuchida et til.. 1994). mice (Fujii et ul.. 1994; Shawlot and Behringer, 1995: Li et til.. 1999). rats (Furuyama et til.. 1994; Kara- vanov et til., 1998). and humans (Dong et ul.. 1997). The biological consequence of the disruption of lim-l HD genes or modifications of their expression patterns have demon- strated the crucial role of these genes in development, especially in the nervous tissue, kidney, and gonads (Shawlot and Behringer. 1995; Shawlot el til.. 1999: Taira et til.. 1997; Carroll and Vize. 1999). In invertebrates, lim-l related genes have been identified in nematodes. fruit flies, and sea urchins. The lin-11 gene of C. elegans (the closest homolog of amphibian and mamma- lian lim-l) is expressed in different subsets of neurons and in the vulva, and it is essential for uterine morphogenesis (Hobert et ai. 1998: Hobert and Westphal. 2000; Newman et al. 1999). In Drosophila. the gene termed dlim I is expressed in the head, the brain lobes, and in neurons of the ventral nerve cord (Lilly et til.. 1999). The ///(/-/-related HD gene of the sea urchin (Hemicentrotiis pulcherrimns). HpLiml, is detected in early embryos and involved in the differentiation of endoderm, mesenchyme, and aboral ecto- derm (Kawasaki etui., 1999). Thus, although expression in neural tissues seems to be a common feature of Lim-l -related HD factors in both ver- tebrates and invertebrates, most of these factors are also characterized by their expression in excretory and reproduc- tive organ systems. These expression phenotypes have been described mainly at the molecular level by analysis of lim-l gene transcription patterns. An alternative approach is the analysis of post-translational Lim-l expression by immuno- chemical methods. This approach makes it possible to mea- sure this factor at the protein level in different cell types and to detect other tissues that express the polypeptide at dif- ferent stages of development and in the adult state (Kara- vanov et til., 1996; Brown et til., 1999; Lilly et at.. 1999; Shimono and Behringer, 1999; Mauch et til., 2000). To our knowledge, no information is available on Lim-l proteins (genes) in marine bivalve molluscs, although such data would be useful for further comparative analysis of Lim-l expression patterns and functions in invertebrates and vertebrates (Hobert and Westphal, 2000). Our study represents the beginning stage of the identification of Lim- 1 -related proteins in bivalve molluscs. We describe the distribution patterns of immunoreactive Lim-l -like proteins in adults and early embryos of the mussel Mytilus gallopro- vincitilis. We also report the first examination of Lim-l antigen signals in sea urchin (Strongylocentrotus purpiirt/- lus) embryos, as well as in different compartments of the chick embryo brain. Materials and Methods Animals and embryos Adult mussels (Mytilus galloprovincialis) and sea urchins (Strongylocentrotus purpuratus) were purchased during the spawning season (April-May of 1999) from commercial suppliers in La Coruiia (Galicia, NW Spain). Published procedures (Sprung and Bayne. 1984; Holland and Holland. 1993; Mikhailov et ai. 1996) were followed for stripping of animals to obtain oocytes and sperm and for the subsequent in vitro fertilization and culture of embryos. At each chro- nological stage, the bulk of the embryos were re-collected, placed on ice, and typed morphologically under a dissecting microscope (Nikon). The embryos, selected according to morphology, were put into centrifuge tubes containing ster- ile seawater and permitted to settle to the bottom of tubes or pelleted by low-speed centrifugation: then the upper solu- tion was discarded. Fertilized chicken (Callus gullus) eggs were obtained from the cooperative chick network hatchery (Ferrol. Province of La Coruna. Galicia) and incubated at 37°C. Different brain regions (forebrain. optic lobes, and cerebellum) were microsurgically isolated in cold minimum essential medium (MEM; Gibco) and pelleted by low-speed centrifugation. Tissue dissection and processing Before use, adult mussels (M. galloprovincialis) were kept in a dry state for 1 h at 4°C. Mussels were opened with the aid of scalpel and placed on ice; a small portion of gonad material was microscopically examined to determine the sex of the individual. Different tissues and organs (gonad, foot, labial palps, hepatopancreas, gill, and fragments of posterior adductor muscle) were excised, rinsed in sterile seawater, and blotted on sterile filter paper. To obtain cell suspensions of sperm or oocyte, gonad follicle biopsy was performed as described in Torrado and Mikhailov (1998). Then, follicle luminal masses were aspirated and resuspended in ice-cold sterile seawater; the released cell suspension was micro- scopically tested for the presence of spermatozoa or oo- cytes. The sperm suspension was then centrifuged ( 100 X i>, 5 min, 2 C). the oocytes were permitted to settle to the bottom of the tubes, and the rinse solution was discarded. For some experiments, gonad collecting tubules (with adja- cent connective tissue) were dissected manually from the ripe male or female gonad and the excised tissue was microscopically examined to definitively determine the ab- sence of gametes (spermatozoa or oocytes) in the tubule lumen. Isolated tissues were additionally shaken in ice-cold sterile seawater for 20 min; after settling, rinse solutions were discarded. Pedal ganglia (see Fig. 4A) were microsur- gically dissected under a stereomicroscope (Nikon), pooled in ice-cold sterile seawater. and pelleted by low-speed cen- trifugation. When the samples were not homogenized im- mediately, they were stored at -85°C for several days. MUSSEL LIM-I RELATED PROTEINS 31 Spent male gonads were sampled in 1998. and the corre- sponding frozen and Bourn-fixed tissue fragments were stored in liquid nitrogen and ethanol (70%). respectively. Sample preparation Mussel and sea urchin eggs and embryos were resus- pended in cooled deionized water containing 2 mM EDTA (Merck). 6 M urea (Merck), and the protease inhibitor cocktail P2714 (Sigma). The supernatants obtained after centrifugation (30.000 X g. 8°C, 30 min) were mixed with SDS sample buffer containing the protease inhibitor cock- tail, kept for 1 h at room temperature, and stored at — 30°C until use. All other tissue samples were first homogenized in 1:5 (v/v) ratio in 100 mM Tris (Sigma). 2 mM EDTA (Merck) solution, containing the protease inhibitor cocktail. After centrifugation (10.000 X g. 2°C. 30 mm), the super- natants were discarded, and the pellets were then re-ex- tracted and assayed as described for embryos. Antibodies Anti-XLim-1 antibodies were a generous gift from Dr. A. A. Karavanov and Prof.. Dr. I. Dawid. These polyclonal rabbit antibodies produced against the C-terminal region of the XLim-1 downstream of the HD (amino acids 265-403) (Taira et a I., 1992) have been characterized and shown to cross-react with Lim-1 proteins of fishes, mice, rats, and humans (Karavanov et al., 1996. 1998). Gamma-globulin fraction was obtained from anti-XLim-1 serum with the aid of the Mab Trap G II Kit for antibody purification (Phar- macia), according to the manufacturer's protocol. The frac- tion was concentrated using concentrator units (Millipore), supplemented by glycerol (Merck) at a final concentration of 50%, and stored at — 20°C in aliquots. Chemicon has recently commercialized these anti-XLim- 1 antibodies. Protein determination Protein concentration was measured (Ultrospec 1000E spectrophotometer. Pharmacia) according to the Bradford method using rabbit immunoglobulin G (Sigma) or bovine serum albumin (Sigma) as standards. SDS -PAGE assays For all separations, the Mini-Protean II electrophoretic cell (Bio-Rad) was used. Samples were electrophoresed using 5% stacking and 10% resolving Tris-glycine SDS- polyacrylamide gels (Bio-Rad). The gels were stained with Coomassie blue R250 (Sigma) or electrophoretically trans- ferred to membranes. The apparent molecular weights of the bands were determined by comparing low and high molec- ular weight calibration kits (Pharmacia) in the same gel. A micro-preparative variant of SDS-PAGE was performed as previously described (Mikhailov et al., 1997) using a Mini- Protean II comb with one reference well. After electro- phoresis, the reference gel strip was stained and used for isolating the Lim-1 -containing fraction in the remaining gel slab. Alternatively, whole gel slabs were stained with Coo- massie solution (0.0004% in 20% methunol and 3% acetic acid), and fractions of interest were cut out (Mikhailov ct til., 1996). The protein was eluted from gel fractions so obtained using a model 442 electro-eluter (Bio-Rad) in accordance with the manufacturer's recommendation: eluted solutions were concentrated using microconcentra- tion units (Amicon, the 30-kDa cut-off membrane). Blotting assays Proteins resolved in 10% SDS-PAGE were transferred to nylon (Nytran. Schleicher and Schuell) or nitrocellulose (Optitran. Schleicher and Schuell) membranes by routine methods (Mikhailov et at.. 1997) using the mini Trans-Blot cell (Bio-Rad). Protein loading and localization of molecu- lar weight standards was verified by membrane staining with amido black (Merck) or Ponceau S (Sigma). For im- munodetection. the blots were incubated in blocking solu- tion containing 20% of normal horse serum (Sigma) at room temperature for 1 h and further assayed as described in Mikhailov and Simirsky (1991). As primary antibodies, rabbit anti-XLim-1 or rabbit pre-iinmune (negative control) gamma-globulin fractions were used at appropriate dilutions. Peroxidase-labeled anti-rabbit immunoglobulins (Sigma) were used as the second-stage reagent, and diaminoben- zidine (Sigma) was used to develop the blots. The relative amounts of antibody-labeled proteins were quantified by densitometry (GS-700 densitometer, Bio-Rad) and image software (Molecular Analysis, Bio-Rad). For total carbohy- drate detection, blots were treated with an Immun-Blot kit (Bio-Rad) for glycoprotein detection as described (protocol 1A) by the manufacturer: chicken egg ovalbumin (Sigma) and rabbit liver carboxylesterase (Sigma) were used as positive controls. For a precise comparison of the position of the glycoprotein signal with that of Lim-1 immunoreac- tivity, the blot membrane was cut (along the direction of electrophoretic separation) at the middle of the run pocket width; one half was treated with Immun-Blot kit and the other with anti-XLim-1 antibodies (see Fig. 3C.D). Deglycosylation ussa\ Extracts and Lim-1 -containing fractions of mussel pedal ganglia and forebrains of 16-day-old chick embryos were desalted (using Microcon units), re-dissolved in 250 mM sodium phosphate (Merck). pH 6.0. and treated with an enzymatic deglycosylation kit (Bio-Rad) according to the manufacturer's denaturing protocol. Briefly, both neuro- minidase (EC 3.2.1.18) and O-glycosidase (EC 3.2.1.97) were first added to the reaction vials; after the incubation and denaturation step, Af-glycosidase F (EC 3.5.1.52) was added to the mixture. To determine deglycosylation effi- ciently, samples (before and after deglycosylation) were 32 M. TORRADO AND A. T. MIKHAILOV subjected to SDS-PAGE followed by Coomassie staining (to detect the shift in band mobility) or blotting. Blots were treated with an Immun-Blot kit (Bio-Rad) for glycoprotein detection to additionally check the efficiency of the degly- cosylation reaction. Bovine fetuin (Bio-Rad) and rabbit liver carboxylesterase (Sigma) were used as positive con- trols. Ultrafiltration procedures Lim-1 -containing fractions isolated from mussel pedal ganglia and forebrains of 16-day-old chick embryos were subjected to subsequent ultratiltration using Microcon mi- cro-units with 100-kDa and 50-kDa cut-off YM membranes ( Amicon) according to the manufacturer's recommendation. Bovine serum albumin (Sigma) and chicken ovalbumin (Sigma) solutions (1 mg/ml) were used as reference pro- teins. To prevent aggregation, urea (Merck) and SDS (Bio- Rad) were added to starting protein solutions at final con- centrations of 6 M and 1%. respectively. Each sample was first applied on the Microcon- 100 unit and centrifuged at 2500 X g for 30 min at 10°C. The resulting filtrate was then introduced into a sample reservoir of the Microcon-50 unit and centrifuged at 12.000 X g for 20 min at 10°C. The volume of each retained and filtered fraction so obtained was adjusted to that of the starting sample, and the solutions were subjected to SDS-PAGE followed by Western blot with anti-XLim-1 antibodies. Immunocytochemistry Mussel pedal ganglia were fixed in 100 mM 3-(/V-mor- pholino) propane sulfonic acid (MOPS; Sigma). 2 mM MgSO4 (Panreac). 2 mM EGTA (Merck), 3.8% formalde- hyde (Panreac) for 1 h at room temperature. Standard his- tological techniques were used for sample dehydration, em- bedding in paraffin, sectioning at 6 jam, deparaffmization, and rehydration (Mikhailov and Simirsky. 1991). The slides were precoated with 3-aminopropyltriethoxysilane (Sigma). Prior to immunostaining. sections were blocked with 20% normal horse serum in 50 mM Tris-HCl, pH 8.0. Sections were then incubated with different dilutions of the anti- XLim-1 immunoglobulin fraction (3 h at room temperature or overnight at 4°C) and secondary antibodies conjugated to alkaline phosphatase (Boehringer-Mannheim). Antibody di- lutions were prepared in blocking solution. All incubations were followed by six washes (10 min in each) in 50 mM Tris-HCl. pH 8.0. Staining was developed using 5-bromo- 4-chloro-2-indolyl-phosphate (Sigma) and 4-nitro blue tet- razolium chloride as substrates (Sigma) as described in Karavanov et al. (1996). The sections were mounted in Permount (Fisher) and examined under the Nikon Micro- phot microscope. Control experiments were included (1) omitting anti-XLim-1 antibodies, (2) replacing the latter by normal rabbit immunoglobulins (Sigma), and (3) using anti- XLim-1 antibodies preadsorbed by fixed pedal ganglia or by foot tissue of M. galloprovincialis. Fixation was done in 3.8% formaldehyde as above, followed by a methanol wash. Using micro-forceps, fixed tissues were ground, rehydrated in 50 mM Tris-HCl, pH 8.0, blocked in blocking solution for 2 h. pelleted by a low-speed centrifugation, resuspended in anti-XLim-1 antibody solution (at 1/50 or 1/200 dilution), and incubated overnight at 4°C. For some experiments. anti-XLim- 1 antibodies were depleted prior to staining by incubation at 1/50 dilution with hyperfixed Xenopus laevis embryos (this was performed by Dr. A. A. Karavanov and Dr. A. T. Mikhailov in the laboratory of Prof. I. Dawid). Histological analysis A portion of male ripe and spent gonads of M. gallopro- vincialis was fixed in Bouin's solution, embedded in paraf- fin, cut into 6-ju.m sections, and stained with hematoxylin- eosin; the gonadosomatic index was estimated from the sections and expressed as the percentage of the gonad occupied by follicle structures (Mikhailov et al., 1996; Torrado and Mikhailov. 1998). Results and Discussion In this study we used rabbit polyclonal antibodies against a C-terminal region (as an immunogen) of the Xenopus laevis Lim-1 protein (i.e., anti-XLim-1 antibodies). It has been shown that these antibodies detect the Lim- 1 protein in X. laevis and also cross-react with Lim- 1 polypeptides of the mouse (Karavanov et al.. 1996; Shimono and Behringer. 1999) and rat (Karavanov et al.. 1998). These studies have also demonstrated the high specificity of the antibodies to Lim-1 proteins and the absence of any discrepancy between the expression patterns of protein and mRNA. Note that the transcriptional activation domain of Xlim-1 resides in its carboxyl terminus (Breen et al., 1997). Although it is generally accepted that the C-terminal peptide is a good choice for the production of antibodies specific to a protein of interest (Hancock and Evan, 1992), we decided to test, additionally, the degree of "specificity" of the Xlim-1 C-terminal region (used as an immunogen) for Lim-1 proteins. Using the BLAST program (Altschul el al.. 1997), we performed alignments of the C-terminal sequence of Xlim-1 with all the protein sequences listed in the SW1SSPROT database (Bairoch and Apweiler, 2000). The most similar (similarity 80%-90%; Fig. 1) sequences, which were aligned first, are those of the fish, chick, mouse, and human Lim-1. Frog and fish Lim 5 factors are charac- terized by significantly lower primary structural similarity (47% and 46%. respectively) to the Xlim-1 C-terminus sequence. It is significant that the regions of homology reside only in the C-terminus of the sequences mentioned above. Other sequences returned by the BLAST program displayed values of similarity with the C-terminus of Xlim-1 (used as a query) that are not distinguishable from those LIM domain MUSSEL LIM-1 RELATED PROTEINS Xenopus laevis Lim-1 LIM domain HOMEOBOX 265 C-terminal region Immunogen 25 I • 33 403 G T P L GB1B3H PQPGHHP S G T P L GgMD HPIPGHHPS GT P LG AIW3H PQPGHHPQ LIM1-XENLA LIM1 -MOUSE LIM1 -HUMAN LIM1-BRARE 1M1-CHICK G E AQR F T D I AQR FJT D I DIMS HflP G I pITM^H pHc D S P S P E P ;HSMSAEVFG HSMSAEVFG H S M sEBlv F G SPPFSSLSVNGG YGNHLSHPP - EMNEJTAVW YGNHLSHPP~ VGNHLSHPP Y0N H L S H P P YGNHLSHPP H QJ3 L L S N U A FJ3o TGLGI I GQGGQ - G V AflT M R V l~GH!KTp S S D L sT ILIM3-XENLA G I Figure 1. Multiple alignment of the deduced amino C-terminal sequence of Xlini-l with those identified from BLAST search comparisons. (A) Schematic structure of the frog Lim-1 deduced from the previously published Xliin-l sequence (Taira eral.. 1992). The C-terminal region, which was used to generate anti-XLim-1 antibodies (Karavanov el ai, 1996), is shown in black. (B) The complete sequence of the Xennpiix (XENLA) C-terminus is shown aligned with those of mouse, human, /.ebrafish (BRARE), and chick Lim-ls as well as with Xenopus Lim-5 and Lim-3 and zebrafish Lim-5. All the protein sequences were obtained from the SWISSPROT database (Bairoch and Apweiler, 2000). Black — identical amino acid residues. Dash — gaps. Comparison shows that both the size and the sequence of the XLim-1 C-terminus are highly similar (80%-90% of similarity; extent internal homology above seven amino acid residues) to that of Lim-1 proteins from other species. At the same time, the XLim-1 C-terminal sequence reveals no more than 50% of similarity (extent internal homology below seven amino acid residues) with that of Lim-5 proteins. No significant similarity was observed in the case of the .\V»i'/'ii.s Lim-3. expected by chance (Fig. 1. see Xenopus Lim-3 as an example). It is generally accepted that short peptides (below about seven amino acid residues) are of insufficient size to func- tion as immunogenie and antigenic epitopes (Hancock and Evan. 1992). Using the CLUSTAL W program (Thompson i't ul.. 1994), we performed a multiple sequence alignment of the XLim-1 C-terminal region with that of the Lim-1 and Lim-5 proteins identified from BLAST searches (see above). As shown (Fig. 1), the XLim-1 C-terminus shares a high sequence homology with a number of Lim-1 proteins but not with Lim-5 factors. Given the above criteria, it is probable that the XLim-1 C-terminus (used as immunogen) could generate successful antibodies characterized by a high cross-reactivity with Lim-1 proteins in other species. At the same time, it may be predicted that antibodies against the XLim-1 C-terminus possess a much lower cross-reactivity with Lim-5 proteins. Note that anti-XLim-1 antibodies used in this study cross-react with Lim-1 factors from various species but do not cross-react with the closely similar XLim-5 protein on tissue sections (Karavanov et til., 1996). Collectively, the data indicated that the Xlim C-terminal sequence (used as an immunogen to generate anti-XLim-1 antibodies) is highly conserved among most of other known Urn- 1 genes and seems to be diagnostic for their protein products. This would in turn account for the use of the corresponding antibodies in selective (discriminative) im- munochemical screening of Lim-1 -related proteins in dif- ferent species. This suggestion is supported by the results of application of anti-XLim-1 antibodies for immunocyto- chemical Lim-1 protein detection in frog, mouse, and rat tissues (Karavanov ct ul., 1996. 1998; Shimono and Behringer. 1999). Anti-Xlim-1 initihoilies cross-react witli mussel, sea urchin, cind chick tissue antigens SDS-PAGE followed by immunoblot analysis of a pro- tein extracted from isolated pedal ganglia of Mytilus gullo- pnn-inciiilis. mature oocytes of Strongylocentrotus /yiir/ni- ratus. and brain tissues of chick embryos revealed a single band with an apparent molecular weight (MW) of approx- imately 65. 70, and 65 kDa, respectively (Fig. 2). Note that the open reading frame of the chicken (Tsuchida et ul.. 34 M. TORRADO AND A. T MIKHAILOV v 1 2[]3 45 6 7 [8 L9 94- 67- 43- 30- 70 65 -170 -116 -76 -53 Figure 2. Cross-reactivity of anti-XLim-1 antibodies with mussel, sea urchin, and chick tissue antigens. Equal amounts (about 100 fig/pocket) of total protein extracted from different tissues were resolved in a 10% SDS-PAGE. blotted on membranes, and probed with anti-XLim-1 antibod- ies at 1/500 dilution. Membrane strips containing electrophoretically sep- arated molecular weight markers were stained with Ponceau S. Mytiliis galloprovincialis organs and tissues: Lane 1 — male pedal ganglia; Lane 2 — female pedal ganglia; Lane 3 — male muscle adductor posterior; Lane 4 — male gills; Lane 5 — male foot; Lane 6 — male hepatopancreas; Lane 7 — male labial palps. Lane 8 — Strongylocentrotus purpuratus eggs. Lane 9 — forebrain tissues of 16-day-old chick embryos. LMW and HMW — low and high molecular weight calibration kit proteins (30-170 kDa), respec- tively. 65 and 70 — molecular weights of antibody-labeled proteins. kDa. 1994) and sea urchin (Kawasaki ct id.. 1999) lim-1 gene encodes a protein with a predicted MW about of 45 kDa. The high MW value of the Lim-1 antigens is apparently not due to aggregation with other molecules, because it did not change when the antigens were extracted and electro- phoretically analyzed in the presence of 6 M urea. In addi- tion, observed low migration of mussel and chick Lim-1 antigens in SDS-PAGE is not due to their interactions with non-polymerized products of polyacrylamide gel (data not shown). The discrepancy between the theoretical (45 kDa) and apparent (65 kDa) MWs of the Lim-1 antigens could be due to post-translation modifications in the protein molecules. In X. laevis, three Lim-1 bands were detected (by SDS-PAGE followed by Western blot with anti-XLim-1 antibodies) in embryos injected with the full-length synthetic Xlini-1 mRNA. The fastest band of the "triplet" co-migrated with the protein product obtained from the same mRNA in a cell-free translation system, whereas other fractions were characterized by a lower electrophoretic migration. The latter suggested that a portion of the protein could be subject to post-translational modifications in the embryo (Kara- vanov et «/., 1996). Using the ScanProsite tool (Hofmann et til.. 1999), we found that the Xlim-1 sequence contains three potential sites for glycosylation. one of which resides in the C-terminus of the protein. By analogy with the XLim-l protein, we pro- posed that mussel and chicken Lim-1 antigens run more slowly than predicted in SDS-PAGE, probably due to a glycosylation of the corresponding proteins. To investigate this option, we performed two experiments. First. Lim-1 - containing fractions isolated from mussel pedal ganglia and chick embryo brain tissues (Fig. 3A, B) were electropho- resed on SDS-PAGE, blotted onto nylon membrane, and treated with the Immun-Blot kit for glycoprotein detection. Although a portion of each fraction displayed positive stain- ing, the zones corresponding to Lim-1 antigens were abso- lutely negative (see Fig. 3C, D). Next, the same fractions were treated with the Bio-Rad deglycosylation kit, which enzymatically cleaves all AMinked and most O-linked oli- gosaccharides from glycoproteins. Treated and untreated Lim-1 -containing fractions were subjected to SDS-PAGE followed by Western blot. All comparisons failed to identify any change in electrophoretic mobility of treated Lim-1 antigens (Fig. 3E, F). Thus, the difference between the predicted (45 kDa) and the apparent (65 kDa) MW of the Lim-1 antigens studied is apparently not due to the post- translational glycosylation of protein products. To begin characterizing effective size values, Lim-1 - containing fractions of mussel pedal ganglia and chick embryo brains were subjected to a subsequent ultratiltration using 100- and 50-kDa cut-off membranes (Fig. 3G). Since mussel proteins tend to aggregate during ultrafiltration (Mikhailov ct al., 1997), the SDS and urea were added to starting Lim-1 and reference protein (i.e., bovine albumin and chicken ovalbumin) solutions. Using SDS-PAGE fol- lowed by Western blot, we found that about 50% of the Lim-1 immunoreactivity, characteristic of Lim-1 -containing fractions, is retained by the 100-kDa cut-off membrane, whereas no more than 40% of the immunoreactivity is detected in the filtrate. The latter is completely retained by the 50-kDa cut-off membrane. Such retention and recovery patterns are more similar to those of bovine serum albumin (MW 67 kDa) than to those of chicken ovalbumin (MW 43 kDa). In particular, about 60% of the bovine albumin was retained by the 100-kDa cut-off membrane, whereas more than 70% of the chicken ovalbumin passed through the membrane (data not shown). This raises the possibility that the effective MW (size) of the Lim-1 antigens studied could be larger than the theoretical one (45 kDa). Taken together, the results indicate that the apparent MWs of the Lim-1 polypeptides, immunochemically de- tected in M. galloprovincialis pedal ganglia. S. r>itrpi;-* ••. F '< F &4 B 65- 60- -65 -60 Figure 5. Analysis of Lim- 1 antigen distribution in male gonads of A/vr;7ii\ galloprovincialis. Histological sections of ripe male gonad before (Al and after (B) complete spawning (spent gonad). Note that the gonad samples are characterized by the same patterns of follicle (F) morphogen- esis and gonadosomatic index values (in both samples, about of 90% of gonad volume was occupied by follicles) (scale bar — 100 jxm). (C) Ex- tracts prepared from complementary gonad (Lane I and Lane 3; arrows) of the same animals, as well as from somatic tissues (Lane 2) and sperm cells (Lane 4) of the other ripe gonad before spawning, were subjected to SDS-PAGE followed by Western blot with anti-XLim-l antibodies. Lane 1 — gonad biopsy containing somatic tissue, gonad ducts, fluids, and sperm; Lane 2 — gonad tube-free sample containing the mantle connective tissues only; Lane 3 — spent gonad; Lane 4 — mature sperm cells; Lane 5 — optic lobe of 16-day-old chick embryos (reference). 60 and 65 — apparent molecular weight values of Lim-1 antigens. kDa. 65- 40- -65 -40 Figure 6. Analysis of Lim-1 antigen distribution in female gonads of Mvtilus galloprovincialis using SDS-PAGE followed by Western blot with anti-XLim-l antibodies. Lane 1 — gonad biopsy containing somatic tissues, gonad ducts, fluids, and oocytes; Lane 2 — gonad collecting ducts; Lane 3 — immature oocytes obtained by biopsy of gonad follicles; Lane 4 — spawned mature eggs; Lane 5 — cerebellum of 16-day-old chick em- bryos (reference). 40 and 65 — apparent molecular weight values of Lim-1 antigens. kDa. appear to be an artifact of degradation caused by sample processing. Detection of the 40-kDa Lim-1 antigenic polypeptide in the mussel female gonad is perhaps not surprising, because the X. luevis ovary contains the 2.7-kb maternal Xlim-1 mRNA that is smaller than the larger zygotic 3.4-kb transcript found in the adult brain (Taira et ul.. 1992). It should be noted that in immature oocytes (obtained by biopsy of female gonad follicles), neither 65- kDa nor 40-kDa antigens were found (see Fig. 6. Lane 3). It is clear from these results that the 65-kDa Lim-1 form in the female is associated with somatic tissues of the gonad just like the Lim-1 variants detected in the male gonad. At the same time, in the female gonad the 65-kDa antigen is also characteristic of mature eggs. The 40-kDa antigen, despite our uncertainty regarding its precise nature, is spe- cific to the female germ line. It is likely that neither of the two antigens are expressed at early phases of oocyte differ- entiation, but are expressed and accumulated in eggs at terminal stages of their maturation. Therefore, the M. gal- loprovinciulis female gonad pattern obtained by Western immunoblot analysis for Lim-1 antigens may be interpreted as a compound profile of the 65-kDa variant, which origi- nates from both somatic tissues and eggs, and of the 40-kDa form, which seems to be specific to mature oocytes only. Dynamics of Lint- 1 -like immunoreactivity during earl\ development Observations on female gonads have led us to examine the patterns and timing of maternal expression of the 65- kDa and 40-kDa Lim-1 variants during early development of M. galloprovincinlis. Both antigen signals, already seen in unfertilized and fertilized eggs, persist in embryos during cleavage. At the beginning of the blastula stage, the inten- 38 M. TORRADO AND A. T. MIKIIAII.OV sity of immunostaining of both the 65-kDa and the 40-kDa antigens decreases. In stereoblastulae, instead of these two Lim-1 antigenic variants, only 45-kDa immunoreactivity was observed. The latter was first detected in 8-cell em- bryos, and its intensity reached a maximum in blastulae (Fig. 7A). Data from a variety of sources are consistent with the fact that zygotic transcription of //;;;-/ genes begins before gas- trulation at or very shortly after the midblastula transition (Taira et ai, 1992; Rebbert and Dawid, 1997; Curtiss and Heiling, 1998; Kawasaki ft ai, 1999). It may be speculated that in mussels, the 65- and 40-kDa signals are due to Lim-1 -related maternal molecules stored in the full-grown oocytes, whereas the 45-kDa protein reflects zygotic activ- ity of the gene. Such an interpretation may explain why multiple Lim-1 antigen variants have been detected in em- bryos of M. galloprovincialis at early blastula stages, but there is no definitive proof. It remains to be established how the compound profile of Lim-1 protein variants relates to maternal and zygotic gene expressions as well as to possible post-translation modifications of the primary gene product (Karavanov et a/., 1996) or to the so-called premature termination of translation processes involving both untrans- lated and coding regions of the zinc-tinger transcriptional factors (Klenova el ai. 1997). In sea urchin embryos, expression of the liin- /-related gene (Hpliiu-1) has been studied at the transcriptional level (Kawasaki et ai. 1999), so we decided to examine the Lim-1 antigen dynamics in the course of sea urchin early embryogenesis. Levels of Lim-1 immunoreactivity during S. purpuratus development are shown in Figure 7B. A relatively high-abundance signal of the Lim-1 antigen, al- 1 65- 40- -65 -45 -40 B 70- Figure 7. Patterns and timing of Lim-l antigen expression in the course of earK development of Mylilnx galloprovincialis and StrrwKylu- centrotus purpiirntii.-. The extracts from eggs and embryos were subjected to SDS-PAGE followed by Western blot with anti-XLim-l antibodies. (A) M. t'tilliipriivim'itili', l.ane I -unfcrtili/cd eggs; Lane 2 — fertilized eggs; Lane 3 — 4- and 8-cell embryos; Lane 4 — 16- and 32-cell embryos; Lane 5 — early blastulae; Lane fi — stereohlustulae. (B) S. />/II/>M/I//».V Lane I — unfertilized eggs; Lane 2 — fertili/.ed eggs; Lane 3 — 8- and 16-cell embryos; Lane 4 — unhatched hlastulae; Lane 5 — hatched blastulae; Lane 6 — prism larvae. 40, 45, 65, and 70— apparent molecular weight values of Lim-1 antigens, kDa. ready seen in eggs, persists in embryos to the blastula stage, decreases dramatically in unhatched blastulae, and increases again in late (post-hatched) blastulae. In more advanced embryos (i.e., at prism and pluteus stages), trace amounts of the Lim- 1 antigen were detected. Although the developmen- tal kinetics of the Lim-1 protein in S. purpuratus is quite similar to that of the Hplim-1 mRNA in H. pulcherrimus (Kawasaki et ai, 1999), the most interesting finding re- vealed by Western blot is that the Lim- 1 antigen is present at relatively high levels very early during development. Note that H. pulcherrimus fertilized eggs and cleavage embryos contain a trace amount of the Hplim-1 mRNA that becomes abundant only at the blastula stage just after hatch- ing (Kawasaki et ai. 1999). Thus, Lim-1 -like polypeptides, which share common epitopes with the C-terminus of the frog XLim- 1 protein, have been detected in both M. galloprovincialis and 5. purpuratus. In these species, characterized by very different modes of early embryogenesis, the similar developmental kinetics of the Lim-1 antigens has been demonstrated. Whether this likeness leads to similar developmental con- sequences remains to be elucidated. In sea urchin embryos, ectopic expression of the Hplim-1 inhibits endoderm and mesoderm differentiation, directing all embryonic cells to form oral ectoderm (Kawasaki et ai, 1999). It is widely accepted that maternally expressed gene products, stored in the egg, establish initial differences within the early embryo that, in turn, could contribute to further regionalization of the embryo body (Raff, 1996). The work described here particularly highlights the fact that in the marine inverte- brates studied the Lim-1 -like proteins maternally accumu- lated in the egg could persist after fertilization and be present in the early embryo long before zygotic expression of the genes is activated. As mentioned above, the present study represents the first step in the identification and characterization of Lim-1 -like proteins in marine bivalves. If it were accepted that the antibodies used recognize epitopes of the XLim-1 C-termi- nal sequence, then their cross-reactivity would appear to be specific for Lim- 1 -related proteins in many species (see Fig. I ). The corresponding immunochemical data obtained on rats (Karavanov et ai, 1996, 1998) and mice (Shimono and Behringer, 1999) confirm this assumption. Moreover, there is similarity between Lim-1 antigenic patterns observed in bivalves and those detected with the aid of the same anti- bodies in other species. This involves ( 1 ) the immunodetec- tion of the Lim-1 protein in both ganglia and somatic gonads (bivalves — this work; rats — Karavanov et ai. 1996), and (2) the nuclear localization of Lim-1 immuno- reactivity in tissue sections (bivalves — this work; frog and rats — Karavanov et ai, 1996; mice — Shimono and Behringer. 1999). In addition, the developmental dynamics of the Lim-1 antigen (this work) and Hplim-1 mRNA (Ka- wasaki et ul., 1999) in sea urchin embryos appears to be very similar. MUSSEL LIM-1 RELATED PROTEINS 39 On the basis of the HD sequence similarity, vertebrate Lim-1 proteins, as well as Lim-5 and Lim-6 factors, have been included in the so-called LIN- 11 class of LIM-HD proteins (Hubert and Westphal. 2000). Two lim- /-related genes have been recently identified in sea urchins (Ka- wasaki et nl., 1999) and fruit flies (Lilly et til.. 1999), and we suggest that they may be added to the same LIN- 11 group. To the best of our knowledge, this study is the first report that describes Lim-1 -like protein patterns in bivalve mollusc, sea urchin, and chick embryo tissues. Clearly, much remains to be learned about the corresponding factors involved, especially in bivalves. 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L. HARVEY Department of Biology. Acadia University; Wolfi'ille, Nova Scotia. Canada BOP 1X0 Abstract. The spionid Pygospio elegans reproduces both asexually and sexually. Using scanning electron and bright field microscopy, we examined morphogenesis following asexual reproduction to determine how "lost" body regions were regenerated after a worm spontaneously divided. Asexual reproduction occurred through transverse fission and divided the parent worm into 2 to 6 fragments (archi- tomy). All fragments retained their original anterior-poste- rior polarity. Regeneration in all fragments followed a spe- cific series of events: wound healing (day 1 ); extension of the blastema to generate lost body regions — specifically, the head and thorax for posterior fragments and the tail and pygidium for anterior fragments (days 2-3); segmentation (days 3-6); and differentiation of segment- or region-spe- cific structures (days 4-8). This pattern occurred regardless of where the original division took place. Subsequent growth occurred through addition of terminal setigers ante- rior to the pygidium followed by differentiation of tail setigers into abdominal setigers, leaving the tail region about 6 to 10 setigers in size. Division rates were compared in worms from three populations in Nova Scotia, Canada. Worms from two populations (Conrad's Beach, Starr's Point) divided more frequently (about 1.2 and 1.3 weeks between divisions, respectively) than worms from Bon Por- tage Island (3.5 weeks between divisions). Fragments con- taining the original head (original mouth intact, generally much larger fragment) had a higher survivorship than frag- ments containing the original tail. Introduction Asexual reproduction is the process of forming two or more offspring from one parent body without involving Received 23 July 1999; accepted 4 May 2000. * To whom correspondence should be addressed. E-mail: glenys. gibson@acadiau.ca gametes, or cells with a meiotically reduced chromosome number (Balinsky, 1975; Solomon et al. 1993). Although polychaetes reproduce sexually, asexual reproduction, through fission or budding, also occurs in many families including spionids, cirratulids, syllids, and sabellids (Barnes, 1980). In the spionid Pygospio elegans, asexual reproduc- tion occurs through transverse fission of the parent body into fragments, each of which will regenerate "lost" body regions (Rasmussen, 1953). Asexual reproduction has been widely reported in P. elegans. with most authors reporting its occurrence or testing environmental factors that may influence rates of division (Anger, 1984; Wilson. 1985). Despite the prevalence of asexual reproduction in this spe- cies, morphogenesis during post-fission regeneration has not been described. Pygospio elegans is a tubiculous polychaete that is com- mon on mud and sand flats and has a cosmopolitan, tem- perate distribution (Anger, 1984; Wilson, 1985). Adults grow to be 12 mm long, and feed on detritus (Wilson. 1985) and phytoplankton (Anger et al.. 1986). Rasmussen (1953) first described asexual reproduction in this species. He re- ported that both females and males could divide anywhere in the body and generally formed three to four fragments. Each fragment stayed in the original tube until regeneration was complete, about 8 d after division (20°C). Subse- quently, several authors reported asexual reproduction in P. elegans from populations from the eastern seaboard of the United States (Hobson and Green, 1968). Washington State (Wilson, 1985), and the Baltic Sea (Anger, 1984; Gud- mundsson, 1985). P. elegans also reproduces sexually, and it exhibits considerable flexibility in reproduction, as both planktotrophic and adelphophagic (a form of lecithotrophy) larval development have been reported in worms from dif- ferent populations (e.g., Thorson, 1946; Hannerz, 1956; Hobson and Green, 1968; Anger, 1984; Anger et al.. 1986; Schlotzer-Schrehardt. 1991; Morgan et al.. 1999). 41 42 G. D. GIBSON AND J. M. L. HARVEY Our objective is to describe morphogenesis during post- fission regeneration in P. elegans. We use bright field and scanning electron microscopy (SEM) to describe morpho- genesis after spontaneous divisions to determine ( 1 ) if an- terior and posterior body regions show similar patterns of regeneration; and (2) how subsequent growth occurs. We use the term regeneration to refer to the replacement of lost body regions (e.g., the head, thorax, tail) and growth as the addition of setigers to increase size, once the major body regions have formed. Also, we examine the rates of fission and fragment mortality in laboratory-maintained worms originating from three populations. No sexual reproduction was observed during the present study. Materials and Methods Adult specimens of Pygospio elegans were collected be- tween May and September from three sites: Bon Portage Island, Starr's Point, and Conrad's Beach, Nova Scotia. At each site, sediments containing worms were sieved (500-;u,m mesh), and tubes were brought into the laboratory. P. elegans was identified following Bromley and Bleakney (1984). Worms were placed in either 250-ml Pyrex crystal- lizing dishes or 150-ml custard dishes, with seawater and defaunated sand. Dishes containing stock cultures were submerged in larger trays of seawater and aerated. Cultures were maintained at 20°C on a photoperiod of 16 h light. Worms were fed a mixture of dehydrated, ground Entero- morplui and Tetramin fish food suspended in seawater twice weekly. Seawater was changed once a week. Stock cultures were sieved daily, and worms were iso- lated if they could be identified as having divided on that day (presence of a clean, smooth blastema) or showed signs that fission was about to occur (constriction of the body wall). Isolated worms were cultured separately to prevent movement of worms among culture dishes. Regeneration was observed with bright field and scanning electron mi- croscopy. Fragments, anesthetised in 1% MgCl:, were ex- amined and photographed daily from fission to the comple- tion of regeneration (8 d post-fission) using bright field techniques (n =- 25 worms). Fragments at each stage of regeneration (2 to 3 fragments per stage for both anterior and posterior fragments) were prepared for SEM by fixation in 2.5% glutaraldehyde followed by post-fixation in !%• osmium tetraoxide. both in 0.1 M cacodylate buffer and seawater (Gibson ft ai. 1999). After fixation, regenerates were dehydrated in an ascending series of ethanol. critical point dried with a Bio-Rad E3000 critical point drier, coated with gold-palladium with a Hummer II sputter coaler, and observed with a JEOL JSM-25S or JEOL T330A scanning electron microscope. Growth was followed in additional worms that had completed the regeneration process (;; = 1 2 worms). After the head and thorax or tail and pygidium had been regenerated, growth was examined by counting the number of setigers in each body region for a 17-d period. Intact worms that showed no signs of a recent asexual event were cultured in isolation to determine rates of regen- eration. Worms were observed from Bon Portage Island (/; =: 15). Starr's Point (;; = 15), and Conrad's Beach (;; = 10). Dishes were sieved weekly over a 6-week period. Original worm size was determined as the number of seti- gers at the beginning of the experimental period. Each week, the number of fragments per dish was noted, as well as the size of the fragments (number of setigers) and the degree of regeneration. Data were compared among the three study populations using one-way ANOVA in Stat- works 1.2 (Cricket Software). Where significant differences were noted, a post-hoc Scheffe comparison was also per- formed using SPSS 8.0 (SPSS Inc.). Results Adult morphology The overall body plan of Pygospio elegans is divided into four regions: the head, thorax, abdomen, and tail. The head is characterized by two ciliated palps, a prostomium with two or three pairs of eyes and paired nuchal organs (Fig. la). The thorax contains 10 to 12 abranchiate setigers, each with a single dorsal ciliary band, capillary notochaetae. and a lateral tuft of cilia. Neurochaetae are simple capillary on setigers 1 to 8 and hooded hooks on setigers 9 to 12 (Fig. Ib). The abdomen is 25 to 35 setigers in length. Each abdominal segment has paired branchiae and either a single (first few abdominal setigers) or double ciliary band, with two closely apposed bands of tufted cilia. Abdominal seti- gers also have capillary notochaetae, a lateral tuft of cilia, and neurochaetae that are hooded hooks (Fig. Ic). The tail contains 6 to 12 abranchiate setigers. Tail setigers have capillary notochaetae, neurochaetae that are hooded hooks, and a lateral tuft of cilia. There is a reduced ciliary band on the first few tail setigers only. The pygidium consists of four cirri, each with tufts of cilia on the inner surface (Fig. Id). Male P. flegans have a pair of branchiae on the second setiger (Fauchald, 1977) and dorsal organs on each setiger (Schlotzer-Schrehardt, 1991). Only tour males (/; = 200 worms) were observed during the present study. No mor- phological differences were noted (SEM) between worms from the three study populations. Morphogenesis following fission In all cases, fragments retained their original anterior- posterior polarity. Posterior fragments regenerated only the head and thorax, and anterior fragments regenerated only a new tail and pygidium. Subsequent growth involved elon- gation of the tail by the addition of terminal setigers. We based our description on division into two fragments, as that ASEXUAL REPRODUCTION IN PYGOSP1O 43 Figure 1. Scanning electron micrographs of adult Pygospio elegans. (A) Head and anterior thorax including the reduced first setiger. (B) Thorax, showing both anterior setigers with capillary neurochaetae and posterior setigers with neurochaetae that are hooded hooks. (C) Abdomen, characterized by branchiate setigers and a double dorsal ciliary band. (D) Tail and pygidium. b = branchus, c = cirrus, d = dorsal ciliary band, h = hooded hook, n = nuchal organ, cp = capillary chaetae, p = palp, pr = prostomium. t = tuft of cilia. Scale bar = 100 /am. was the most common form of fission observed in the present study. The maximum number of fragments observed per division was six, and regeneration in all fragments followed the same basic pattern. Table I provides a list of the structures that were observed during regeneration and the time at which the regenerated structures were first ob- served. On day 1, transverse fission began as a muscular con- striction in the body wall, usually in the abdominal region located at a point about two-thirds along the length of the worm. Constriction of the body wall continued until the gut separated and the two fragments, each anchored to the substrate v/a mucous, pulled apart. The anterior fragment consisted of the head, thorax, and most of the abdomen (about 25 or more pairs of branchiae), while the posterior fragment consisted of the tail, pygidium, and usually about five or fewer branchiate abdominal setigers. The epidermis healed quickly and formed a smooth surface the same day as division occurred (Fig. 2a). On day 2, the blastema of both the anterior and posterior fragments showed a small amount of new tissue with tiny, scattered tufts of cilia on an other- wise smooth epidermis (Fig. 2b). Regeneration on day 3 is characterized by rapid develop- ment of the blastema and formation of lost body regions (Table 1). As the anterior blastema increases in size, the regenerated head and thorax are readily distinguished (Fig. 2c). The head has palp buds, small dorsal depressions indi- cating formation of the nuchal organs, and a slightly rounded prostomium. The thorax shows the initial forma- tion of 3 to 6 setigers, visible with both SEM and bright field microscopy. The gut, visible with bright field microscopy, has extended into the thorax near the parental abdomen. The tail blastema is smaller than the anterior blastema and shows 2-3 slight wrinkles, suggesting early segmentation. Bright field microscopy also revealed the formation of segments and as well as the extension of the gut into the tail region. Cirri buds are also visible (Fig. 2d). On day 4, regeneration is characterized by further seg- mentation and early differentiation of region-specific struc- tures. The anterior blastema has 8 to 12 well-defined seti- 44 G. D. GIBSON AND J. M. L. HARVEY Table 1 Siimmaiy of morphogenesis during regeneration in Pygospio elegans Structure Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Anterior blastema + Head + Eyes (no. pairs) 1-2 2-3 2-3 Mouth * #* ** ** ** Nuchal organ # *# #* ** ** #* Palps # ** ** ** ** ** Prostomium * ** ** ** ** ** Thorax + Setigers (no.) 3-6 8-12 10-12 10-12 10-12 10-12 Notopodial lobe * ** ** ** Notochaetae * ** ** ** Ciliary tuft * ** ** ** Neuropodial lobe * ** ** #* Neuropodial capillary chaetae * ** #* Neuropodial hooded hooks * *# ** Posterior blastema + Tail + Setigers (no.) 2-3 3-6 3-6 5-7 5-7 5-7 Notopodial lobe * #* ** ** Notochaetae * ** ** ** Ciliary tuft * ** ** ** Neuropodial lobe * ** *# *# Neuropodial hooded hooks * #* ** Pygidium + Cirri * #* *# ** #* ** Ciliary tufts * *# ** *# ** + = body region recognizable, * = structure visible as a bud or rudiment, ** structure well developed but smaller than in parent. gers in the thorax (Fig. 2e, f), each with two dorsal tufts of cilia. The mouth and prostomium are visible on the regen- erating head, and the nuchal organs have small cilia. The gut has extended from the original abdomen to the head (Fig. 2f). Segments are further developed in the posterior blas- tema as well, with 3 to 6 well-defined setigers, each with paired lateral pits in the region of the presumptive noto- and neurochaetae. Differentiation of the pygidium involves ex- tension of the cirri and the appearance of small tufts of cilia on the inner surface (Fig. 2g). On the fifth day post-fission, the anterior blastema has regenerated the entire thoracic region and shows early dif- ferentiation of segment-specific structures. The number of thoracic setigers (10 to 12) that regenerated in the anterior blastema is similar in all specimens regardless of where fission occurred in the parent worm. The head has an elongate prostomium. The thoracic setigers develop neu- ropodial and notopodial buds, with a few small capillary notochaetae and a small tuft of cilia between the neuropo- dium and the notopoditim (Fig. 3a). The gut extends through the thorax, and the mouth is complete (Fig. 3b). On the same day, the 3 to 6 setigers of the posterior blastema also develop parapodial buds, a few notopodial capillary chaetae on setigers nearest the abdomen, and small lateral tufts of cilia. The pygidium has larger cirri with tufts of cilia (Fig. 3c). Regeneration on day 6 involves greater differentiation of segment-specific structures and addition of posterior seti- gers to restore the parental organization of the tail. The regenerated head has elongate, ciliated palps, a blunt pros- tomium (Fig. 3d), and 1 to 2 pairs of subdermal eyes (Fig. 3e). The thorax has dorsal bands of cilia on each setiger and well-developed notopodial chaetae throughout. Also in the thorax, the neuropodia exhibit short capillary chaetae on setigers 1-8 and a single hooded hook per setiger from setiger 8 posteriorly. The tail blastema has the 5 to 7 setigers characteristic of this region, with capillary notochaetae and notopodial hooded hooks that decrease in number from three on the proximal, earliest-forming setiger. to one on the later-developing terminal setiger (Fig. 3f). Lateral tufts of cilia are present on all setigers. The pygidium has cirri that are mature in size and have well-developed tufts of cilia. By day 7, the anterior blastema has regenerated a head and thorax that are identical to those of the parent worm except in setiger size and number of chaetae (Fig. 3g). Subsequent development in this region involves an increase in setiger size but not number. In the tail, setiger size and chaetae number also increases (Fig. 3h). By day 8, the ASEXUAL. REPRODUCTION IN PYGOSPIO 45 Figure 2. Early regeneration in Pygospio elegtinx following spontane- ous transverse fission. (A) Day 1 post-fission, anterior fragment. (B) Day 2. posterior fragment with blastema. (C) Day 3. anterior blastema, showing regenerated head, thorax, and evidence of early segmentation. (D) Day 3, posterior blastema, showing regenerated tail and buds of cirri. (E) Day 4, regenerated head and thorax with segments. The head has a regenerated mouth, palp buds, and a rounded prostomium. (F) Day 4, regenerated head and thorax showing extension of the gut into regenerated tissue. (G) Day 4, regenerated tail and pygidium. A-E. G are scanning electron micro- graphs. F is a bright field micrograph, bl = blastema, c = cirri bud. n = nuchal organ, p = palp bud. pr = prostomium. t = tuft of cilia. Arrows indicate setigers. Scale bar = 100 /j.m for A and E. 50 ^m for B-D and G. Figure 3. Completion of regeneration in Pygospio elegans. (A) Day 5 post-fission, anterior regenerate. (B) Day 5. anterior regenerate showing development of the gut. mouth, and setigers. (C ) Day 5. posterior blastema. (D) Day 6, anterior regenerate. (El Day 6, regenerated head with two pairs of eyes. (F) Day 6, regenerated tail and pygidium. (G) Day 7, anterior regenerate. (H) Day 7, posterior regenerate. A. C, D, F-H are scanning electron micrographs, B and E are bright field micrographs, c = cirrus, cp = capillary chaetae, d = dorsal ciliary band, e = eyes, g = gut. h = hooded hooks, m = mouth, ne = neuropodium, no = notopodium. t = tuft of cilia. Scale bar = 100 /j.m, anterior and posterior fragments for each day are shown at the same magnification. regenerated thorax and tail have an increased number of chaetae, and are similar to the pre-fission organization ex- cept for setiger size. Also on day 8, the gut extends through the new tail to the pygidium. In all fragments, regeneration produces only specific body regions, regardless of where fission occurred in the parent. Anterior fragments regenerate only the pygidium and the 6 to 12 abranchiate setigers of the tail. Posterior fragments regenerate only the thorax ( 10 to 12 setigers) and head. Mid-worm fragments concurrently regenerate both anterior and posterior regions as described above, with the result that these fragments regenerate the head and thorax and tail and pygidium but not the abdomen, regardless of the size of the original fragment (Fig. 4). After regeneration, worms grow to their pre-fission size by increasing setiger size and setiger number. During the growth phase, new setigers will only form immediately anterior to the py- gidium; new setigers do not form in the thorax or abdomen once regeneration is complete. Newly formed terminal se- tigers develop chaetae and parapodial lobes typical of the Figure 4. Scanning electron micrograph of a specimen of Pygospio elegcins regenerating from a mid-worm fragment, about 6 days after fission. The larger, parental setigers originated from the abdominal region and have branchiae. Both the anterior (head, thorax) and posterior (tail, pygidium) regions have regenerated. Scale bar = 100 ^im. 46 G. D. GIBSON AND J. M. L. HARVEY tail region (Fig. 5). As the tail region increases in setiger number, anterior tail setigers differentiate into abdominal setigers by forming dorsal ciliary bands and branchiae buds. About one-half of a setiger is added each day during the growth phase (n = 11 worms, mean ± SD 0.52 ± 0.23). Occasional anomalies were noted in this general pattern. For example, Figure 6 shows a P. elegtms that regenerated two thoracic regions and heads, both containing extensions of the gut. Such anomalies, although rare, reinforced the general pattern of regeneration described above. For both heads, the blastema gave rise to a specific number of seti- gers, and segmentation was followed by differentiation. Population comparison Frequency of spontaneous division and mortality were compared in P. cleganx originating from the three popula- tions. Specimens from all three populations were roughly the same size at the start of the experiment and ranged from 25 to 62 setigers overall (Table 2). Worms from Starr's Point and Conrad's Beach divided about once per week ( 1.3 and 1.2 weeks between divisions, respectively), while worms from Bon Portage Island divided less frequently (3.6 weeks between divisions; Table 2). although sample sizes were low for the Starr's Point and Bon Portage Island worms. Most worms divided into two fragments, but up to six fragments per division were observed. Conrad's Beach worms divided at the smallest size (average of 34 setigers), whereas those from Starr's Point and Bon Portage were, on average, larger before undergoing fission (42 and 45 seti- gers, respectively; Table 2). Mortality was also compared among regenerating frag- Figure 5. Growth in Pv.i.vi.v/i/n <7ri; 4 P — r(2.52l *--^' r 0.10 i, = 17 I! = 1 1 n = 26 Mortality (no. dead/no, fragments per type) % Mortality Anterior fragment 6/26 0/11 3/29 13% Middle fragment 1/2 1/2 2/24 14% Posterior fragment 14/14 7/8 11/18 80% Data are means, standard errors, and sample sizes (n) for traits indicated, in a comparison between laboratory-maintained worms from three populations. The final column gives results of a one-way ANOVA among populations and results of a post-hoc Scheffe comparison among populations, where significant differences were found. men increased in size only during the growth phase as tail setigers differentiated into abdominal setigers by develop- ing branchiae and dorsal cilia. In all fragments, regeneration produced only specific body regions, regardless of where fission occurred in the parent. For example, one worm divided in the original thoracic region and, after fission, had only a head and nine thoracic setigers. This individual regenerated only a tail and pygidium; abdominal setigers redifferentiated from tail setigers as growth proceeded. Many worms were observed to undergo a second asexual event before growth was complete (often 8 to 10 days after fission), and several individuals divided almost immediately after fission (days 1-3) as evidenced by the presence of fragments at different stages of regeneration in a single culture. Spionids, in general, are not as well known for their ability to regenerate as are some other polychaetes, such as sabellids. Asexual reproduction by regeneration is common, however, in the spionids Polydora tetrabranchia (Camp- bell, 1955) and throughout the genus Polydorella (Rada- shevsky, 1996). In Polydorella, unlike P. elegans, new individuals are formed by paratomy, resulting in a chain of clones. Otherwise, morphogenesis during an asexual event in Polydorella dawydoffi is similar to architomy in P. el- egans: the new individual forms through development of a growth zone (similar to the blastema reported here), elon- gation to form specific anterior body regions (i.e., head and thorax), segmentation resulting in a specific number of thoracic setigers, and differentiation to form region-specific structures such as the chaetae, eyes, and branchiae. Once the new head has formed in Polvdorella. transverse fission occurs and the two daughter worms separate (Radashevsky, 1996). Although asexual reproduction does not appear to be widespread in spionids, regeneration as a response to tissue loss (e.g., palps or the tail) occurs frequently in Polydora cornuta (Zajac, 1985, 1995), Boccardia proboscidea (Gib- son, pers. obs.), and Streblospio benedicti (Harvey, pers. obs.). Further work may reveal whether the restricted po- tential for asexual reproduction within the spionids could have arisen by decoupling regeneration and reproduction, as has been suggested in the oligochaete Paranais litoralis (Bely, 1999). Although the mechanisms leading to the restoration and differentiation of body regions are not known, it seems likely that the regulatory genes important in embryogenesis may play a role. For example, distal-less is known to be important in the development of parapodia in polychaete embryos (Panganiban et ai. 1997) and possibly is reacti- vated during regeneration, although this remains to be dem- onstrated. In an asexual race of Dugesia tigrina (platy hel- minth), lost body regions are defined during regeneration by Hox genes that have sequences very similar to those found in annelids (Bayascas etal., 1998). Interestingly, in this race of D. tigrina. Hox genes were found to be permanently expressed in adults, perhaps contributing to the impressive regenerative capabilities of this species (Bayascas et ai., 1998). There were no differences among populations in original size and number of fragments per asexual event in Pygospio elegans, although time between divisions and size at divi- sion did vary. Rasmussen found that rates of division in- creased at low temperatures, and Wilson ( 1985) found that 48 G. D. GIBSON AND J. M. L. HARVEY division rates increased at low worm densities. Anger (1984) observed that the number of individuals (in an asex- ual population) increased at low salinity and temperature. In the present study, fission was observed in isolated worms that were maintained under constant conditions (34 ppt, 20°C and with an abundance of food); therefore, these conditions were unlikely to contribute to the differences in division we observed among laboratory cultures. Posterior fragments (original tail) had a higher mortality than did anterior fragments (original head). Posterior frag- ments were much smaller than anterior fragments; had few branchiate, abdominal setigers (5-6 on average, vs. 25 for anterior fragments); and lacked a mouth until day 5 post- fission and therefore were unable to feed immediately after division. Differences in mortality could be due to fragment size (e.g., energy reserves or number of neoblasts available) or lack of a mouth. However, the few mid-worm fragments observed during the present study had a high survivorship, despite their small size. Despite the high mortality of pos- terior fragments, extensive laboratory culturing by others indicates a net population growth through asexual reproduc- tion (Anger, 1984; Wilson. 1985). Although P. elegans is known to reproduce sexually (Thorson, 1946; Hannerz, 1956; Anger, 1984; Anger et al., 1986; Morgan et al., 1999), only asexual reproduction was noted in the worms observed in the present study (more than 200 in total). This suggests that asexual reproduction is the dominant reproductive mode in these populations during the study period (May-September). Anger (1984) reported a population in the Kiel Bight, Baltic Sea, that reproduces exclusively through asexual reproduction; two additional populations were predominantly sexual, although occa- sional fragmentation was noted. Anger (1984) attempted to induce specimens of P. elegans from these three populations to switch between sexual and asexual reproduction by vary- ing culture conditions (temperature and salinity) but found that worms retained the reproductive mode of their original population, leading her to suggest the potential for cryptic species. Other investigators have reported seasonal differ- ences in reproductive mode within a single population, with asexual reproduction being dominant in the spring or sum- mer, and sexual reproduction prevalent in the fall or winter (Rasmussen, 1973; Hobson and Green, 1968; Wilson, 1985). Rasmussen (1953) also noted that fission could be induced in P. elegans by temperatures of 4°-5°C. In addition to asexual reproduction, P. elegans exhibits considerable flexibility in sexual reproduction, including both planktotrophic and adelphophagic larval development (e.g., Thorson, 1946; Hannerz, 1956; Hobson and Green, 1968; Anger, 1984; Anger et al.. 1986). This suggests the potential for reports of P. elegans to include cryptic species, but Morgan et til. (1999) clearly demonstrated that poecil- ogony does exist in this species, based on a molecular (allozyme) comparison of populations with planktotrophic or adelphophagic development. Poecilogony in P. elegans is, in several regards, similar to that of the spionids Boc- cardia proboscidea (Blake and Kudenov, 1981; Gibson, 1997) and Polydora connita (Mac Kay and Gibson, 1999), which also reproduce by means of planktotrophic and adel- phophagic larval development. Such flexibility makes P. elegans a valuable model for tests of the ecological conse- quences of life-history variability, as well as for understand- ing the developmental mechanisms underlying a change in development mode. Acknowledgments We thank H. Smith for general and field assistance, and H. Taylor for assistance with the SEM. P. Allen-Wojtas and S. Carbyn of the Electron Microscopy and Imaging Labo- ratory, Agriculture and Agri-Food Canada, Kentville, Nova Scotia, provided facilities and assistance with SEM. The manuscript has benefited from comments from M. Gibson and I. Paterson. This research was funded by an NSERC grant to G. Gibson and Nova Scotia Summer Career Place- ment to J. Harvey. 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Sublethal predation on Polydora conuita (Poly- chaeta: Spionidae): patterns of tissue loss in a field population, predator functional response and potential demographic impacts. Mar. Biol. 123: 531-541. Reference: Biol. Bull. 199: 50-5S. (August 2(1 Factors Influencing Spawning and Pairing in the Scale Worm Harmothoe imbricata (Annelida: Polychaeta) G. J. WATSON1-*, F. M. LANGFORD2. S. M. GAUDRON2, AND M. G. BENTLEY2 1 Institute of Marine Sciences, Ferry Road, Eastney, University of Portsmouth, Portsmouth, P04 9LY, UK; and " Department of Marine Sciences and Coastal Management , Faculty of Agriculture and Biological Sciences. Ridlev Building. University of Newcastle, Newcastle-upon-Tyne, NE1 7RU, UK Abstract. Endocrine and environmental factors control reproduction of the polynoid scale worm Harmothoe imbri- cata. We confirmed that the rate of vitellogenesis was greater in winter specimens transferred from ambient re- gimes of photoperiod and temperature to a light:dark (LD) photoperiod of 16:8 at 10°C and showed that the number of females spawning was significantly greater than for those transferred to LD8:16 at I()°C. The endocrine mediation of this response was investigated using prostomium implanta- tions. Significantly more LD8:16 females implanted with prostomia from LD16:8 conditioned females spawned than LD8:16 females implanted with LD8:16 prostomia. Fe- males without prostomia failed to spawn. LD16:8 exposure may increase levels of a possible "spawning hormone" in the prostomium. Spawning proceeded in these LD16:8 fe- males and allowed spawning to occur in LD8:16 females implanted with LD16:8 prostomia. In LD8:16 prostomia. liters of the spawning hormone reached the threshold in significantly fewer individuals, so that significantly fewer females implanted with LD8:16 prostomia spawned. Using Y-maze choice chambers, pair formation was shown to be under pheromonal control, with males being attracted to mature females but not to females carrying fertilized oocytes or to LD8:I6 conditioned females. Pro- duction of this attraction pheromone can, therefore, be ma- nipulated through photoperiodic control, suggesting a link between oogenesis. spawning, and pheromone production. Received 23 November 19W; accepted 31 May 200(1. * To whom correspondence should he addressed. E-mail: gordon. watson@port.ac.uk Introduction The polynoid scale worm Harmothoe imbricata is a com- mon inhabitant of temperate intertidal rocky shores, where it lives under rocks and small stones. It is an active carni- vore that preys upon other small invertebrates. The repro- ductive biology of this species is relatively well understood. It is a dioecious, iteroparous species with an annual cycle of reproduction (Daly. 1972, 1974; Garwood. 1980). Females develop two cohorts of oocytes: the first is grown slowly during the winter months to be spawned in March; the second is produced rapidly and spawned about 30 days after the first. During the breeding season individuals pair, a behavior in which a male lies closely along the dorsal surface of the female (Daly. 1972). Cohorts of oocytes, once spawned, are fertilized and earned under the female's dorsal elytra (scales) during embryogenesis. Embryos are released as trochophores after about 16 days. Oogenesis is initiated in late September without any specific environmental input. During the autumn, the first cohort grows under conditions of decreasing environmental temperature, and growth is accelerated by exposure to low temperatures (Garwood, 1980). The stabilization of oocyte development also requires exposure to a light-dark (LD) cycle with a photophase less than 13 h for between 42 and 55 days during the late autumn period. If these photoperiods are not experienced, oocyte development is aborted (Clark. 1988). Once the winter solstice has passed, natural popula- tions respond to a second photoperiodic input. An exposure to LD cycles with a photophase above 10 or 11 h increases oocyte growth rate and may synchronize oogenesis and spawning among individuals (Garwood and Olive, 1982; Clark. 1988). Although environmental manipulation affects both oo- genesis and spawning, the endocrine role in mediating these 50 REPRODUCTION IN HAKMOTHOE IMBRICATA 51 influences has received comparatively little attention. Bent- ley et til. ( 1994) and Lawrence ( 1996) confirmed the pres- ence of a gonadotrophic hormone from the prostomium that probably mediates oogenesis, and it seems likely that the hormone is secreted only under the appropriate regimes of photoperiod and temperature. Nevertheless, the role of the endocrine system in spawning is unknown in this species. In a number of other polychaete species, the implantation or injection of prostomia into individuals has been used to investigate their endocrine control of reproduction and spawning (Golding. 1983: Bentley et al., 1984; Pacey and Bentley, 1992; Bentley et al., 1994; Watson et al., 2000). In the work reported here, transplantation experiments estab- lish the role of the endocrine system in mediating photope- riodic influences, and environmental manipulation is used to investigate the effect of photoperiod on spawning and oo- genesis of the first cohort of oocytes. Pherornones coordinate and control reproduction in a number of marine invertebrates, including species of the opisthobranch mollusc Aplysia (Zeeck et al.. 1990, 1996; Painter et al., 1998). by inducing spawning and by attracting other individuals towards spawning animals. In this paper we provide evidence of an attraction pheromone produced by female Harmothoe imbricata to attract males for mating, and we examine the influence of environmental manipula- tion on its production. The results presented here provide the first link between environmental conditioning, endocrine activity, and phero- monal control of attraction for pairing and spawning in a polychaete species. Harmothoe imbricata is thus likely to become a model species for the investigation of environ- mental-endocrine-pheromone interactions. Materials, Methods, and Results Photoperiodic control of ooc\te growth and spawning mediated b\ the endocrine svstem Collection anil maintenance of animals. Specimens of Harmothoe imbricata were collected from the intertidal zone of three rocky shores in E. Scotland, UK: St Andrews (56°20'N, 2°47'W), Kingsbarns (56°18'N, 2°38'W) and Fife Ness (56°16'N, 2°35'W). Individuals were maintained in glass crystallizing dishes containing 100 ml of TFSW (twice-filtered seawater, pore size 0.34 jam) and provided with a cleaned Patella vulgata shell for shelter. All worms were hand-fed pieces of Mytilus edulis muscle once per week, after which the water was changed. All individuals were collected in December and January and kept at 10°C and ambient photoperiods until the experiments commenced on 9 February 1999. To assess their state of maturity, individuals were narco- tized in 5% ethanol in seawater and a small incision was then made in the lower edge of the 16th setiger. For micro- scopic analysis, a small sample of coelomic fluid and blood vessel was removed using a 20-;u.l glass micropipette. The diameters of about 30 oocytes from five randomly chosen females from each treatment group were measured using a compound microscope. Sperm activity was assessed after dilution. Experimental protocol. On 9 February 1999, half of the females were transferred to conditions of 16 h light and 8 h dark (LD16:8) at 10°C. The individuals that remained in LD8:16 conditions were termed LD8:16 controls while those transferred to the LD16:8 were termed LD16:8 con- trols. Oocyte diameters were measured from five randomly selected individuals from the two groups on this day and subsequently once per week for 3 weeks. Prostomium (PM) transplants were performed on 26 Feb- ruary 1999, 2 weeks after photoperiod manipulation had commenced. Individuals from the LD16:8 control and the LD8:16 control were narcotized as described above to pro- vide prostomia. They were termed LD16:8 donors and LD8:16 donors, respectively. The PM was accessed by removing the first two scales and then excised with iridec- tomy scissors. Once removed, the PM was trimmed of excess flesh and tentacles and then placed in seawater on ice until implanted. Both sets of PM donors were then returned to seawater to recover from the narcotization. Individuals designated to received the implanted PM were also narcotized, and a PM was inserted through an incision in the 2()th setiger. This was far enough from the head to prevent any interference with movement. Twenty- five LD16:8 control females were used as PM donors and their PMs were implanted into females from the LD8:16 control group. They were termed LD8:16PM(LDI68) females (the superscript notation refers to the conditions that the female, from which the prostomium used for implantation was removed, was exposed to). As a control, eight LD8: 16 control females were also implanted with LD8:16 control PMs, and these were termed LD8:16PM(LD8:I6'. After im- plantation, all PM recipients and PM donors were returned to their respective photoperiod treatments. All individuals were examined daily for evidence of spawning (the pres- ence of oocytes under the elytra). The diameters of about 30 oocytes were measured from individuals that had spawned. Statistical aitalvses. The nature of the oocyte diameter data (unbalanced nesting and sample sizes) precluded anal- ysis using a multiple ANOVA for all data. Instead, mean oocyte diameters for each female were obtained, and these data were then assessed using one-way and two-way ANOVAS. Subsequent pairwise comparisons were per- formed using Tukey tests. The numbers of spawning indi- v iduals were analyzed for independence by using an R X C contingency table and the chi-square statistic f^2); pairwise comparisons were performed subsequently using a modified Tukey test. Results: photoperiodic control of oocyte growth. Mean oocyte diameters of LD8:16 and LD16:8 controls and PM 52 G. J. WATSON ET AL. recipients (LD8:16PM|LDI ' and LDS:16PM(L ' are shown in Figure 1. Analysis, using a two-way ANOVA. of the mean oocyte diameters of the LD8:16 and LD16:8 controls for weeks 1 to 3 shows that significant differences were present between weeks (F = 28.64, P < 0.001 ), but not between treatments. There were also significant inter- action effects (F == 7.407. 0.05 > P > 0.001). Pairwise comparisons using a Tukey test confirm that the mean diameters of LD16:8 and LD8:16 controls were not signif- icantly different from each other in week 1. By week 2, LD16:8 control diameters increased significantly when compared to week 1, whereas LD8:16 control diameters did not increase. By week 3, LD8:16 control diameters in- creased significantly when compared to week 2. At the same time, all mean oocyte diameters of all treatments were not significantly different from each other when analyzed with a one-way ANOVA. (F = 0.1 1. P > 0.05). Analysis of all treatments in week 4 shows that there were significant differences between the treatments (F = 9.56, 0.05 > P > 0.001 ). However, pairwise comparisons reveal that only the LD16:8 control mean diameter was significantly greater than the LD8:16 control and LD8:16PM(LD8 lftl. No other pairwise comparisons were significantly different. Results: photoperiodic control of female spawning medi- ated by the endocrine system. During the experimental period, all individuals in all treatments were monitored daily for spawning; the cumulative percentage of individual spawning females is shown in Figure 2. Over the duration of the total experimental period (9 February- 14 March), 96% (24 individuals) of the LD16:8 controls spawned, with the majority (16 individuals) spawning on 23 February and another 8 spawning between 24 and 28 February. In com- parison, only 33% of the LD8:16 control females spawned during the experimental period, one on 19 February, two on 23 February, and another on 25 February. During the experimental period for prostomial manipula- tion (26 February-14 March), 80% of the LD8:16PM(LD168) treatment group spawned, with 10 individuals spawning on 120- 10- u o o 90 - 80 n=5 5=? n=5 LD16:8 control LD8:16 control LD8:U/N"""""' LD8:16 PM (U>X- Ih) n=5 zL?"3 />vnt 0=3 9-Feb(Wcckl) 16-Feb (Week 2) 23-Feb (Week 3) Week 2-Mar (Week 4) Figure 1. Mean weekly oocyte diameter ( ±95% confidence limits) of female Hannothoe imbricata exposed to various treatments at 10"C: LD16:8 control, females maintained in long-day photoperiod (LD16:8); LD8:16 control, females maintained in short-day photoperiod (LD8:16); LD8:I6PM|LD">I", females maintained in I '• 16 each implanted with one prostomium from un LDI6:H control female; LD8:16PM|LDS "". females maintained in LD8:16. each implanted with one prostomium from an LD8:16 control female. All individuals were collected in December and maintained in ambient photoperiod at 10°C prior to the start of the experiment on 9 Februar> I'l'W (week 1 ). All transplantations were performed on 26 February 1999 (week 3); n = number of females sampled, 30 oocytes were counted per female. REPRODUCTION IN HARMOTHOE IMBKICATA 53 00 c 'S o eo u 1 00 -, 80- 60- 40- 20- 0- •— LD16:8 control o — LD8: 16 control ._LD8: ,„""">"«' ._LD8:16™ <'•""« o — LD8: 16 donor x — LD16:8 donor •B-B(n=S) u, 6 c3 a rt 2! 2 S Date Figure 2. Cumulative percentage of spawning female Harmothoe imhricata after exposure to various treatments at 10°C. LD16:8 control, females maintained in long-day photoperiod (LD16:8); LD8:16 control, females maintained in short-day photoperiod (LD8:16): LD8:16PM|LD168'. females maintained in LD8:lft and each implanted with one prostomium from an LD16:8 control female; LD8:16PM|LD8 lf", females maintained in LD8:16 and each implanted with one prostomium from an LD8:16 control female; LD8:16 donor, LD8:16 control females used as prostomial donors; LD16;8 donor. LD16:8 control females used as prostomial donors. All individuals were collected in December and maintained in ambient photoperiod prior to the start of the experiment on 9 February 1999, start of photoperiodic conditioning. All transplantations were performed on 26 February 1999. n is the number of females in each treatment group. 27 February and a further 10 spawning between 1-8 March. In contrast, only 37.5% (3 individuals) of the LD8: 16PM|LDS l6' treatment group spawned, and none of the LD8:16 or LD16:8 donors spawned. Statistical analysis using an R X C test of independence and the \2 statistic reveals highly significant differences between the total proportion of spawning to nonspawning females between all treatments (^2 = 68.409, P < 0.001 ). Statistical analysis of pairwise comparisons, using a modi- fied Tukey test, shows that all pairwise comparisons were significantly different from each other, except the following treatments: LD8:16 control compared with LD8:16PM" Ds 16'. and LD8:16 donor compared with LD16:8 donor. Pheromone influence on pairing behavior Y-maze behavioral bioassays. Six Y-mazes. the dimen- sions of which are shown in Figure 3A, were constructed from 5-mm-thick clear acrylic plastic. Each was sealed with silicon sealant and allowed to cure by soaking in seawater for several days prior to use. Each arm had a removable acrylic partition with 0.4-mm-diameter holes drilled through to allow pheromonal diffusion. All experiments were performed at 10°C in ambient illumination. Animals. Animals were collected from the Fife Ness site during March and April and maintained as described above. Individuals collected in March and April (classed as "old" and "new," respectively) were sexed, and their state of maturity assessed as described above. Of the old individuals collected, 10 females were carrying fertilized oocytes and 7 females still had fully grown oocytes in the coelomic cavity. Of the males collected, 18 had active sperm in their coelo- mic cavity. Of the new individuals collected, 10 females had not spawned and 10 males had sperm in their coelomic fluid. Experimental protocol. The basic experimental protocol is summarized in Figure 3B. Before each set of experiments, each maze was washed in fresh water before air-drying. During a run of experiments each maze was washed in TFSW between tests. After each maze was filled, a potential stimulus animal was placed in one arm of the maze behind the partition. After 1 min, a test male was placed in the base of the maze. To minimize handling, a glass tube was used in positioning the test animals. Each test was run for 30 min (except the first directional bias test with old males, see below), after which the position of the test male was noted. At the end of the 30 min, a response was considered positive if the test male was in the arm with the stimulus animal. 54 G. J. WATSON ET AL. Wash maze in freshwater and allow to dry Fill maze to 5cm depth I Place stimulus animal in left or right arm behind gate (position X) Leave for 1 minute I Place test male at start position (*) of maze i Allow 30 minutes before recording position onses: ( + ) Animal is in arm with stimulus (-) Animal is in opposite arm to stimulus (NC) No choice, in bottom on "Y" B Figure 3. (A) Schematic diagram of Y-maze for the investigation of pheromonal attraction of male Harmothoe imbricata to various stimuli. Dashed lines represent removable partitions. Depth of seawater in Y-maze: 5 cm. (B) Summary flow diagram of the experimental protocol. negative if it was in the opposite arm, and a no choice if it was in the base of the Y. During an experimental run, all potential stimulus individuals were alternated between each arm in consecutive experiments and cross-tested with all test males. Tests performed. All tests were performed within 2 weeks of collection. To assess for any directional bias ("handed- ness") and for chance levels of attraction in the maze, two sets of experiments were performed in which males were placed in the Y-maze with no stimulus animal. In the first experiment, nine old males were each run three times in the Y-maze and the position of the animal was recorded after 10 min. From observations of the males, it was suspected that this time period was not sufficient for the test animals to complete exploratory behavior before settling. A further six old males were tested without a stimulus six times each, and their position was recorded after 30 min. All subsequent tests were run for 30 min. Old males were tested against each of the following: old mature females (two sets) and old females carrying fertilized oocytes. New males were tested against each of the following: new mature females, new females maintained in short days, and old males. All statis- tical significance was assessed using \2 analyses. Results. The first set of experiments was performed with old (collected in March) males and females (Fig. 4). To assess for any directional bias and for chance levels of attraction, the positions of the males were recorded 10 min after being introduced into the maze. Fifty-three percent of Figure 4. Percentage response of old (collected March 1999) male Hunmiihoe imhricata to various stimuli in a Y-maze run at IO°C. SW. no stimuli added (number of runs = 36 [6 males x 6 tests]): Mature Fem. I, mature old females (number of runs = 27 [9 males X 3 females]); Mature Fem. II, second group of mature old females (number of runs = 63 [7 males X 9 females]); Pert. Females, females carrying fertili/ed oocues under their elytra (number of runs = 27 [9 males x 3 females]). The position of each male was recorded 30 min after introduction to the maze. Responses were classed as positive if the male was in the arm containing the stimulus (or designated as stimulus if seawater only), negative if in the arm with no stimulus, or no choice if it moved to neither arm. REPRODUCTION IN HARMOTHOE IMBRICATA 55 these males moved to the arbitrarily labeled positive arm and 47% moved to the negative arm. However, this exper- iment was omitted from the graph and from statistical analysis beeause the males had not completed their explor- atory behavior within the 10-min time frame. The experi- ment was, therefore, repeated with a further six old males whose positions were recorded 30 min after introduction. In the 36 tests performed, 28% of the males moved to the arbitrarily positive arm and 19% to the negative arm, while 53% made no choice. This result was not significantly different form an expected value of 33.3% moving to each arm or making no choice (^2(2) = 2.60. P > 0.05). These nine old males were then tested against three old mature females, and a significantly higher level of attraction (X2(2) = 23.07, P < 0.001) was observed: 81% of the males moved to the positive arm and the other 1 9% moved to the negative arm (without the females). To confirm this attrac- tion response, a further test was performed with 1 1 males and 7 old mature females. Sixty-one percent of the males moved to the positive arm. 13% moved to the negative arm, and 26% made no choice. This level of attraction was also significantly higher than shown for the seawater 30-min control (x2(2) = 10.58. 0.05 > P > 0.001). An additional experimental run was performed using the nine old males and three old females that had spawned in the field and were carrying fertilized oocytes under their elytra. In the 27 tests performed, 48% of the males moved to the positive stimulus arm, 37% moved to the negative arm. and 15% made no choice. Statistical analysis of this data shows that this was a significantly higher level of attraction than shown for the seawater control (^2(2) = 9.69. 0.05 > P > 0.001). However, if the no choice and negative results are combined and compared statistically with the positive stimulus, there was no significant differ- ence between the treatments (^(1) = 2.74, P > 0.05), confirming that the differences were due to the decrease in no choices and not to an increase in positive results. Statis- tical analysis also shows that there was a significantly lower level of attraction for fertilized females than for both mature female I (^2(2) = 6.87. 0.05 > P > 0.001) and mature female II experiments (\2(2) = 7.97. 0.05 > P > 0.001). The second set of experiments was performed with new animals collected in April (Fig. 5). To assess again for directional bias, six new males were tested with no stimulus, and their position was recorded after 30 min. Thirty-three percent and 36% of the males moved to the arbitrary posi- tive and negative arms respectively, with 31% making no choice. This was not significantly different from an ex- pected value of 33.3% moving to each arm or making no choice ()C(2) = 0.593, P > 0.05). These new males were then tested against six new mature females. A significantly higher level of attraction was ob- served when compared to the seawater test, with 61% of the males moving to the positive arm, 22% to the negative arm. LDIS lf> K'nuiks Stimulus Figure 5. Percentage response of new (collected April 1999) male Harmothoe imhricata to various stimuli in a Y-maze run at 10°C. SW. no stimuli added (number of runs = 36 [6 males x 6 tests]): Mature Females, mature new females (number of runs = 36 [6 males x 6 females]); Males: new males tested against themselves (number of runs = 36 [6 males x 6 males]): LD8:16 Females, females maintained in short days (LD8:16) for 3 months (number of runs = 36 [6 males x 6 females]). The position of each male was recorded 30 min after introduction into the maze. Responses were classed as positive if the male was in the arm containing the stimulus (or designated as stimulus if seawater only), negative if in the arm with no stimulus, or no choice if it moved to neither arm. and 17% making no choice (/(2) = 12.95. 0.05 > P '- 0.001). These six new males were also tested against themselves. Twenty-five percent moved to the positive arm. 33% moved to the negative arm. and 42% made no choice. Statistical analysis of these data shows that this was not a significantly different level of attraction when compared to the seawater control (,\2(2) = 0.506. P > 0.05), and males were signif- icantly less attractive than mature females <^2(2) = 12.61, 0.05 > P > 0.01). These males were also tested against six mature females that had been maintained in LD8:16 cycles for 3 months. Twenty-five percent moved to the positive arm, 50% moved to the negative arm, and 25% made no choice. Levels of attraction for this test also did not differ significantly from seawater (^2(2) = 0.7, P > 0.05). and LD8:16 females were significantly less attractive than mature females (^2(2) = 10.5. 0.05 > P > 0.001). Discussion Photoperiodic control of oocylc u The rate of oogenesis of the first oocyte cohort in Hur- motlioe imbricata can be altered by manipulation of tem- perature and photoperiod (Garwood. 1980; Garwood and Olive. 1982; Clark. 1988). A period of exposure of 42-55 days of less than 13 h (winter conditions) photophase is 56 G. J. WATSON ET AL required to prevent the first cohort of oocytes from being aborted. In experiments in this study, females had been maintained in ambient photoperiods prior to the experi- ments commencing in February. They were, therefore, not exposed to either LD16:8 or LD8:16 conditions until after the critical minimum number of days had been reached, thus preventing resorbtion. In the field, once females have been exposed to this critical period of LD8:16 cycles, an increasing ambient photophase (after the winter solstice) allows oogenesis to proceed normally. Above a critical photoperiod of 10-1 1 h photophase, oogenesis is accelerated; however, an increased number of winter condition cycles (between 55 and 73) is required for this response to be exhibited (Garwood, 1980; Garwood and Olive. 1982; Clark, 1988). The results pre- sented in Figure 1 confirm that an increase in photoperiod (LD16:8 conditions) accelerates oogenesis. The results also show that the response to LD16:8 conditions was rapid, with effects occurring within I week from exposure. However, oocyte growth continued under LD8: 16 conditions, as by 23 February both LD16:8 and LD8:16 conditions had oocytes that were not significantly different from each other. Incubating prostomium homogenate with oocytes in vitro significantly increases their uptake of radiolabeled amino acid and their subsequent protein synthesis (Bentley et nl., 1994; Lawrence, 1996). These authors suggested the pres- ence in the prostomium of a gonadotrophic hormone that promotes and controls oogenesis. The manipulation of pho- toperiod and temperature may, therefore, be acting directly on the levels of this hormone and it is this hormone level that subsequently mediates oogenesis. Ambient conditions (increasing photophases) in January and February may sus- tain oogenesis by increasing the circulating tilers of this substance. The exposure of females to LD16:8 conditions may have triggered a burst of secretion of the hormone (above the levels normally experienced in February), induc- ing an increase in mean oocyte diameter that occurred in week 2. Females exposed to continued LD8:16 conditions had no burst of secretion; instead, oogenesis continued at a constant rate, resulting in the delayed increase (week 3) in mean oocyte diameter. By week 3, oocytes were fully grown — approximately 120 jam (Daly, 1972); data from Figure 1 show that, in these experiments, oogenesis was complete by 23 February. Data also show that implantation of prostomia had no effect on oocyte diameter and did not induce oocyte degeneration. The diameters of spawned oocytes from females implanted with LD8:16 or LD16:8 prostomia were not significantly different from each other, from the LD8:16 controls in weeks 3 and 4. or from the LD16:8 control in week 3. In week 4, the mean oocyte diameters of LD 16:8 controls were significantly higher than those in any other treatment, al- though this is more likely to be due to the low numbers of females sampled than to any effect of the treatment. Photoperiodic control of spawning mediated b\ the endocrine system Early transition through the critical photoperiod can ad- vance the time of spawning; long days or photoperiods with greater than 1 1 h photophase can cause the first cohort of oocytes to be spawned about 1 month earlier than the natural date (Garwood and Olive, 1982). In this study, females carrying fertilized oocytes were collected from the field on 3 March 1999. We calculated that their natural spawning data was late February to early March. From the results presented in Figure 2, exposure to a LD16:8 photo- period did not induce notably earlier spawning dates when compared to a LD8:16 photoperiod, and neither was differ- ent from the natural date. Instead, when compared to LD16:8 exposure, LD8:16 exposure actually prevented spawning in the majority of females. Ninety-six percent of the LD16:8 females spawned, as opposed to only 33% of the LD8:16 females. We conclude that the presence of the prostomium is required for spawning to occur in H. imbricata, as none of the females that were used as LD16:8 or LD8:16 donors spawned. Examination of the oocytes form these donors also showed that they had not increased in diameter after prostomium removal and had begun to degenerate (data not shown). The prostomium is, therefore, required for the maintenance of oogenesis and for spawning to occur. Nev- ertheless, a failure to spawn after prostomium removal cannot, at present, be attributed solely to a loss of endocrine function because it may also be due to the severance of nervous connections controlling spawning. To investigate whether the inhibition of spawning through exposure to LD8:16 photoperiods is endocrine me- diated, we implanted LD8:16 control females with prosto- mia from females maintained in LD16:8 or LD8:16 photo- periods for 2 weeks. Although the implantation of prostomia is an established technique for investigating the role of endocrine substances in polychaetes (see Golding, 1987), this is the first time that it has been used successfully for H. inihriciiui. Previous attempts with this species re- sulted in the degeneration of the implanted prostomium (P. J. W. Olive, University of Newcastle upon Tyne, pers. comm.). Prostomia implanted during our experiments showed no obvious degeneration with light microscope analysis up to one month after implantation (data not shown). It should be noted that implantation of prostomia is not an ideal technique for identifying the putative spawning hormone in this species. Future investigations will focus on the development of in vitro bioassays to reduce the numbers of prostomia used and to meet the levels of sensitivity and reliability that are essential for purification studies. The results presented in Figure 2 show that 80% of the LD8: 16PM(I m" Nl females spawned, as opposed to 37.5% of theLD8:16 I'MlI 1)S:I6) 4.- " females. These results confirm that the REPRODUCTION IN HAKMOTHOE IMBRICATA 57 implanted prostomia were still functioning as endocrine organs. We suggest the following hypothesis for the endo- crine control of spawning. A spawning substance present in the prostomium is required for spawning to occur. The liters of this substance, as with the gonadotrophic hormone, are mediated by photoperiod. The exposure of females to lengthening photophases (ambient conditions in February) increases the liters of this spawning substance to a level above which spawning can occur. Exposure of females to LD16:8 conditions also allows liters of the spawning sub- stance to reach the threshold level, so that nearly all the females spawn. In contrast, only a small proportion of the females exposed to LD8:16 conditions have liters of the spawning substance that reach the threshold, so significantly fewer spawn. Implanting the prostomia of an LD16:8 ex- posed female into an LD8:16 female also provides a source of higher levels of spawning hormone and thus increases the total circulating liters, enabling the LD8: 16 females to reach the threshold required for spawning to occur. Implanting the prostomia of an LD8:16 exposed female into an LD8:16 female provides a second source of the hormone, but at lower concentrations. In most females, the combined level of hormone is lower than the threshold, so most fail to spawn. The nature and action of a spawning substance in H. imbricata may take two forms. It could be a "true" spawn- ing substance like that found in Nephtys Iwmbergii. In that species, a hormone released from the supraesophageal gan- glion induces spawning by acting on the musculature to allow the release of gametes through the anus (Bentley el al., 1984). The maturation of the gametes is independent of the spawning hormone; they mature once released into seawater (Olive, 1976; Olive and Bentley, 1980). The other form of spawning substance induces gamete maturation and subsequent spawning either directly or in- directly. This form occurs in Arenicola marina. Oocytes mature through a two-step system involving a substance from the prostomium and then a second substance in the coelomic fluid. The latter, termed the coelomic maturation factor (CMF), induces the oocytes to mature; these oocytes are subsequently spawned (Watson and Bentley, 1997). It is unclear whether CMF also acts on the musculature to facil- itate spawning. However, in male A. marina, the sperm maturation factor (8, 11, 14 eicosatrienoic acid) not only induces maturation of the sperm but also produces specific behavioral changes associated with spawning (Pacey and Bentley, 1992). In H. imbricata, oocytes are released from the ovaries at prophase of the first meiosis a few days before spawning. They mature to metaphase of meiosis I in the coelomic Huid, are collected by the nephridia, and spawned (Daly, 1972). Further experiments are required to elucidate whether the putative spawning substance from the prostomium can in- duce spawning of immature oocytes or actually induces the maturation of the oocytes that are subsequently spawned. Pheromone influence on /minni; behavior Initial observations of the test males in the Y-maze indi- cated that 10 min was not sufficient time for them to complete their exploratory behavior. After 10 minutes most of the males were still actively searching. In all subsequent experiments we allowed 30 min for the males to settle before their position was recorded. This time period was sufficient for the males to settle and complete their explor- atory behavior. Results presented in Figures 4 and 5 provide the first evidence that pheromones are involved in the reproductive behavior of H. imhncata. Specifically, these data indicate that a mature female with fully grown oocytes in its coelo- mic cavity releases a waterborne substance or substances that attracts significantly more mature males than are at- tracted by seawater. males, or females that are carrying fertilized oocytes. Harmothoe imbricata is a solitary species outside the breeding season, but it reproduces by forming single copu- lating pairs (Daly, 1972). This method of reproduction requires mature individuals to locate each other, but at low population densities, chance encounters may be infrequent. An attraction pheromone released by a mature female in- creases the chances that a male will find her and, therefore, increases the number of successful fertilizations. The pher- omone may also maintain the pair bond and could suppress the cannibalistic tendencies of both individuals, allowing pairing to proceed. Once the female has fertilized oocytes, she stops releasing the pheromone and becomes unattractive to the male; this is confirmed by the data shown in Figure 5. One of the best-studied attraction pheromones in marine invertebrates is attractin, a peptide found in the egg cordon of the opisthobranch mollusc Aplysia spp. The function of this 58-residue peptide is to attract other individuals to the mating aggregation and to induce mating (Painter et ai, 1991, 1998). Pheromones are also a component of a number of polychaete reproductive strategies, particularly in some nereid species (for review, see Zeeck et ai, 1996). How- ever, these pheromones have been isolated only from spe- cies such as Platvnereis dumerlii and Nereis succinea that swarm en masse in the water column (Zeeck et al., 1988. 1996; Hardege et al., 1998). The results presented here are. therefore, the first report of a waterborne cue being used as an attraction pheromone from a polychaete that reproduces following pair formation. Although the evidence for pheromones in marine inver- tebrates is steadily growing, the environmental control of pheromone production has not been investigated. The effect of environmental manipulation (particularly photoperiod) on oogenesis is confirmed and the influence of photoperiod 58 G. J. WATSON ET AL on spawning in Harmothoe imbrictitei has been described for the first time (Garwood and Olive. 1982; Clark, 1988). Establishing a link between the photoperiodic input and the production of a pheromone that regulates spawning behav- ior is an important step. Results presented in Figure 5 show that females maintained in LD8:16 photoperiods were only as attractive to mature males as seawater or other males. Just as LD8:16 exposure may prevent the production (or the attainment of threshold levels) of the putative spawning hormone and thus prevent spawning, it may also prevent the production of the attraction pheromone. The relationship between the spawning hormone and pheromone production requires further investigation, but the production of the two may be intricately linked, and they may be the same or similar substances. Acknowledgments The authors acknowledge the support of NERC grant GR3/ 10521 to MGB. The authors declare that the experi- ments comply with the current laws of the country in which the experiments were performed. Literature Cited Bentley, M. G., P. J. W. Olive, P. R. Garwood, and N. H. Wright. 1984. The spawning and spawning mechanisms of (Vc/i/jm caeca (Fabricius, 1780) and Nephrys hombergi (Savigny, 1818) (Annelida: Polychaeta). Santa 69: 63-68. Bentley, M. G., J. Boyle, and A. A. Pacey. 1994. Environmental influ- ences on endocrine systems controlling reproduction in polychaetes. Mem. A/H.V. Nat/. Hist. Nat. 162: 37-44. Clark, S. 1988. A two phase photoperiodic response controlling the annual gametogenic cycle in Harmothoe imbricala (L.I (Polychaeta: Polynoidae). linvrtehi. Reprod. De\: 14: 245-266. Daly, J. M. 1972. The maturation and breeding of Harmothoe iinhricatu (Polychaeta: Polynoidae). Mar. Biol. 12: 53-66. Daly, J. M. 1974. Gametogenesis in Harmothoe imbricata (Polychaeta: Polynoidae). Mar. Binl. 25: 35-40. Garwood, P. R. 1980. The role of temperature and day length in the control of the reproductive cycle of Harmothoe iinhricutu (L.) (Polychaeta: Polynoidae). 7. E.\p. Mar. Biol. Ecol. 47: 35-53. Garwood, P. R., and P. J. W. Olive. 1982. The influence of photopenod on oocyte growth and its role in the control of the reproductive cycle in the polychaete Hunnothoe inibriciita (L.). Int. ./. lm-crlebr. Reprod, 5: 161-165. (iolding, D. W. 1983. Endocrine programmed development and repro- duction in Nereis. Gen. Comp. Endocrmol. 52: 456 — 166. Gnlding. D. VV. 1987. Brain-body interactions in Nereis. Deactivation of the cerebral neuroendocrine system by ganglion transplantation. Int. J. Im-ertebr. Reprod. 12: 281-294. Hardege J. D., C. T. Muller, and M. Beckmann. 1998. A waterborne female sex pheromone in the ragworm. Nereis succinea (Annelida: Polychaeta). Polychaete Res. 17: 18-21. [ISSN 0961-320X. Available from C. Mettam, Univ. of Wales Cardiff, PO Box "15, Cardiff CF1 3TL. UK] Lawrence, A. J. 1996. Environmental and endocrine control of repro- duction in two species of polychaete: potential bio-indicators for global climate change. J. Mm: Biol. Assoc. UK 76: 247-250. Olive, P. J. W. 1976. Evidence for a previously undescnbed spawning hormone in Neplnys hombergii (Annelida: Polychaeta). Gen. Comp. Em/ocrinol. 28: 454-460. Olive, P. J. VV., and M. G. Bentley. 1980. Hormonal control of oogen- esis, ovulation and spawning in the annual reproductive cycle of the polychaete Nep/ilys hombergii Sav. (Nephtyidae). Int. ./. Invertehr. Rcprml. 2: 205-22 1 . Pacey, A. A., and M. G. Bentley. 1992. The fatty acid 8. II. 14 eicosatrienoic acid induces spawning in the male lugworm Arenicola manna. J. Exp. Biol. 173: 165-179. Painter, S. D., M. G. Chong, M. A. Wong, A. Gray. J. G. Cormier, and G. T. Nagle. 1991. Relative contributions of the egg layer and egg cordon to pheromonal attraction and the induction of mating and egg-laying behavior in Aplysiti. Binl. Bull. 181: 81-94. Painter, S. D., B. dough, R. W. Garden, J. V. Sweedler, and G. T. Nagle. 1998. Characterization of Aplysia attractin, the first water-borne peptide pheromone in invertebrates. Biol. Bull. 194: 120- 131. Watson, G. J., and M. G. Bentley. 1997. Evidence for a coelomic maturation factor (CMF) controlling oocyte maturation in the polychaete .4 renk -olu marina (L.). Invertebr. Reprod. De\: 31: 297-306. Watson, G. J., M. E. Williams, and M. G. Bentley. 2000. Can syn- chronous spawning be predicted from environmental parameters'? A case study ol the lugworm Arenicola marinti. Mar. Biol. (in press). Zeeck, E., J. D. Hardege, H. Bartels-Hardege, and G. Wesselmann. 1988. Sex pheromone in a marine polychaete: determination of chem- ical structure. J. Ev/i. Zt>ol. 246: 285-292. Zeeck, E., J. D. Hardege, and H. Bartels-Hardege. 1990. Sex phero- mones and reproductive isolation in two nereid species. Nereis suc- cinea and Plutyncrci* dumerilii. Mar. Ecol. Prog. Ser. 67: 183-188. Zeeck, E., T. Harder, M. Beckmann, and C. T. Muller. 1996. Marine gamete release pheromones. Nature 382: 214. Reference: Biol. Bull. 199: 59-67. (August 2000) Fertilization in Callochiton castaneus (Mollusca) JOHN BUCKLAND-NICKS1 * AND ALAN N. HODGSON- lSt. Francis Xavier University, Department of Biology, Antigonish. Nova Scotia. Canada B2G 2W5: and ^Rhodes University, Department of Zoology and Entomology. Graluumtiwn f>l40. South Africa Abstract. A fine-structural study of fertilization in Callo- chiton castaneus has revealed that the mechanism of sperm penetration into the egg is intermediate between the primi- tive condition found in members of the order Lepidopleu- rida and the more derived condition found in the Chitonida. C. castaneus sperm have the long needlelike nuclear fila- ment and reduced acrosome that characterizes all Chitonida. but they have retained several plesiomorphic features such as an unspecialized mid-piece and a lack of flagellar rein- forcement. As in some Lepidopleurida but unlike any Chi- tonida, the egg hull in this species comprises a thick, smooth jelly coat permeated by pores that permit sperm rapid access to the vitelline layer. The jelly coat is delicate and quickly dissolves when a sperm concentrate is used, suggesting that excess acrosomal enzymes may be responsible. Once the sperm have penetrated the vitelline layer, the long nuclear filament bridges the gap to cups in the egg membrane. However, once the fertilization membrane is raised, the perivitelline space exceeds the length of the nuclear fila- ment, preventing other sperm from penetrating the egg. A fertilization cone forms around the nuclear filament of the penetrating sperm, but it does not appear to engulf the body of the sperm. Rather, the nuclear chromatin is injected into the egg as a long thread. The remaining sperm organelles are apparently abandoned on the egg surface. If this is the case, it would be a significant departure from fertilization in other molluscs and many other metazoans, in which sperm organelles, such as centrioles and mitochondria, enter the egg. New sperm and egg characters, as well as significant differences in fertilization, indicate that Callochitonidae are basal to all other members of the order Chitonida and may Received 7 December 1999; accepted 25 May 2000. * To whom all correspondence should be addressed. jbucklan@stfx.ca E-mail: warrant separation as the sister taxon to the suborders Chi- tonina and Acanthochitonina. Introduction Sirenko ( 1997) recently classified extant chitons into two orders. Lepidopleurida (suborders; Lepidopleurina and Choriplacina) and Chitonida (suborders; Chitonina and Acanthochitonina). Lepidopleurida are considered basal to chitons in general, because they possess many primitive traits. For example, shell valve structure and articulation is simpler and ties in well with the fossil record (Sirenko. 1997): also they are the only chitons known to have typical aquasperm with prominent acrosomes, and eggs with smooth hulls (Hodgson et al., 1988; 1989; Eernisse and Reynolds. 1994; Buckland-Nicks. 1995: Pashchenko and Drozdov, 1998). Presumably, in the ancestor of all members of Chitonida. the sperm acrosome became reduced to a minute vesicle atop a long needlelike extension of the nucleus, since this arrangement is found in all extant Chi- tonida (Buckland-Nicks et al.. 1990) but not in any Lepi- dopleurida examined to date (Hodgson et al., 1988; Pash- chenko and Drozdov, 1998). The intermediate condition of a prominent acrosome and short nuclear filament was re- cently discovered in the lepidopleurid Deshayesiella cur- vata (Pashchenko and Drozdov. 1998). The term egg "hull" is used here instead of "chorion" as in previous publications (Buckland-Nicks et al.. 1988a. b: Buckland-Nicks, 1993. 1995), to describe the noncellular envelope enclosing the egg and its vitelline layer, because Richter ( 1986) showed that both the vitelline layer and the hull of chitons are formed by the egg and not by the follicle cells as in other Metazoa (for review of terminology, see Eernisse and Reynolds, 1994). Chiton eggs evolved from having smooth unspecialized hulls, with fertilization presumably occurring anywhere on the surface, to having elaborate spinous or cupulous hulls that focus sperm to specific regions of the egg surface 59 60 J. BUCKLAND-NICKS AND A. N. HODGSON FC FERTILIZATION IN CALLOCH1TON 61 (Buckland-Nicks, 1993. 1995). Among Lepidopleurida. Leptochiton asellus has a smooth hull comprising a thick homogeneous jelly coat (Hodgson et til., 1988). whereas D. ciin'ata egg hulls are composed of a jelly coat that is penetrated by regularly spaced pores (Pashchenko and Drozdov, 1998). The mechanism of fertilization must be quite different in these lepidopleurids compared to Chito- nina such as Stenoplax conspicua (Buckland-Nicks. 1995). or Acanthochitonina such as Tonicella lineata (Buckland- Nicks et «/., 1988b). because of basic differences in sperm acrosomes and egg hull structure. Sperm structure of dif- ferent species has been found to be an accurate indicator of phylogenetic relationship and sometimes is used as grounds for reclassifying species to alternative taxa (Jamieson, 1987; Healy. 1988; Hodgson et al, 1996). The family Callochitonidae, although placed within the order Chitonina and regarded as "evolutionarily advanced" (Sirenko. 1997). exhibits several plesiomorphic traits (Buckland-Nicks. 1995) and is therefore one of several groups that are important to investigate in clarifying the phylogeny of chitons. This study describes in detail, for the first time in Callochiton castanens, the structure of the egg hull and the mechanism of fertilization. The information gained is discussed in relation to the evolution of mecha- nisms of fertilization in chitons, as well as to chiton phy- logeny in general. Materials and Methods Specimens of Callochiton castanens Wood, 1815, were obtained in breeding condition from beneath intertidal rocks at East London (33° 03' S; 28° 03' E) and Port Alfred (33° 52' S; 26° 53' E), South Africa, from August to October 1999. The animals were brought back to the laboratory and placed individually in 60-mm petri dishes half-rilled with 0.45-;um filtered seawater (FSW). Some individuals of each sex had spawned by the second day. Alternatively, eggs or sperm were obtained by removing the foot and digestive gland and puncturing the dorsal gonad. If the specimen was male, the white "dry" sperm concen- trate would ooze from the punctured testis and could be easily aspirated into a pipette. For a "concentrated sperm suspension." one drop of dry sperm was diluted in 5 ml of FSW. For a "dilute sperm suspension." one drop of the latter was further diluted in 5 ml of FSW. If the animal was female any free-spawned eggs were collected from the petri dish; otherwise the eggs were flushed from the ovary by aspirating a stream of FSW into the gonad. Eggs were then pipetted individually into another clean petri dish containing FSW, to reduce debris. Batches of about 50 eggs were removed into six small glass vials containing 4 ml of FSW. Two vials served as unfertilized controls. Two vials re- ceived three drops of diluted sperm suspension, and in two vials the FSW was replaced with concentrated sperm sus- pension. The eggs in one set of vials were transferred to primary fixative (see below) after 30 s, and the eggs in the second set of vials were fixed after 10 min. Light microscopy A few eggs were removed from control and experimental vials during the experiment to monitor the progress of fertilization and to be photographed using an Olympus BX50F-3 light microscope equipped with bright field and DIG optics. Slides were made by pipetting a few eggs onto a clean glass slide, placing pieces of coverslip around to make a well, then adding a whole coverslip, drying off excess water, and finally sealing the coverslip with nail varnish. Sperm activity was noted to be highly variable. We tested the effects of serotonin (5-hydroxytryptamine) on sperm activity by mixing a drop of sperm concentrate with 1 ml of serotonin solution, to give a final concentration ranging from 1 to 10 jii/W serotonin. One drop of this sperm solution Figures 1-6. Micrographs of unfertilized eggs of Callochiton castaneus. Figure 1. Ripe egg with intact layer of follicle cells (FC) dissected from the ovary. Note regular arrangement of pores in hull (arrows) visible beneath follicle cells. Scale bar = 100 /urn. Figure 2. Light micrograph of l-/j.m section of egg removed from the ovary, showing a layer of intact follicle cells (FC) and regularly spaced pores (P) in the jelly hull; the pores penetrate to the vitelline layer (VL) overlying the egg membrane cups (MC). Note meshwork of fibers (arrowheads) supporting pore structure. Scale bar = 10 /im. Figure 3. Similar to Figure 1 except the egg has been rolled on sticky tape, which removed follicle cells and pore openings (arrowhead), thus revealing pores in the jelly hull (arrows). Scale bar = 100 /Mm. Figure 4. Close-up of a spawned egg in which the follicle cells have retracted, revealing the arrangement of pores in naked jelly hull. Pore entrance (PE) is usually one-third of the diameter of the pore itself (double arrow), thus restricting sperm entry. A sperm (Sp) is visible at the entrance to one damaged pore. Scale bar = 2 /^m. Figure 5. Vitelline layer (VL) of the egg has been rolled off on sticky tape, revealing a regular series of membrane cups (arrows) that match up with pores in the hull. Scale bar = 15 ^m. Figure 6. Close-up of Figure 5, showing egg membrane cups with microvilli that are prominent on the raised edges (arrows) but sparse in the bases of cups. Scale bar = 5 /j.m. 62 J. BUCK.LAND-NICKS AND A. N. HODGSON was placed on a slide next to a separate drop of control sperm in filtered seawater, and observed periodically. were examined and photographed in a JEOL JSM 840 scanning electron microscope. Electron microscopy The primary fixative was made by mixing I ml of 25% glutaraldehyde with 9 ml FSW and adding this to 10 ml of 0.2 M Na cacodylate buffer (pH 7.4). This gave a final concentration of 2.5% glutaraldehyde in 0.1 M Na cacody- late buffer and FSW. to which was added 0. 1 M sucrose. The fixative was refrigerated before use. Samples were fixed overnight and then washed in two changes of 0.1 M Na cacodylate buffer in FSW (pH 7.4) before post-fixing for 1 h in 1.5% osmium tetroxide in the same buffer. Fixed eggs were rinsed in distilled water and dehydrated in an ethanol series to 100%. At this stage about half of the eggs in each vial were removed to a second series of vials containing 100% ethanol for preparation for scanning electron micros- copy (SEM). In samples destined for transmission electron micros- copy, ethanol was replaced with propylene oxide and then the eggs were transferred through infiltration media consist- ing of mixtures of propylene oxide and TA AB 8 1 2/Araldite CY212 resin (75:25. 50:50. 25:75) for 2 h in each mixture in capped vials (after Cross, 1989). Samples were ex- changed into pure resin and left overnight in uncapped vials in a desiccator. The next afternoon, with the aid of a dissecting microscope, groups of five to six eggs were aspirated into BEEM capsules half filled with resin. Eggs were allowed to sink and then were arranged into the center of each mold using a stainless steel insect pin. Labels were added, and the BEEM capsules were placed in a 60"C oven for 40 h. Thick sections were cut with glass knives in an LK.B 8800 ultratome, transferred to glass slides, and stained with 1% toluidine blue for about 20 s before rinsing with distilled water and air drying. Thin sections, with silver/gold interference color, were cut on a diamond knife (Diatome) and picked up on naked 150-mesh copper grids. Sections were reverse stained with aqueous lead citrate for 1 min, followed by aqueous uranyl acetate for 2 min, after the method of Daddow ( 1986). Stained sections were examined and photographed in a JEOL 1210 transmission electron microscope operated at 80 kV. Samples destined for SEM were exchanged through an amyl acetate series to 100% and then aspirated under a dissecting microscope into Teflon flow-through specimen vials (Pelco) before capping and critical point drying. Sub- sequently, individual Teflon vials were uncapped and in- verted on an SEM stub coated with a carbon sticky tab. The eggs stuck fast but could be rolled with an insect pin to remove the egg hull and expose either the vitelline layer or the egg membrane itself. Some eggs were cut in two with a Kesei rnicroknife (Japan). Stubs prepared in this way were coated with gold in a Polaron E5100 sputter coaler. Stubs Results Morphology of the egg The unspawned ripe eggs of Callochiton castaneus are about 220 ju,m in diameter and surrounded by a vitelline layer and a smooth, 20-/u,m-thick egg hull enclosed by a single layer of follicle cells, usually hexagonally disposed (Figs. 1, 2). The egg hull is made up of a delicate jelly coat supported by a fibrous matrix that is permeated by a series of pores spaced at regular intervals of 9 ju,m all over the surface (Figs. 3. 4). If unspawned eggs are rolled on sticky tape to remove the follicle cells, the regular array of pores in the jelly coat becomes visible (Fig. 3). At its entrance, each pore is about 2 /urn in diameter (Fig. 4), but below this the diameter enlarges to about 8 /-im (Fig. 2). Furthermore, there is a network of fibers that criss-cross each pore from apex to base (Fig. 2). Opposite, but below, the point at which the pores contact the vitelline layer, the egg mem- brane is formed into a series of cups (Figs. 5, 6). The depression in each cup usually coincides with a pore, and the lip of each cup coincides with the division between two pores (Fig. 2). The raised edges of the cups are rich in microvilli that penetrate into the vitelline layer (Figs. 2, 6); in the base of the cups, microvilli are sparse (Fig. 7). The mechanism of fertilization Soon after spawned eggs contact seawater, any remaining follicle cells retract, thus exposing the pores in the hull (Figs. 4. 8). In dilute sperm suspensions, sperm quickly locate the entrance to these pores and swim down to the egg surface (Fig. 8). If a concentrated sperm suspension is used, many sperm arrive at the egg surface simultaneously, over- riding any potential block to polyspermy (Fig. 9). In these cases the jellylike hull is dissolved in 1 or 2 min, leaving the exposed vitelline layer of the egg coated with thousands of penetrating sperm, many of which induce fertilization cones (Figs. 9, 10). The fragile jelly coat degenerates in about an hour even under natural conditions, and it was not preserved intact by routine fixation. When a fertilizing sperm penetrates the vitelline layer, the needlelike nuclear filament bridges the perivitelline space and egg cup to reach and fuse with the egg membrane (Figs. 1 1-13). The distance between the base of each egg cup and the vitelline layer varies from 1.5 -4 /im (Fig. 2), but the elongate nuclear filament permits sperm-egg fusion up to a distance of 6 /urn (Fig. 12). Contact between sperm and egg results in their fusion and the formation of a narrow tube (< 0. 1 /xm in diameter) through which the threadlike chromatin is injected into the egg cortex (Fig. 14). This is visible as a thin white thread when stained with Hoechst's 33358 DNA FERTILIZATION IN CALLOCHITON 63 Figures 7-10. Micrographs of fertilized eggs of Callochiton castaneus: SEM = scanning electron micro- graph: DIC LM = differential interference contrast light micrograph. Figure 7. SEM of an unfertilized egg split in half with a Kesei microknife to show egg membrane cups (arrowheads). The vitelline layer has been removed. Scale bar = 10 (nm. Figure 8. SEM view of broken edge of the jelly hull, showing regular arrangement of pores above (arrowheads) and penetrating sperm (Sp) on vitelline layer below. Scale bar = 2 ju.ni. Figure 9. DIC LM of a polyspermic egg showing numerous fertilization cones (arrows) beneath the elevated vitelline layer (VL). Scale bar = 70 /A in. Figure 10. SEM of a polyspermic egg showing fertilization cones (arrowheads) beneath the vitelline layer, which has been partly removed by rolling the egg on sticky tape. Note also numerous sperm (Sp) on region of intact vitelline layer (VL). Scale bar = 10 /am. stain and viewed under UV light (Fig. 12 inset). A fertili- zation cone is raised up around the penetrating nuclear filament from the surrounding egg cortex, as well as from fusion of adjacent egg microvilli (Fig. 13). However, the fertilization cone remains below the hairier of the vitelline layer and engulfs only the nuclear filament (Figs. 12. 14). The vitelline membrane raises up and forms the fertilization membrane (Figs. 9, 12). The raised edges of the egg mem- brane cups retract from the vitelline layer, creating a larger (6-7 jam) peri vitelline space that excludes late-arriving 64 J. BUCKLAND-NICKS AND A. N. HODGSON 14 Figures 11-17. Micrographs of sperm and fertilized eggs of Ciillix'liiiuii ftiMnni'its: SEM = scanning electron micrograph; TEM = transmission electron micrograph; DIG LM = differential interference contrast light micrograph. Figure 11. SEM of polyspermic egg that has been rolled on sticky (ape. stripping the vitelline layer (VL) next to a penetrating sperm (Sp) and revealing the fertilization cone (FC) beneath it. Scale bar = 2 jj.ni. Figure 12. DIG LM of polyspermic egg showing one sperm (Sp) that has penetrated the vitelline layer (VL) and induced a fertilization cone, and a second, late-arriving sperm that cannot reach the egg membrane with Us nuclear filament (arrowhead). Note also sperm flagellum (F) exhibiting large amplitude beat. Inset: Same as Figure 12 except that the penetrating sperm has been labeled with Hoechst's 33358 DNA stain and photographed under UV epifluorescence. revealing nucleus (N) injecting chromatm (arrowhead) into the egg cortex through the fertilization cone (FC). Scale bar = 6 /urn. Figure 13. TEM of a fertilization cone (FC) with part of a penetrating sperm (Sp) visible above the vitelline layer (VL). Note that the fertilization cone incorporates elevated cytoplasm as well as microvilli (arrowheads). Scale bar = 2 /am. Figure 14. TEM of penetrating sperm injecting chromatin (arrowhead) into egg cortex. Note that the vitelline layer (VL) is intact except for a small pore through it. Sperm mitochondria (M) and remnants of glycogen (arrow) have collected in the membrane bag posterior to (he nucleus (N). Compare with Figure-Id. Scale bar = 1 jum. VL FERTILIZATION IN CALLOCHITON 65 sperm (Fig. 12). The sperm organelles — including the cen- trioles, flagellum, and mitochondria, as well as some resid- ual glycogen granules — collect in a bag of membranes above the fertilization membrane and do not appear to enter the egg cortex (Figs. 11, 14). Morphology of the sperm The sperm of Callochiton castaneus (described by Hodg- son et ul., 1988) has a bullet-like nucleus extending into a long nuclear filament tipped by a minute acrosome, but it has retained a relatively unspecialized mid-piece (Figs. 15- 17). Hodgson et al. (1988) believed that C. castaneus sperm did not possess an acrosome. Careful reexamination of their sections, however, revealed the presence of the minute acrosomal vesicle, which is separated from the nuclear extension by a basal plate (Figs. 15, 17). It was not possible to discern any subdivision of the acrosomal vesicle in this species. The main body of the nucleus is 3 /J,m long, and the nuclear filament is a further 6 /J.m. The mid-piece comprises five oblong mitochondria arranged fairly symmetrically around the centrioles (Figs. 15, 16). Sperm dissected from some males were inactive even after being placed in seawater. However, all became active within 1 min following the addition of 1 ju,M serotonin. The degree of activity increased with increas- ing concentrations of serotonin, up to the maximum tested concentration of 10 Discussion Morphology of the sperm Except in the suborder Lepidopleurina, the acrosome of all chitons examined has been reduced to a small vesicle at the tip of a needlelike nuclear filament, which is an exten- sion of the main body of the nucleus (see review by Buck- land-Nicks, 1995). Partial reduction of the acrosome is noted among species of Lepidopleurina such as De- shayesiellu cun'iitn (Pashchenko and Drozdov, 1998). How- ever, sperm of a number of families in this basal suborder have not been examined; these include Hanleyidae, Choriplacidae, and Nierstraszellidae. Acrosomes are fully reduced in Cullochitonidae, although the mid-piece of sperm in this family has retained the primitive state, in which mitochondria are symmetrically disposed around the centrioles and there is no reinforcement of the flasellum (Buckland-Nicks, 1995). In most Chitonina the mitochon- dria are asymmetrically distributed around the centrioles and, in addition, the flagellum is reinforced near the annulus (Buckland-Nicks, 1995). The activation of sperm by serotonin, which was ob- served here for C. castaneus, has not been previously re- corded in chitons, although this response is well known from bivalves (Juneja et ul., 1993) and has also been noted in limpets (Bucklund-Nicks and Howley, 1997; Buckland- Nicks and Hodgson, unpubl. data). In bivalves, serotonin has been shown to initiate sperm motility and egg matura- tion, as well as to improve fertilization success (Juneja et ul., 1993). This preliminary evidence for a function of serotonin in chiton reproduction brings hope that induction of spawning also may be possible. Until now, the inability to predict spawning has hampered studies of chiton repro- duction. Morphology of the egg In Chitonida the egg hull is resilient, easily preserved, and elaborated into spines or cupules that not only slow the sinking rate but direct sperm to specific locations on the egg surface (Buckland-Nicks, 1993, 1995). The egg hulls of most Chitonina have elaborate spines with narrow bases and highly variable tips (Eernisse, 1984; Sirenko, 1993). Eer- nisse ( 1984) first suggested developing independent charac- ter sets based on hull spine structure and gill placement to test the validity of phylogenies based solely on shell valve morphology (Smith, 1960; Van Belle, 1983). Sirenko's sub- sequent investigations proved that these characters (1993). as well as variation in the articulamentum ( 1997) are useful in the analysis of chiton phylogeny. The new sperm and egg characters described here for C. castaneus will be important in future cladistic analyses of the Chitonina because they indicate that Callochitonidae are basal to Chitonina. The egg hull of C. castaneus differs in some key respects from that of other chitons studied; in particular, it is unlike that of any other Chitonina. The vitelline layer is enclosed by a fragile, smooth jelly coat that is permeated by large pores. However, this type of hull may also occur in De- shayesiel/a cun'uta (Lepidopleurina); drawings of this spe- cies show a similar jelly coat containing regularly spaced pores (Pashchenko and Drozdov, 1998), although no micro- graphs of this feature have been published. In fertilization experiments with polyspermic eggs of D. cun'ata, it was Figure 15. SEM of sperm, showing acrosome (A) at tip of nuclear filament (NF). main body of nucleus (N). mid-piece (MP). and flagellum (F). Scale bar = 1 /urn. Inset: TEM of apex of sperm revealing acrosomal vesicle (AV) separated from nuclear filament (NF) by basal plate (BP|. Scale bar = 0.7 /j,m. Figure 16. TEM of sperm nucleus (N) and mid-piece showing mitochondria (M), centrioles (C). and glycogen granules (G). Note portion of nuclear filament (arrowhead). Scale bar = 0.7 ^im. Figure 17. TEM of apex of sperm revealing acrosomal vesicle ( AV) separated from nuclear filament (NF) by basal plate (BP). Scale bar = 0.7 /urn. 66 J. BUCKLAND-NICKS AND A. N HODGSON observed that the jelly coat disintegrated, much like that of C. castaneus (Buckland-Nicks and Sirenko, unpubl. re- sults). This phenomenon of a fragile jelly coat is a plesi- omorphy shared between Lepidopleurina and Callochi- tonidae. which excludes all other Chitonina studied thus far. In species that have egg hulls with closed cupules. sperm penetrate the hull exclusively between the cupules, usually where their hexagonal bases meet (Buckland-Nicks. 1995). Eggs of some Acanthochitonina. such as Lepidochitona dentiens and L. femuldi. have micropores in this region. These permit easier access to the vitelline layer, although sperm have two granules in the acrosome, suggesting that the hull may still represent a barrier (Buckland-Nicks et al., 1990). C. castaneus appears to have a simpler acrosome structure, which would correlate with the provision of direct access to the vitelline layer by large pores in the jelly hull. In summary, C. castaneus gametes and those of the Lepidopleurina share plesiomorphic characters such as a simple arrangement of mitochondria in the sperm mid-piece and a smooth egg hull. Yet C. custuneus shares the derived characters of nuclear extension and reduction of acrosome with all other Chitonida. A previous cladistic analysis of chitons, which was largely based on sperm and egg char- acters, predicted that Callochitonina was distinct from Chi- tonina (Buckland-Nicks, 1995). The present study corrobo- rates this prediction and indicates that C. castaneus is unique and perhaps should be placed in a sister taxon to both Chitonina and Acanthochitonina. The mechanism of fertilization in chitons Fertilization in C. castaneus and other Chitonida bears some similarity to that in other molluscs, in the sense that a sperm acrosome releases enzymes that digest a pore in the egg envelope, enabling the inner acrosomal membrane to fuse with the egg membrane (Buckland-Nicks et al.. 1988. this study). However, in many other respects the mechanism of fertilization in these chitons is highly derived when compared with those of other molluscs and of metazoans in general. Firstly, there is no extrusion of an acrosomal process or "perforatorium" by the polymerization of actin, as occurs throughout molluscs and other metazoan groups (see review by Tilney. 1985). Rather, in all members of Chitonida the permanent needlelike nuclear filament has replaced the per- foratorium (Buckland-Nicks et al.. 198Sb. 1990). The inter- mediate condition of a short nuclear extension found in Deshayesiella cunata (Pashehenko and Drozdov, 1998) suggests that reduction in acrosome size among lepidopleu- rids may be linked with an increase in the length of the nuclear filament. Furthermore, all lepidopleurids examined have a subacrosomal granule, which in other metazoans is composed of actin for extruding the perforatorium during fertilization. A second important difference observed between Chi- tonida and other molluscs is that the sperm organelles, as well as most of the nuclear membrane, apparently remain on the surface of the egg (Buckland-Nicks el al., 1988b; Buck- land-Nicks. 1995; this study). No chiton sperm has been observed becoming completely engulfed by a fertilization cone. The probable reason for this is that the vitelline layer, disturbed only by a minute pore permitting penetration of the nuclear filament, remains a barrier to the envelopment of the sperm by the fertilization cone. In other molluscs such as bivalves, as well as in many other metazoans, the vitelline layer is breached and the fertilization cone raises up through it to engulf the entire sperm, including part of the flagellum (see reviews by Tilney. 1985: Longo, 1987). In these spe- cies there is an initial paternal contribution of centrioles and mitochondria to the egg at fertilization, although, with the exception of some bivalve molluscs (Hoeh et al.. 1991 ), the paternal mitochondria degenerate and do not contribute to the zygote. Furthermore, in sea urchins and some other metazoans. a sperm centriole contributes to the movement of the pronuclei as well as to the formation of the mitotic spindle prior to first cleavage (see review by Gilbert, 1999). Chitons may be unique among molluscs if. in addition to the exclusion of paternal mitochondria, the centrioles that form the mitotic spindle are also maternally derived. Confirma- tion of this derivation will require appropriate labeling of sperm centrioles and mitochondria before and after fertili- zation. To better understand how the mechanism of fertilization has evolved in chitons, it will be important to examine fertilization in a species like Leptocliiton iixellus, which has a typical molluscan acrosome (Hodgson et al., 1988) and perhaps a mechanism of sperm entry more similar to that of limpets or bivalves. Acknowledgments We thank Robin Cross and Shirley Pinchuk for assistance in setting up electron microscopes and ancillary equipment, as well as Marvin Randall for doing most of the darkroom work. We are grateful to Doug Eernisse for important and helpful criticisms. This research was supported by a Hugh Kelly Fellowship from Rhodes University and an NSERC of Canada research grant, both to J.B-N.. as well as a Rhodes University research grant to A.N.H. Literature Cited Buckland-Nicks. J. 1993. Hull cupules ot chiton eggs: parachute struc- tures and sperm Incusing devices'7 Biol. Bull. 184: 269-276. Buckland-Nicks. J. 1995. infrastructure of sperm and sperm-egg inter- action in Aculifera: implications for molluscan phytogeny. Pp. 129- 153 in Ail\'unci'\ in Si>criiiati>;onl f'li\lnf(cii\' uiul Taxonomy. B. G. M. Jamieson, J. Ausio. and J.-L. Justine, eds. Mem. Mus. Natl. Hist. Nat. 166. Paris. FERTILIZATION IN CALLOCHITON 67 Buckland-Nicks, .)., and B. Hovvlev. 1997. Spermiogenesis and sperm structure in relation to early events of fertilization in the limpet Teetura testitdinalix (Muller, 1776). Bwl. Bull. 193: 306-314. Buckland-Nicks, J., R. Koss, and F.-S. Chia. 1988a. The elusive ac- rosome of chiton sperm. Int. J. Invertebr. Reprod. 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The importance of the development of articulamen- tum for taxonomy of chitons (Mollusca. Polyplacophora). Ruthenica 7: 1-24. Smith, A. G. 1960. Amphmeura. Pp. 141-176 in Treatise on Inverte- brate Paleontology. Pan I. Mollusca I. R. C. Moore, ed. Geological Society of America, New York. Tilney, L. G. 1985. The acrosomal reaction. Pp. 157-2 1 3 in The Biology of Fertilisation. Vol. 2, C. Metz and A. Monroy, eds. Academic Press. New York. Van Belle, R. A. 1983. The systematic classification of chitons (Mollus- ca: Polyplacophora). Informations Jc la Societe Beige de Malacologie. Serie 11: 1-164. Reference: Bwl. Bull. 199: 68-75. (August 2000) Early Development of Zooxanthella-Containing Eggs of the Corals Pocillopora verrucosa and P. eydouxi with Special Reference to the Distribution of Zooxanthellae M. HIROSE', R. A. KINZIE III2, AND M. HIDAKA1 * 1 Marine Environmental Science, Department of Chemistry, Biology and Marine Science, University of the Rviikvus, Nishihara. Okinawa 903-0213, Japan; and 2 Department of Zoology and Hawaii Institute of Marine Biologv. University of Hawaii, Honolulu. Hawaii 96822 Abstract. Some hermatypic corals spawn eggs that con- tain zooxanthellae. We followed development of zooxan- thella-containing eggs of two such species, Pocillopora verrucosa and P. eydouxi. We also documented changes in the distribution pattern of zooxanthellae during develop- ment. Oocytes of both species took up zooxanthellae 3 to 4 days before spawning. At first, zooxanthellae were evenly distributed in oocytes, but they later moved to the hemi- sphere that contained the germinal vesicle. After fertiliza- tion, early cleavage events were holoblastic, progressing by furrow formation. The first cleavage furrow started at the hemisphere that contained zooxanthellae. dividing the zoo- xanthellate complement of the zygote about equally into the two blastomeres. The second division divided each blas- tomere into one zooxanthellae-rich cell and one with few zooxanthellae. With continued cell division, blastomeres containing zooxanthellae moved into the blastocoel. The blastocoel disappeared at about 5 h after the first cleavage, and the central region of the embryo was filled with cells containing either zooxanthellae or lipid droplets, forming a stereogastrula. Our results suggest that only blastomeres that had been determined to develop into gastrodermal cells receive zooxanthellae during cleavage. This determination appears to take place, at the latest, by the second cell division at the four-cell stage. Received 22 October IWJ; accepted 17 May 2000. * To whom correspondence should be addressed. E-mail: hidaka @sci.u-ryukyu.ac.jp Introduction Reef-building corals harbor intracellular symbiotic dinotlagellates, zooxanthellae, in their endodermal cells. Some hermatypic corals acquire their symbionts from their mother colony before fertilization (Kojis and Quinn, 1981; Babcock and Heyward, 1986; Tomascik and Sander, 1987; Yeemin, 1988; Glynn et al., 1991. 1994; Heyward et til.. 1987; Kinzie. 1993, 1996; Sier and Olive, 1994; Kruger and Schleyer, 1998). It is not known how zooxanthellae are delivered to oocytes and how their distribution relates to their eventual restriction to the endodermal cells in adults. Early development of scleractinian corals has been de- scribed in various species (e.g.. Szmant-Froelich ct a/.. 1980, 1985; Babcock and Heyward, 1986; Harrison and Wallace, 1990). However, early development of corals with oocytes containing zooxanthellae has been described only in the spawning species Montipora effusa (Yeemin, 1988) and M. verntcoxa (Mate et al., 1998) and the brooding species Porites porites (Tomascik and Sander, 1987). Although zooxanthellae are generally restricted to the gastrodermis of adult corals, they are at least temporarily observed in the ectoderm of planulae of some corals and soft corals (Szmant-Froelich, 1985; Benayahu ft al.. 1988; Benayahu, 1997; Benayahu and Schleyer. 1998; Sehwarz et al.. 1999). This is probably because infection first occurred in the ectoderm cells of embryos or early planulae (Szmant- Froelich ct al.. 1985) or because dividing cells of these stages transferred the multiplying symbionts to their daugh- ter cells, including presumptive ectoderm cells (Benayahu. 1997; Benayahu and Schleyer, 1998). In these cases, zoo- xanthellae were transferred from ectoderm to endoderm 68 ZOOXANTHELLATE EGGS OF CORALS 69 across the mesoglea before larvae develop into mature plan- ulae (Benayahu, 1997; Benayahu and Schleyer. 1998). Montgomery and Kremer (1995) also found that in the larvae of a scyphozoan, Linuche ungiiiculata. the algae were found mostly in the ectodermal cells, and suggested mech- anisms by which zooxanthellae could be transferred from ectoderm to endoderm of planulae. The corals Pocillopora eydonxi and P. verrucosa release zooxanthellate eggs, which display an uneven distribution of algal cells (Hirose et 2: 145-151. Kruger, A., and M. H. Schleyer. 1998. Sexual reproduction in the coral Pocillopora vernicosa (Cnidaria: Scleractinia) in KwaZulu-Natal, South Africa. Mar. Bio/. 132: 703-710. Mate, T. J. L., J. Wilson, S. Field, and E. G. Neves. 1998. Fertilization dynamics and larval development of the scleractinian coral Mwitipurn vernicosa in Hawai'i. Univ of Hawaii. Hawaii Institute of Marine Biology. Technical Kepuri 42: 27-39. Montgomery. M. K., and P. M. Kremer. 1995. Transmission of sym- biotic dinoflagellates through the sexual cycle of the host scyphozoan Linuche iin^iiiciilata. Mar. Bio/. 124: 147-155. Schwarz, J. A., D. A. Krupp, and V. M. Weis. 1999. Late larval development and onset of symbiosis in the scleractinian coral Fungia scutaria. Biol. Hull 196: 70-79. Sier, C. J. S., and P. J. W. Olive. 1994. Reproduction and reproductive variability in the coral Pucillopnra vernicosa from the Republic of Maldives. Mar. Kiol. 118: 713-722. Szmant-Froelich, A., P. Yevich, and M. E. Q. Pilson. 1980. Gameto- genesis and early development of the temperate coral Astrangia danae (Anthozoa: Scleractinia). Biol. Bull. 158: 257-269. Szmant-Froelich, A., M. Reutter, and L. Riggs. 1985. Sexual repro- duction of Favia fragum (Esper): lunar patterns of gametogenesis. embryogenesis and planulution in Puerto Rico. Bull. Mar. Sci. 37: 880-892. Titlyanov, E. A., T. V. Titlyanova, V. A. Leletkin, J. Tsukahara, R. van Woesik, and K. Yamazato. 1996. Degradation and regulation of zooxanthellae density in hermatypic corals. Mar. Ecol. Prog. Ser. 139: 167-178. Titlyanov, E. A., T. V. Titlyanova. Y. Loya, and K. Yamazato. 1998. Degradation and proliferation of zooxanthellae in planulae of herma- typic coral STyluphora pistillata. Mar. Biol. 130: 471-477. Tomascik, T., and F. Sander. 1987. Effects of eutrophication on reef- building corals. III. Reproduction of the reef-building coral Porites porites. Mar. Biol. 94: 77-94. Yeemin, T. 1988. A comparative study of reproductive biology in four congeneric species of scleractinian corals (Montipora) from Okinawa. Master's thesis. University of the Ryukyus, Okinawa. Reference: Bio/. Bull. 199: 76-84. (August 2000) Revised Description of the Fine Structure of in situ "Zooxanthellae" Genus Symbiodinium TIMOTHY S. WAKEFIELD1 *, MARK A. FARMER2, AND STEPHEN C. KEMPF1 ^Department of Biological Sciences, Auburn University, Alabama 36849-5414: and * Center for Advanced Ultrastructural Research, University of Georgia, Athens, Georgia 30602 Abstract. The fine structure of the symbiotic dinoflagel- late genus Symbiodinium has been well described. All of the published descriptions are based on tissue that was fixed in standard aldehyde and osmium fixatives and dehydrated in an ethanol series before embedding. When the technique of freeze-substitution was used to fix tissue from Cassiopeia xamachana, Aiptasia pallida, and Phyllactis flosculifera and prepare it for embedding, thecal vesicles were revealed within the in situ symbionts of all three species. Although these structures have been identified in cultured symbionts, they have never been described in the in situ symbionts. A review of the literature has revealed several instances where thecal vesicles were either overlooked or identified incor- rectly. Thus the formal description of the genus Symbio- dinium, which describes the in xitu symbionts. contains information that is based on artifact and should be revised. A revision of the genus is suggested, and the true nature of these structures and their significance in the symbiotic as- sociation are discussed. Introduction The symbiotic algae found in the tissues of numerous marine invertebrates and often called "zooxanthellae" have included members of the classes Bacillariophyceae, Cryp- tophyceae. Dinophyceae, and Rhodophyceae (Rowan, 1998). In recent years, common usage has relegated this term almost exclusively to the description of dinoflagellate symbionts. Loeblich and Sherley (1979) suggested that many of the ta.xonomically described dinoflagellate symbi- onts should be assigned to a new genus they termed Zoo- Minthella in recognition of very early work on dinoflagellate symbiosis by Brandt ( 1SSI >. However, continued studies in Received 27 September 1999; accepted 18 April 2000. * To whom correspondence should he addressed. symbiotic dinoflagellate taxonomy have shown that there are. in fact, many species from several genera including Amphidiiiiinn (Taylor, 197 la; Trench and Winsor. 1987), Aureodinium (Anderson and Be, 1976), Gymnodinium (Spero. 1987). Gyrodiniimi (Spindler and Hemleben, 1980), Prorocentrum (Yamasu, 1988), Pyrocystis (Alldredge and Jones, 1973), Scrippsiella and Gloeodinium (Banaszak et Jiiunin hciinuJciL'.c within the host Ai/'imui />Jininm hcriinuli'ii.w within host Aiptu.iiu pulliilu. Note the difference in thickness of the cell wall inner layer between points a and b; ch = chloroplast; arrowheads indicate accumulations of symbio- some membrane outside of vegetative cell wall. Scale bar = 500 nm. Figure 3. Dividing Symbiodinium hcnmulfiixc within Aipiasia pa/lulu. ch = chloroplast; nti = nucleus; arrowheads indicate areas of thickening of inner layer of the cell wall along the division furrow. Scale bar = 1 /nm. REVISED DESCRIPTION OF SYMBIODINIUM Table 1 Cell wall layer thickness, total cell wall thickness, and ihecal vesicle thickness lull measurements in nanometers, mean ± sttl. dev.t 79 Symbiont species IL EDL OL Total thickness (in Thecal vesicle thickness («) Symbiodinium sp. S. microadriaticum S. bermudense 96.7 ± 4S.4 40.4 ± 7.7 137.8 ± 65.5(100) IOS.4 ± 37.7(100) 10.6 ± 1.4 147.7 ± 49.9(100) 34.3 ± 7.8(100) 38.8 ± 7.3 (50) 34.9 ±8.5(100) EDL = electron dense layer. IL = inner layer, OL = outer layer; — indicates no visible layers. becomes increasingly thicker in the region of the division furrow (Fig. 3). Symbiodinium sp. from P. flosculifera and S. microadriaticum lacked this three-layered structure, the entire cell wall being similar in consistency to the IL layer of S. bermudense. A typical multilayered symbiosome membrane could be seen around the symbionts of all three host animals, but was most prevalent around S. bermudense (Figs. 1, 2, 4). The membranes that completely surrounded most symbionts seemed to be distributed evenly, but in some cases a dis- proportionate number of membranes were located to one side of the symbiont (Figs. 2. 4). ac *•• 9 \\ ' " nu cw ch Figure 4. Symbiodinium bermudense within Aiptasia pallida. ac = accumulation body; ch = chloroplast; li = lipid vacuole; nu = nucleus; PV = pyrenoid; arrowhead indicates large number of membranes on only one side of symbiont. Scale bar = 2 fj.m. Figure 5. Thecal vesicles in Symbiodinium bermudense. ch = chloro- plast; arrowheads indicate individual thecal vesicles. Scale bar = 500 nm. Figure 6. Symbiodinium sp. within the host Phylactis flosculifera. ac = accumulation body; ch = chloroplast; nu = nucleus; py = pyrenoid. Scale bar = 1 jj.ni. Figure 7. Thecal vesicle in Symbiodinium sp. from Ph\lactis floscu- lifera. ch = chloroplast; cw = cell wall; arrow = algal cell plasma membrane; arrowhead = thecal vesicle. Scale bar = 300 nm. Just inside of the cell wall is the continuous cell mem- brane. Below this cell membrane, the freeze-substitution process has revealed distinct thecal vesicles. These vesicles are most prominent in Symbiodinium bennudense (Figs. 4, 5) and the Symbiodinium sp. (Figs. 6, 7), but they are also present in S. microadriaticum (Figs. 8, 9). In TEM sections, each vesicle is membrane bound and has rounded edges at cw ^> •\ ch Figure 8. Symbiodinium microadriaticum from the host Cassiopeia \iiinachana. ch = chloroplast; nu = nucleus. Scale bar = 500 nm. Figure 9. Thecal vesicles in Symbiodinium microadriaticum. ch = chloroplast; co = calcium oxalate crystal; cw = cell wall; arrowheads indicate thecal vesicles. Scale bar = 100 nm. Figure 10. SynibioJiiiitim heriiuulcine within host Aiptasia palliJa. ch = chloroplast: cw = cell wall; ml = multiple layers of symbiosome membrane; sm = outer symbiosome membrane; arrows identify thecal vesicles; arrowheads indicate linear array of microtubules beneath thecal vesicles. Scale bar = 300 nm. 80 T. S. WAKEFIELD ET AL. Figure 11. Svmbioilininin bermiulfiixi' within heist .4/i/.w'uil ihecal vesicles appear as multiple membranes between the outei - mbiusnme membrane and the vegetative algal cell wall, cw = cell wall; ml = multiple layers of membrane; pin T symbiont plasma mcmlnaue; sm = surface of symbiosome membrane; tv = thecal vesicles. | figure adapted from Hohfeld and Melkonian. 1992.) Colley and Trench, 1983; Blank, 1987; Trench and Blank. 1987; Trench and Winsor. 1987; Palincsar et at.. 1988; Rands et til., 1993). These accumulations of apparently membranous mate- rial were first described by Taylor (19681, who ascribed their origin to both the algal cell and the host, but gave no specific structures from which they might arise. Later, Kevin et al. (1969) redefined the location of the mem- branes surrounding the algal cell, but once again failed to clearly state their origin. This uncertainty as to the origin of these membranes has continued throughout the litera- ture, with some authors attributing it to the host (Tripodi and Santisi. 1982; Colley and Trench, 1983; Palincsar et til.. 1988; Rands et al.. 1993), and others to the algal cell (Schoenberg and Trench. 1980; Trench and Blank, 1987). Specifically, Trench and Blank (1987) reported that the outer layer of the cell wall is periodically "sloughed off" the surface and often produces a "scroll-like" appearance in sections. However, they do not offer any evidence to support this process as the origin of the multiple mem- branous layers, nor do they offer any suggestions about how this sloughed layer might be regenerated outside of the continuous cell wall. Our hypothesis of a continuing in situ ecdysis cycle as the origin of the apparent mem- branes (see Fig. 12) is based on the presence of symbiont thecal vesicles in situ, and on a known event within the life cycle of the symbiont. As such, it does not require the proposal of another "unknown mechanism" to explain how additional membranes would be added to those already present around the symbiont. There is another question that must be addressed if our hypothesis is correct. If a delayed ecdysis cycle is continu- ing within the host cell symbiosome, then in addition to the shedding of the theca and plasma membrane, there must also be a shedding of the cell wall. If the thecal vesicles are retained within the host membrane, what happens to the cell wall material? Although this is a valid question, it is not unique to our hypothesis. The same question can be asked of mitotically active, vegetative cells in situ. It has been assumed that, at the conclusion of a mitotic event, the symbiont daughter cells within the same sym- biosome membrane are separated by invading extensions of symbiosome membrane and host cytoplasm (Reisser, 1992). Following division, each new daughter cell pro- duces a new cell wall within the old cell wall of the parent cell (Taylor. 1968: Kevin ct til.. 1969) Thus, if the "old" cell wall were not degraded in some way, each host symbiosome would contain remnants of cell wall mate- rial from previous mitotic events. Because such remnants have not been observed, the old cell wall material must be degraded, perhaps by enzymes released from the sym- biont itself. As was mentioned previously, the release of motile zoospores from within the parent cell wall is controlled by an unknown mechanism, presumably enzy- REVISED DESCRIPTION OF SYMBIODIN1UM 83 matic in nature. Perhaps this same mechanism is respon- sible for the degradation of the cell wall within the host symbiosome during the mitotic event, with the outer symbiosome membrane retaining active enzymes in the vicinity of the discarded cell wall. In the case of a retarded ecdysis cycle, if the thecal plates differ in com- position from the cell wall and thus are not subject to similar enzymatic breakdown, they could accumulate as the multiple layers of membranous material found be- tween the symbiosome membrane and symbiont cell wall in situ. These membranes contribute to the membranous struc- ture of the symbiosome and are part of the boundary be- tween host and symbiont. All "communication" between host and symbiont. transport of gasses, and translocation of photosynthetically fixed carbon must occur through, and in conjunction with, these membranes (Rands et ai. 1993). Thus their origin and their role in these events is of great importance. Acknowledgments We thank Dr. Daphne Fautin for her aid in identifying the anthozoan species Phyllactis flosculifera. We also thank Dr. William Fitt and the Key Largo Marine Research Labora- tory, Dr. Mike Miller and the Auburn University Biological Electron Microscope Imaging Facility, and the University of Georgia's Center for Advanced Ultrastructural Research for their assistance in this research. This work was sup- ported by NSF #9018698 (SCK), NSF-BIR #9220230 (SCK), ONR #3231114 (SCK), and a grant from the Ala- bama Agricultural Experiment Station (SCK) Journal # 6-996072. 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Symbiosis with dinoflagellates in two pelagic flat worms Amphiscolops sp. and Haplodiscus sp. Symbi- osis 3: 1-22. Tripodi, G., and S. Santisi. 1982. A study on the cell covering of Symbiodinium, a symbiote ot the octocoral Eunicellu. J. Suhniicrosc. Cytol. 14: 613-620. Yamasu, T. 1988. Symbiosis of marine animals and algae. Genetics 42: 12-20. Reference: Binl. Bull. 199: 85-94. (August 21)00) Overgrowth Competition Between Clades: Implications for Interpretation of the Fossil Record and Overgrowth Indices DAVID K. A. BARNES' * AND MATHEW H. DICK2 1 Department of Zoology and Animal Ecology, University College Cork, Cork, Ireland; and 2 Department of Biology, Middh'hitry College, Middlebury, Vermont 05753 Abstract. Overgrowth interactions (2693 in total) were observed among three major groups (arguably clades) of bryozoans — cheilostomatids (57 species), ctenostomutids (3 species), and cyclostomatids (14 species). The bryozoans studied here occur in shallow water at high-temperate polar latitudes where they encrust hard substrata such as rock piles. The main study site was the intertidal and infralittoral zones of Kodiak Island, Alaska, but observations were also made in similar zones of South Georgia Island and the Falkland Islands in the South Atlantic Ocean. Cheilostoma- tids dominated the number of species, individuals, and interactions at all depths. Intraclade interactions formed 73.7% of the encounters for cheilostomatids, 1 .6% for cten- ostomatids, and 5.7% for cyclostomatids. The competitive ranking of the three clades was broadly ctenostomatids > cyclostomatids > cheilostomatids. Significantly, these re- sults contradict all previous quantitative studies of bryozoan overgrowth, in which cheilostomatids are reported to over- grow cyclostomatids at a higher rate. From these studies and the literature, we calculated win indices to vary from 0 to 0.42 for living cyclostomatids, from 0.08 to 0.9 for living cheilostomatids, and from 0.25 to 0.75 for living ctenosto- matids. The win indices of cyclostomatid and cheilostoma- tid clades show significantly more variation in living assem- blages than in fossil assemblages. This disparity may be due to differential preservation (polar and subpolar assemblages last less than 4 years). The diversity was very high in terms of both species richness and interaction types (outcomes between competitor pairs). Comparison with the literature suggests the possibility that nearshore diversity of bryozo- ans may be bimodal (have two peaks) between high arctic Received 21 August 1999; accepted 20 April 2000. * To whom correspondence should he addressed. E-mail: DKAB@ucc.ie and antarctic latitudes. Indices of success in overgrowth competition have been constructed in various ways. For cheilostomatids, the method of calculation had little in- fluence on the ranking of representatives. In contrast, the apparent success of ctenostomatids and cyclostomatids var- ied hugely with how the index was calculated. This incon- sistency is due to the use of very different strategies in overgrowth competition; among the two latter groups, many interactions involve tied outcomes. Introduction Cyclostomatida, Ctenostomatida. and Cheilostomatida are the major groups of the Phylum Bryozoa (the fourth is exclusively fresh water). For convenience, we will use the term "clade" to refer to these major groups of marine bryozoans, but this is not entirely accurate. Although the Cyclostomatida and the bulk of the Cheilostomatida prob- ably represent monophyletic groups, or clades, Ctenostoma- tida is probably a paraphyletic group (Todd, 2000), better referred to as a "grade." Representatives of these groups occur together in most benthic assemblages, where fre- quently the encrusting members are directly competing for space and food (e.g., Stebbing, 1973; Sebens, 1986; Lopez Gappa. 1989). Cheilostomatids generally dominate the bryozoan component of assemblages in space occupied, numbers of species, numbers of colonies, and overgrowth performance. As a result of such dominance, most studies of competition between encrusting benthos have either docu- mented cheilostomatid interactions with representatives of other benthic phyla (Quinn, 1982; Sebens, 1986) or have been restricted solely to cheilostomatid-cheilostomatid in- teractions (e.g., Jackson, 1979a; Buss. 1980; Palumbi and 85 86 D. K. A. BARNES AND M. H. DICK Jackson, 1983; Tanaka and Nandakumar. 1994; Barnes and Rothery, 1996). A few studies have shown that representa- tives of the Ctenostomatida may be high or mid-ranked in overgrowth performance against the cheilostomatid repre- sentatives (Stebbing, 1973; Turner and Todd. 1994). Cy- clostomatids, in contrast, have been found to be almost always overgrown by cheilostomatids in the few studies of Recent (living) competition between the two clades (Harmelin. 1976; Buss and Jackson, 1979; Lopez Gappa, 1989: McKinney, 1992). Analysis of the fossil record has shown that competitive performance has been stable for the last 100 million years, with cheilostomatids overgrowing cyclostomatids in about 66% of encounters (McKinney, 1995a). Although broad trends have been described, many factors contribute to the outcome of interactions between any pair of competitors. Phylum membership is the principal factor determining overgrowth ability, with ascidians > sponges > bryozo- ans > unitary forms such as barnacles, annelids (Buss and Jackson, 1979; Russ, 1982; Sebens, 1986). Growth form is also important, with foliaceous forms > encrusting sheets > stoloniferous types (Buss. 1979; Barnes and Rothery. 1996). and bryozoans that have the capability of frontal budding overgrowing those that lack it (Lidgard and Jackson, 1989; McKinney, 1992; 1995a). To explain the competitive ad- vantage that cheilostomatids have over cyclostomatids, McKinney (1992; 1993; 1995a, b) has described a number of attributes, including higher growth rates and larger col- onies and feeding structures. The crucial features of chei- lostomatids, however, are probably ( 1 ) rapid ontogenetic development resulting in full-sized zooidal skeletons (and feeding structures) at colony margins, (2) labile morphoge- netic responses at colony margins (raised growing edges. frontal budding, stolon production and others), and (3) water excurrents that leave around the colony margin, po- tentially into the area of uptake for a cyclostomatid com- petitor. These features may explain the great radiation that, since the mid-Cretaceous period, the cheilostomatids have undergone relative to the cyclostomatids (Lidgard ct ul.. 1993), or this may be due to unrelated factors such as the acquirement of planktotrophic larvae (Taylor, 1988). It seems likely that an increase in encounters with a superior competitor would contribute to the decline of cyclostomatid species richness from the end of the Cretaceous to Recent periods (Lidgard el ul., 1993; McKinney, 1995a: Sepkoski et ai, 2000). Studies involving interpretation of competitive interac- tions between major groups or clades of the same phylum are rare for both living and fossil assemblages, and this is the case with the Bryozoa. Consequently, our knowledge is biased to the results of the few studies earned out and the limited distribution of the localities of these studies. Many studies are based on relatively few interactions between many species pairs and even fewer between clades, and thus a synoptic interpretation of overall outcomes is difficult. Problems of interpretation are compounded by differences in the way performance is measured (e.g., wins compared to losses, or wins compared to total interactions) and by the way contact matrices are analyzed (that is. by using transi- tivity indices) (see Petraitis, 1979; Rubin, 1982; Tanaka and Nandakumar, 1994). Perhaps the largest barrier to meaning- ful comparison, though, is that the three marine bryozoan clades have not, to date, been evaluated in the same study (at a single locality). In this study we investigate intraspecific and interspecific competitive encounters among representatives of the bryo- zoan clades Cyclostomatida, Ctenostomatida, and Cheilo- stomatida from the intertidal and infralittoral zone of Ko- diak Island, Alaska. The boreal/subpolar region is unusually diverse with respect to many taxa, but particularly bryozo- ans (see Barnes and Dick. 2000; Dick and Ross, 1986; 1988). and provides an opportunity for comparing interac- tions between abundant representatives of the three clades. We compare the outcomes using different methods of com- petitive strength calculation, and we evaluate these meth- ods. We also compare win indices and rankings of the clades with unpublished work involving interactions among the clades at two south Atlantic localities: the Falkland Islands and South Georgia Island. Win indices and rankings from other localities and time periods were extracted from the literature for comparisons with our results. Materials and Methods Study site and species Bryozoan overgrowth interactions were analyzed on 110 rocks from 14 sites at Narrow Strait, Kodiak Island (57° 54'N, 152° 27'W) in the Alaskan boreal-Arctic (see Dick and Ross, 1988, for more detail). Four tidal levels or depths were represented by a number of rock-pile sites: upper midlittoral (2), lower midlittoral (4). upper infralittoral (3). and lower infralittoral (5). Rock surface area was measured using a nonelastic grid of square centimeters as per Barnes and Rothery ( 1996), but percent cover and colony size were not measured. All competitors were identified into the three orders of bryozoans present and to genus or species level where possible. Poor taxonomic resolution in the initial stages of the study led to uncertainty about the particular species involved in interactions within the genera Caulor- lutinpluis (5 species), Microporella (4 species), Celleporella (2 species), and Alcyonidium (2 species). The cyclostoma- tids from NaiTow Strait have not been worked up taxo- nomically and here were identified to ordinal level only, with the exception of a common lichenoporid designated Lichenopom sp. COMPETITION BETWEEN BRYOZOAN CLADES 87 Table 1 Mean number of species per rock and proportion of rocks (in pari'nihi'scx) ci>li>ni:ril hy species occurring rarely ui tilt' Miul\ \ites, Altisku Mean no. species per rock Tidal cover/depth Cyclostomatids Ctenostomatids Cheilostomatids Upper midlittoral Lower midlittoral 0 0.88(0.31) 0.25(0.13) 0.75 (0.09) 4.88 (0.49) 6.81 (0.49) Upper infralittoral Lower infralittoral 1.54(0.72) 4.25 (0.44) 0.54(0.25) 0.73 (0.30) 1(1,7(1.00) 13.0(0.63) Measurement of interactions All colony-to-colony interactions between representa- tives of the three clades of bryozoans were recorded from each rock and site, along with the number of intraspecific and interspecific encounters within clades. When the grow- ing edge of competitor A covered the apertures of compet- itor B, A was determined to have overgrown B. Only "frontal" overgrowth interactions between two living com- petitors, without direct settlement onto one of the compet- itors, was counted as overgrowth for the purposes of this study (see Rubin, 1982; Turner and Todd, 1994; Barnes and Rothery, 1996). The actual scores were tabulated into a competitor-contact matrix (as Turner and Todd, 1994; Barnes and Rothery, 1996). Measurements of overgrowth performance were calculated for each competitor that took part in more than 20 between-clade interactions. Various measurements of overgrowth performance were used: a score system in which a win = 3, a tied outcome = 1 . and a loss = 0 (wins rated much higher than ties because ties often prevent further growth and development of colonies, see Barnes and Clarke [1998]); the number of wins divided by the total number of interactions for that competitor; the number of losses divided by the total number of interactions for that competitor; the number of wins divided by the number of losses for that competitor; and an aggregate measure in which the mean of the rankings from all methods was obtained. The rankings in Table 2 are those calculated from raw data. These may differ from true population rank- ings because the number of encounters with each competitor was not the same for each species, and some potential competitors did not meet. The rankings were standardized by multiplying all pairwise interactions such that each had a total value of 100, then recalculating the total wins, losses, and ties for each competitor identity. Results Between-species interactions A total of 74 species of bryozoans were recorded during the study: 57 cheilostomatids, 3 Ctenostomatids, and the remainder cyclostomatids (later analyzed to be 14 species). The mean number of cheilostomatid and cyclostomatid spe- cies per rock increased with depth of rocks (Table 1), the former dominating the number of species at all depths. The proportion of ctenostome species was small with respect to the total number of bryozoan species, and it varied incon- sistently with depth. The mean number of interactions in- creased with depth for all clades (Fig. 1 ). Cheilostomatids were involved in 2653 interactions, of which 73.7% were within the clade; Ctenostomatids were involved in 367 in- teractions, of which 1 .67r were within the clade; and cy- clostomatids were involved in 384 interactions, of which 5.7% were within the clade. All cheilostomatids that en- countered Ctenostomatids or cyclostomatids on more than 20 occasions are illustrated in a species-contact matrix (Table 2). The remaining cheilostomatid species are pooled because fewer than a total of 20 competitive interactions were not considered to be representative. Certain pairs of competitors had anomalously higher frequencies of encoun- ters, such as Alcyonidium spp. and Porella alba. The chei- lostomatid species Callipora craticiila encountered only Ctenostomatids and cyclostomatids, despite the overwhelm- ing numerical dominance of cheilostomatids and being in- volved in 30 interspecific interactions. Most (99.3%) of the intra-clade encounters observed were interspecific interactions. Over 80% (1 14) of the pos- sible competitor-pair interactions (136 in the matrix Table 2) and 44 of the 45 between-clade interactions were ob- served (but these represented only a small proportion of the 74 X 74 species interactions theoretically possible). The proportion of indeterminate outcomes (neither competitor won all encounters) from competitor pairs was significantly higher within the clade of cheilostomatids (15.9%) than between cheilostomatids and other clades (5.1%) (Mann- Whitney U test, P < 0.01 ). The proportion of tied out- comes or standoffs in competitor pairs was significantly higher between clades than within clades (Mann-Whitney, P < 0.001 ). The proportion of ties was also significantly Lower mid °s = — Upper infra - 0 50 100 150 200 Number of interclade interactions Figure 1. Mean number of interactions per clade with depth. All data are presented as mean with standard error. 88 D. K. A. BARNES AND M. H. DICK Table 2 Matrix of competitive interactions for Alaskan cheilostomatid, ctenostomatid, and cyclostomatid bryozoans (latter nro groups are grav shaded) F-; i~ § C C 5 t-: 3 5 £ I £ •5; 1 254321 121011 3 0 1 3 2 •) 0 \ 4 3 8 1 7 Tegella anmfera 9 16 3 10 1 4 10 13 2 3 2 4 1 4 27 31 13 17 6 1 44 51 10 19 25 0 11 0 0 211 0 0 1 2 0 1 2 2 0 2 0 3 2 8 1 5 Tegella aquilostris 8191 1 471267 2 4 16 19 6 8 3 5 24 29 7 16 24 0 0 251 0 1 3 3 0 4 5 9 5 0 0 5 2 5 0 11 Myrio-oclla plana 3 3 7 14 5 604 0 3 34 43 37 51 1 1 1 1 8 15 3 8 41 024042 9 2 5 13 9 7 3 2 35 5 20 2 9 Caiilorhamphits sp. 1 32639 1 12 21 39 9 25 4 9 1 1 51 2 24 77 43131 1 6 8 12 14 14 14 6 2 6 3 3 1 8 Other Cyclostomatids 372624 1 15 16 42 20 48 5 13 10 19 3 7 39 2 1 2 0 1 3 0 0 0 2 9 2 5 2 2 Microporella califomica 6 9 0 2 3 7 1 1 0 2 16 27 0 7 26 1 1 3 3 2 5 0 0 5 3 14 0 5 Tegella urtica 4 13 19 1 1 1 6 12 2 7 1 21 8 29 0 6 4 20 21 1 16 Lichenopora sp. 5 17 7 15 3 11 5 13 0 5 21 8 53 7 9 58 47 14 11 IS Other Cheilostomatids 49 110 4 20 43 148 9 17 94 6 13 33 42 15 6 34 Microporella sp. 7 26 27 102 6 27 89 9 6 5 3 10 Cribnlina annit lain 5 20 1 9 14 61 24 40 Poi r//<; alba 16 KM) 73 5 1 34 Alcyt tnidium sp. 1 6 39 Celleporella sp. Data are displayed in standard form — see Turner and Todd (1994), Barnes and Rothery ( 1996). If row = competitor A and columns = competitor B. for each cell the top left, top right, and bottom left data give, respectively, the number of ties between species A and B, wins by B (= losses by A) and wins by A (= losses by B). The number in the bottom right of each cell is the total number of observed interactions for that species pair. higher in the Ctenostomatids than in the \uin-Liclu'n<>/>oni cyclostomatids (Mann-Whitney, P < 0.001) but was not significantly different from that in the total cyclostomatids (Mann-Whitney. P = 0.053). Both clades had a signifi- cantly higher proportion of tied outcomes than did the cheilostomatids (Mann-Whitney, P < 0.001 ). Ctenostomatids and cyclostomatids met too infrequently to assess their overgrowth performance against each other. Both of these clades, however, encountered cheilostomatids on many occasions anil won more encounters than they lost. In both the midlittoral to the infralittoral, Ctenostomatids won about 55% of the encounters that had a decided out- come, but the proportion of ties increased from from 4% in the midlittoral to 58% in infralittoral. Cyclostomatids were better competitors against cheilostomatids, winning 87% of midlittoral encounters and 62% of infralittoral encounters. As with ctenostomatid-cheilostomatid encounters, the pro- portion of tied outcomes increased from the midlittoral to the infralittoral, but less dramatically, from 31% to 35%. The overall transitivity of the assemblage measured using the index of Tanaka and Nandakumar ( 1 994) was 0.62. This was 25% lower than the value obtained for interactions just within the clade of cheilostomatids (0.83 — Barnes and Dick, unpubl. data). This value indicates a generally hier- archical system (Buss, 1980; Russ, 1982) but, as predicted by Jackson (1979b). one that is more intransitive between clades than within the clade of cheilostomatids. The competitors involved in interactions could be ranked in a sequence of overgrowth performance from several typically overgrown by others (cheilostomatids) to several typically overgrowing others (also cheilostomatids). Over- growth performance can be and has been measured in a COMPETITION BETWEEN BRYOZOAN CLADES 89 Table 3 Ranking of competitive ability in Alaskan cheilostomatid, ctenostomatid. and cyclostomatid hryo-oans (latter two clades are in bold), with nutnex listed in descending order of initial number of wins Standardized ranking Taxon identity and coding Initial Win-tie scored WAT L/T W/L Aggregate Tegella armifera (Ta) Ta Tq Tq Tq Ta Tq Tegella aquilirostris (Tq) Tq Ta Ta Li Tq Ta Myriozoella plana (Mp) Mp Mp Mp Ta Mp Mp Cauloramphus sp. (Ca) Ca Ca Tr rv Mp p.. Ca Mm Ca Tr Microporella californica (Me) Me Tr v-y Ca L, d A Tr i r Li Tegella arctica (Tr) Tr Me Me Tr Cy Cy Lichenopora spp. (Li) Li Li M Me Me Me Cheilostomatids (C) C M C Cy A A Microporella sp. (M) M C Cn Cn M M Crihrilina awutlata (Cn) Cn A Li Pa C Cn Porella alba (P) P Cn A M Cn C Aleyonidium spp. (A) A P Pa C Pa Pa Cylindroporella tubulosa (Ct) Ct Ce Ce Ce Ce Ce Celleporella spp. (Ce) Ce Ct Ct Ct Ct Ct Rankings are Initial (number of wins) and Standardized (multiplied up so every competitor meets each other on the same number of occasions). Standardized rankings are win-tie scored (wins score 3, ties score 1). W/T (proportion of wins over total number of interactions). L/T (proportion of losses over total number of interactions), W/L (proportion of wins over losses), and aggregate (of W/T, L/T, and W/L). "Cheilostomatids" in the species-identity column refers to the remainder of the cheilostomatid species present but not listed. number of ways (Table 3). A ranking based on the number of wins (actual overgrowth of the competitor) placed some cyclostomatids as intermediate competitors but others (Lichenopora sp.) and the ctenostomatids as poor competi- tors. In contrast, lichenoporid cyclostomatids and. to a lesser extent, ctenostomatids were good competitors when as- sessed by the ratio of losses to total interactions (they were rarely overgrown). Cheilostomatids spanned the whole range of competitor performance, but those that scored highly in the win index also scored highly on the loss index (i.e.. good overgrowers were rarely overgrown; poor over- growers were usually overgrown). The average ranking change for a competitor between these different indices was 1.4 for Cheilostomatids, but 6 for ctenostomatids and 6.5 for cyclostomatids. At the level of species, the performance of selected Cheilostomatids against the combined representa- tives of each clade is illustrated in Table 4. All performed better against other Cheilostomatids than against ctenosto- matids or cyclostomatids, but most performed better against cyclostomatids than against ctenostomatids (e.g., Micro- porella californica), although a few (e.g., Porella alba) did the converse. A good competitor against one clade was generally a good competitor against the other, but some (e.g., Microporella californica) had quite different perfor- mances against competitors from different clades (Table 4). The between-clade win index of the three clades varied (Table 5) between the Alaskan site and others we analyzed from County Cork (Ireland) and the Falkland Islands and South Georgia Island (both South Atlantic). Other literature and unpublished data in Table 5 for which between-clade win scores have been calculated show the overgrowth scores of cyclostomatids in Alaska and County Cork to be the highest recorded. Table 4 Performance of various Alaskan cheilostomatid species in overgrowth interactions with other Cheilostomatids. ctenostomatids and cyclostomatids: values are the probability of a win for competitor A against competitor B Competitor B Competitor A identity Cheilostomatids Ctenostomatids Cyclostomatids Tegella aqid/ostris 0.76 0.44 0.55 Tegella armifera 0.71 0.53 0.65 Myrio-oel/a plana 0.66 0.38 0.41 Laganicella neosocialis 0.60 — 0.29 Caidoramphis spp. 0.52 0.08 0.07 Tegella arcticu 0.51 0.30 0.38 Microporella californica 0.46 0.00 0.33 Cribrilina anituUitu 0.34 0.09 0.13 Microporella spp. 0.32 0.22 0.25 Porella alba 0.29 0.16 O.I I Cel/epore/la spp. 0.22 0.14 0.12 Callipora craticn/u 0.06 0.14 90 D. K. A. BARNES AND M. H. DICK Table 5 overgrowth competition performance scores and rankings in the three bryo-oan clades (all other rankings are cheilostomatids) Cyclostomatids vs. Cheilostomatids vs. Ctenostomatids vs. cheilostomatids Cyclostomatids cheilostomatids vs. Ctenostomatids* vs. Ctenostomatids* Location Latitude Win index Ranking Win index Ranking Win index Alaska (USA)" 57 N 0.24 7/12 0.42 (0.25*) 6/13 0.21 (0.18*) Scotland 56 N 0.5 5/18 — — (0.32*) Ireland""0 ' 51.5 N — — 0.41 6/8 0.56 Ireland""7 2t 51.5N 0.56 1/6 — — (0.37*) England SON 0.75 1/5 — — (0.08*) Croatia 43 N — — 0.08 — 0.78 Jamaica 18 N — — Low 10/10 -0.9 Mozambiquet 12 S — — 0.2 13/15 0.60 Australia 38 S — — 0.0 8/8 0.74 Argentina 47 S — — 0.33 9/12 0.62 Falkland Is 52 S — — 0.32 7/10 0.44 South Georgia'!' 54 S 0.4 1/7 0.18 (0.08*) 6/7 0.6 (0.32*) Signy 1st 60.5 S — — 0.1 ~> ">/">"> 0.73 Data are taken from Stebbing ( 1973). Buss and Jackson ( 1979). Russ ( 1982), Lopez Gappa (1989). McKinney ( 1992), Turner and Todd ( 1994), Maughan and Barnes (in press), Barnes and Lehane (unpubl. data), present study (") and unpublished sources (t). Discussion Amongst the major groups of the phylum Bryozoa, chei- lostomatids are generally the major space occupiers, the most speciose, and the superior overgrowth competitors (see, for example. Buss and Jackson, 1979). Sometimes they may even locally dominate the macrobenthic community— for example, on shells (Kay and Keough, 1981), on reef rubble (Jackson and Winston. 1982), on floating debris (Barnes and Sanderson, 2000). in mid-latitude shelf waters (James et aL. 1992). and on Antarctic shallow-water hard substratum (Barnes, 1995). When present, the rarer Cteno- stomatids may effectively compete against cheilostomatids (Stebbing. 1973), but Cyclostomatids are typically over- grown in meetings (Buss and Jackson, 1979). Overgrowth of calcified benthos by soft-bodied forms such as ctenosto- matid bryozoans or ascidians may not. however, always prove lethal to the overgrown competitor (Todd and Turner, 1988). In this study, the first to encompass all three "clades." at the same localities, cheilostomatids were the major space occupiers, were involved in most interactions, and were the most speciose of the three clades, as found elsewhere (Table I ). They were, however, outcompeted by Ctenostomatids at all three localities, and at Kodiak Island, Alaska, they were uniquely outcompeted by Cyclostomatids (Table 5). McKinney analyzed living and Recent relict (1992; 1995b) and fossil ( 1995a) cyclostomatid and cheilostomatid interactions and found that living and Recent relict assem- blages in Rovinj. Croatia, were similar to those over the last 105 million years. The win index of fossil cheilostomatids oscillated around a value of 0.66 (66% win rate), and that of Cyclostomatids at around 0.29. In the Alaskan assemblage studied here the win index of Cyclostomatids was nearly 1.5 times greater and the win index of cheilostomatids 3 times lower. The win index of clades varied with site (Table 5) or possibly latitude (Fig. 2). In the living assemblages studied here (from Alaska, the Falkland Islands, and South Georgia Island) and other indices taken or calculated from the liter- ature, the win indices of Cyclostomatids varied from 0 to 0.42. The win indices of living cheilostomatids varied be- g nj E 2 05 - g ° 03 - 1.1 -20 0 20 Latitude ("South) Figure 2. Magnitude of overgrowth C7r I of Cyclostomatids by cheilo- stomatids in Meso/oic and Ceno/oic periods, adapted from McKinney (1 995 1. COMPETITION BETWEEN BRYOZOAN CLADES 91 tween 0.08 and 0.9 and those of living ctenostomatids from 0.25 to 0.75 from the few studies carried out (Table 5). There is significantly more variation (Fig. 3) in the living assemblages that have been studied than in the fossil as- semblages that have been studied in both the cyclostomatid clade (homogeneity of variance test. F = 7.6, P < 0.01 ) and the cheilostomatid clade (F = 24.0, P < 0.01 ). The sample size in both the living and fossil assemblages is reasonable (though not high in the former). McKinney (1995b) has shown that, through careful consideration of differential preservation of overgrower and overgrown, analysis of fossil assemblages probably gives an accurate representation of conditions at the time of preservation. Either there is more variation today than over the past 100 million years or we are getting some information from living assemblages that we are not getting from fossil as- semblages. There is some evidence for both of these expla- nations. The highest values of cyclostomatid win index and the lowest values of cheilostomatid win index are from high (50°-60°) latitude localities (this study and Maughan and Barnes, unpubl. data). The high values for the cheilostoma- tid win index are generally from studies in mid to low latitudes (and typically from more sheltered sites) (e.g., Buss and Jackson, 1979; McKinney, 1992). Thus the high range of modern index values is probably partly due to the range of exposure of localities. Antarctic (high latitude) O) I o 'o c o '•e o Q. O D. 9 Fossil assemblages 0 Living assemblages .. :? Cyclostomatids i or- § Cheilostomatids -100 -80 -60 -40 -20 0 Time (Million yrs) Figure 3. Non-cheilostomatid (ctenostomatid and cyclostomatid) spe- cies richness of rock-pile habitats with latitude. Data points are from Powell and Crowell ( 1 967 ), Gordon ( 1 980 ), Winston 1 1 982 ), Cook ( 1 985 ). Rao and Ganapati ( 1985). McKinney ( 1992), Barnes el ul. ( 1996). Barnes and Arnold ( 1999). Maughan and Barnes (unpubl. data), present study, and unpublished sources. •60 -40 •20 0 20 Latitude (degrees) Figure 4. Cyclostomatid win index with latitude. Dala points are from Buss and Jackson (1979). Russ (1982). Lopez Gappa (1989), McKinney (1992), Barnes and Rothery (1996). Maughan and Barnes (in press), present study, and unpublished sources. values (from data from Barnes and Rothery. 1996; Barnes, unpubl. data), however, also showed high values for the cheilostomatid win index (Fig. 4). The geologically recent succession of glaciation periods, cooling of the poles, and separation of continents has probably resulted in higher levels of disturbance through wave action and ice-scour than before. So the range of nearshore conditions may be greater than in the past 100 million years. Depth is a potential confounding factor because most interaction data that has been recorded from fossil communities is from deeper water shelf environments (see Taylor and Allison, 1998). whereas most modern data sets of similar nature are from shallow water (see Buss and Jackson. 1979: Russ. 1982; Turner and Todd. 1994; Barnes and Rothery. 1996). However, studies of fossil assemblages such as those by McKinney ( 1992) are largely from mid latitudes, suggesting that conditions for fossilization of communities seem to be most prevalent in mid-latitude conditions (Taylor and Alli- son. 1998). The survival of encrusting communities (and. more specifically, bryozoan colonies) becomes progres- sively shorter with increasing latitude within the Southern Ocean, such that the longest survival time of any individual (even skeletal material) at 68° S is just 4 years (Barnes and Arnold, 1999). Additional support is provided by the high win-index values, similar to those in the fossil record, of cheilostomatids in mid to low latitudes and in sheltered localities (Buss and Jackson, 1979; McKinney. 1992). Thus it is possible, and even probable, that a broad range of index values have occurred throughout the last 100 million years but have not been preserved because the very conditions that yield extreme values prevent preservation (Lescinsky. 1993; McKinney, 1995b). This situation makes judgments about the evolutionary ecology and historical position of clades and the non-escalation of competition problematical (Liddell and Brett, 1982; McKinney (1992; 1995a. b). 92 D. K. A. BARNES AND M. H. DICK The diversity of the Alaskan assemblages studied here, with respect to both species richness and variety of species- pair interactions, was high — more so than at any compara- ble site studied to date (Barnes and Dick. 2000). The next highest value of both non-cheilostomatid and cheilostoma- tid species richness in intertidal or shallow subtidal rock- pile habitat is from the temperate zone of the southern hemisphere (Russ, 1982). James et al. (1992) have also shown bryozoan-rich carbonates peaking in mid-latitude shelf environments. This finding raises the possibility of two peaks in nearshore bryozoan species diversity centered around temperate shores (Fig. 4). though clearly more data would be needed to test this hypothesis. If such a pattern is mediated through frequency of disturbance, one would ex- pect the peak in the northern hemisphere to be at a higher latitude than that in the south because the continental effect of Antarctica increases the range of latitude influenced by ice scour in the southern hemisphere. Species richness typ- ically increases towards the tropics (Thorson, 1957; Kendall and Aschan. 1993), but may peak around subequatorial levels (Silva, 1992). Bryozoans (along with polychaetes) are one of the few taxa that have a diversity center in Antarctic waters (Clarke, 1992; Hayward. 1995). The definition of a win or tie in overgrowth competition has undergone some evolution (Jackson, 1979a). A tied outcome has been found to represent a variety of situations including a cessation of growth (e.g., Stebbing, 1973), mi- nor overgrowth (Russ, 1982), mere stalling of a future win for one of the two competitors (Sebens, 1986), mutual overgrowth (McKinney, 1992), fusion of colonies (intraspe- cific meetings), and redirection of growth (Barnes and Roth- ery, 1996). Tanaka and Nandakumar (1994) argued that a tied outcome was a result equal in importance to a win or a loss and should be included in index calculations. The method of win index tabulation, analysis, and interpretation has also changed dramatically, but for bryozoans has typi- cally been based around cheilostomatids (Petraitis. 1979; Buss and Jackson. 1979; Rubin, 1982; Tanaku and Nanda- kumar, 1994). In this study, the various methods of ranking competitors in overgrowth competition indicate that the type of index of success used has little influence on the relative or absolute positions of cheilostomatids. In contrast. the apparent success of ctenostomatids and cyclostomatids varies hugely with how the index is calculated because they use a very different strategy in overgrowth competition; many inici -:iions result in tied outcomes. The strategy is essentially del .nsive rather than offensive, involving not many wins but not many losses. Such a strategy may pay off better where encounters and superior competitors are rare, because either a win or a tie may result in persistence, hut a loss can be lethal (though not necessarily to the genet). In very exposed environments, such as that in the present study and in Antarctic assemblages, encounters and good compet- itors are rare due to habitat ephemerality, so even poor competitors may dominate assemblages (Barnes and Clarke, 1998). The non-lichenoporid cyclostomatids have a higher number of wins, but also a higher number of losses, than lichenoporids. Most cheilostomatids also have mostly de- terminate interactions, which may be a better strategy where habitats are more stable and competition is more intense. To tie with one competitor would not ensure persistence, as sooner or later an even better competitor will arrive. Al- though the assemblages studied here have not been followed through time, ties involving cyclostomatids cannot be con- sidered likely to be delays on eheilostomatid wins (see Rubin, 1982; Sebens, 1986). This is partly because the majority of decided outcomes between these clades in- volved a win for the cyclostomatid competitor and partly because the majority of tied outcomes observed in other similarly exposed latitudes (e.g.. South Georgia Island [Barnes and Arnold, 1999]; Signy Island [Barnes and Roth- ery. 1996]) had remained as "standoffs" for a period of years. The disadvantages of cyclostomatids compared to cheilostomatids. in functional body plan and feeding or water flow dynamics (McKinney. 1992), may be reasons for achieving a defensive rather than aggressive strategy in overgrowth competition. There may, however, be other explanations, such as differential growth rates or budding patterns between environments; and other selective forces, such as the frequency of disturbance, may be more impor- tant. Although the cyclostomatids are marginally superior to the cheilostomatids in overgrowth competition at Kodiak island, Alaska, the other study locations and literature sug- gest that this is atypical. The clades on aggregate are ranked ctenostomatids > cheilostomatids > cyclostomatids, but all, particularly the cheilostomatids, have a range of com- petitors with widely varying overgrowth strength and strat- egy. This study suggests, however, that locality, method of measurement, and number and identity of clades included in competition studies have important influences on and im- plications for the result obtained. Acknowledgments We thank those who assisted with data collection: Pat Chan and Clare Lehane (University College Cork) for the County Cork and Falkland Islands data; Rod Arnold (British Antarctic Survey) for the South Georgia Island data; and the Society for Environmental Exploration, FRONTIER, for the Mozambique data. We also wish to thank Irene O'Sullivan for help with preparation of Table 2. Literature Cited Barnes, D. K. A. 1995. Sublittoral epitaunal communities at Signy island. Antarctica: II. Below the ice foot /one. Mar. Bio/. 121: 565- 572. Barnes, I). K. A., and R. J. Arnold. 1999. Possible latitudinal clines in COMPETITION BETWEEN BRYOZOAN CLADES 93 Antarctic intertidul and suhtidal /.one communities encrusting ephem- eral hard substrata. J. Biogeog. 26: 207-213. Barnes, D. K. A., and A. Clarke. 1998. The ecology of an assemblage dominant: the encrusting bryozoan Fenestriilinu rugula. Invertebr. Bioi 117: 331-340. Barnes. D. K. A., and M. H. Dick. 2000. 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A faster paced world? Contrasts in biovolume and life process rates in cyclostome (Class Stenolaemata) and cheilo- stome (Class Gymnolaemata) bryozoans. Paleobiology 19: 335-351. McKinney, F. K. 1995a. One hundred million years of competitive interactions between bryozoan clades: asymmetrical but not escalating. Biol. J. Linn. Soc. 56: 465-481. McKinney, F. K. 1995b. Taphonomic effects and preserved overgrowth relationships among encrusting marine organisms. Pa/aios 10: 279- 282. Palumbi, S. R., and J. B. C. Jackson. 1983. Aging in modular organ- isms: ecology of zooid senescence in Steginoporclla sp. (Bryozoa: Cheilostomata). Biol. Bull. 164: 267-278. Petraitis, P. S. 1979. Competitive networks and measures of intransivity. Am. Nat. 114: 921-925. Powell, N. A., and G. D. Crowell. 1967. Studies on Bryozoa (Polyzoa) of the Bay of Fundy region. I. Bryozoa from the intertidal zone of Minas Bay and Bay of Fundy. Call. Biol. Mar. 8: 331-347. Quinn, J. F. 1982. Competitive hierarchies in marine benthic commu- nities. Oecologia 54: 129-135. Rao, K. S., and P. N. Ganapati. 1986. Distribution pattern and abun- dance of Bryozoa on rocky shores at Visakhapatnam. Bay of Bengal. India. Pp. 563-578 in Indian Ocean Biology of Benthic Marine Or- ganisms. M. F. Thompson, R. Sarojini. and R. Nagabhushanam. eds. Oxford and 1BH Publishing, New Delhi. Rubin. J. A. 1982. The degree of intransivity and its measurement in an assemblage of encrusting cheilostome Bryozoa. J. Exp. Mar. Biol. Ecol. 60: 119-128. Russ, G. R. 1982. Overgrowth in a marine epifaunal community: com- petitive hierarchies and competitive networks. Oecologia 53: 12-19. Sebens, K. P. 1986. Spatial relationships among encrusting marine or- ganisms in the New England subtidal zone. Ecol. Monogr. 56: 73-96. Sepkoski, J. J., F. K. McKinney, and S. Lidgard. 2000. Competitive displacement among post-Paleozoic cyclostome and cheilostome bryo- zoans. Pa/eobiology 26. (in press). Silva, P. C. 1992. Geographic patterns of diversity in benthic algae. Puc. Sci. 46: 429-437. Stebhing, A. R. I). 1973. Competition for space between the epiphytes of /••//cii.v serratns. L. J. Mar. Biol. Assoc. UK 53: 247-261. Tanaka, M., and K. Nandakumar. 1994. Measurement of the degree of D. K. A. BARNES AND M. H. DICK intransitively in a community of sessile organisms. J. E.\p. Mar. Biol. Eco/. 182: 85-95. Taylor. F. D. 1988. Major radiation of cheilostome bryozoans: triggered by the evolution of a new larval type? Hist. Biol. 1: 45-64. Taylor, P. D., and A. Allison. 1998. Bryozoan carbonates through time and space. Geology 26: 459. Thorson, G. 1957. Bottom communities (sublittoral and shallow shelf). Pp. 461-534 in Treatise on Marine Ecology and Paleoecology Vol. 1. Ecology, i. W. Hedgpeth, ed. Geological Society of America Memoir 67, New York. Todd, J. A. 2(1(10. The central role of Ctenostomes in bryozoan phylog- eny. Pp. 104-125 in I 1th International Bryozoology Assoc. Confer- ence. A. Herrera Cubilla and J. B. C. Jackson, eds. Smithsonian Tropical Research Institute, Panama City, FL. Todd, C. D., and S. J. Turner. 1988. Ecology of intertidal and sublit- toral cryptic epitaunal assemblages. II. Nonlethal overgrowth of en- crusting bryozoans by colonial ascidians. ./. Exp. Mar. Biol. Ecol. 115: 113-126. Turner, S. J., and C. D. Todd. 1994. Competition for space in en- crusting bryozoan assemblages: the influence of encounter angle, site and year on outcome variability. J. Mar. Biol. Assoc. UK 74: 603-622. Winston, J. E. 1982. Marine bryozoans (Ectoprocta) of the Indian river area (Florida). Bull. Am. Mas. Nat. Hi\t. 173: 99-176. Marine Biological Laboratory Woods Hole Massachusetts One Hundred and Second Report for the Year 1999 One Hundred and Eleventh Year Officers of the Corporation Sheldon J. Segal, Chairman of the Board of Trustees Frederick Bay. Co-Vice Chair Mary J. Greer, Co-Vice Chair John E. Dowling, President of the Corporation John E. Burris, Director and Chief Executive Officer Mary B. Conrad. Treasurer Robert E. Mainer, Clerk of the Corporation Contents Report of the Director and CEO Rl Report of the Treasurer R7 Financial Statements RN Report of the Library Director R19 Educational Programs Summer Courses R21 Special Topics Courses R25 Other Programs R32 Summer Research Programs Principal Investigators R35 Other Research Personnel R3(i Library Readers R38 Institutions Represented R39 Year-Round Research Programs R43 Honors R55 Board of Trustees and Committees R62 Administrative Support Staff R66 Members of the Corporation Life Members R69 Members R70 Associate Members R80 Certificate of Organisation R84 Articles of Amendment R84 Bylaws R84 Publications . R89 Photo credits: B. Armstrong . . . .R5. R21, R22, R23, R24. R25, R27. R30, R31, R35, R36. R37. R69, R84 J. Atema R44 M. Dobbins R46 M. Dornblaser Rl L. Eckelbecker R58 L. M. Colder R3 (bottom) R. Howard R55 T. Kleindinst R19 B. Liles R2 G. G. Lower R7 A. C. Malier R43 P. Presley R4 R. Silver R48 (bottom) Tsoi/Kobus R3 (top) L. Amai-al Zettler . R62 Report of the Director and Chief Executive Officer I am pleased to share with you this report as I have each year for the past seven years. 1999 is especially meaningful as it marks my last full year serving as Director and CEO of this wonderful institution. In August of 2000 I will leave the Laboratory to serve as the President of Beloit College, a small liberal arts institution in Wisconsin. This is an exciting opportunity for my family and me, but leaving the MBL. the community that we call home, and our friends and colleagues, will be difficult. I have been proud to serve as Director of the Marine Biological Laboratory. The MBL is a remarkable and special place, thanks largely to the dedication and commitment of its scientists and staff. Curiosity, camaraderie, and thoughtfulness abound here. Throughout my tenure I have been impressed by the innovation and technical expertise at the Laboratory, which enables awe- inspiring advances in our knowledge. 1999 was a landmark year of growth and prosperity for the Marine Biological Laboratory. I am pleased to report that our finances are sound, our educational programs exceptional and expanding, and our research efforts increasingly exciting and novel. More and more, public awareness of the MBL's importance to biology, biomedicine. and environmental science is growing. I am excited about what the future holds for the Marine Biological Laboratory. Thanks to the generosity of the many donors to the Discovery Campaign, we have already enhanced our educational program, strengthened our resident and summer research programs, made major strides towards building a new research facility for The Ecosystems Center, and begun shoring up our physical plant. Although we have work yet to do to complete the Campaign by December 2000, the Laboratory is now well positioned to continue its leadership role in the biomedical and environmental sciences well into the 2P' Century. The Marine Resources Center One of the many gratifying gifts of this campaign was made recently by a long-time, dear friend of the Marine Biological Laboratory. Late in 1999. Honorary Trustee Ellen Grass made a historic gift when she endowed the director's chair of the Marine Resources Center. This is the first time in the history of the Laboratory that a research center director's chair has been endowed. This far-sighted gift will enable us to expand our research projects in the Marine Resources Center, while ensuring the MBL's ability to attract high-quality leadership for this key facility in perpetuity. The MRC is one of the world's most advanced facilities for maintaining and culturing aquatic organisms essential for biological, biomedical, ecological and aquacultural research. As I reported last year, the Ryan- Dowling Program in Scientific Aquaculture has been established at the Marine Resources Center. We are in the final stages of conducting a national search for a scientific aquaculturist who will oversee this exciting effort. The MRC is already actively culturing organisms for biomedical research. Currently the MRC is host to a colony of zebrafish. These tiny freshwater fish have become an exciting and important research model used by embryologists, geneticists and developmental neuroscientists. Interestingly, zebrafish and humans share much of the same genetic material. These two-inch natives of India may hold the key to understanding how all vertebrates — including humans — develop from an embryo into a whole organism. What we learn from a zebrafish ultimately may help us understand — and perhaps Rl R2 Annual Report treat — a wide range of birth defects, among other disorders. MRC Director Roger Hanlon and his colleagues made substantial progress in 1999 in adapting and applying DNA fingerprints to test sexual selection processes in squid. Their results are being used by fishery managers as they decide whether to continue to allow targeted fishing on spawning squids off Cape Cod. In another area of research at the MRC, studies have progressed on understanding the mechanisms and function of polarization vision in cephalopods. MRC investigators recently found that cuttlefish are able to overcome the counter-shading camouflage of silvery fishes by detecting polarization patterns that are reflected by fish scales. The result is that cuttlefish and squid can easily detect and prey upon species that are otherwise camouflaged to most predators. The Ecosystems Center One of the most crucial objectives remaining to be met in our Discovery Campaign is building the new Environmental Sciences Building to house the MBL's Ecosystems Center. Founded 25 years ago, the Center is home to an interdisciplinary group of scientists whose expertise covers the fields of terrestrial and aquatic ecology, microbiology, chemistry, botany, zoology, physiology, hydrology, mathematics, and genetics. Their goal is to study the impact of humankind on the environment and discover what must be done to sustain and manage the earth's resources. Among key environmental issues being examined are the ecological consequences of global warming, the effects of tropical deforestation, how trees in northeast forests are handling excess nitrogen, and how pollution and habitat destruction are damaging coastal ecosystems. The problems are global and so are the Center's research sites. MBL scientists have been conducting more than 30 projects around the world — in Brazil, Alaska, Sweden, Russia, and East Africa, as well as closer to home in the woods of northern New England and along coastal estuarine systems at both Plum Island, north of Boston, and here in Waquoit Bay on Cape Cod. This summer, for example. Senior Scientist Bruce Peterson will travel to Siberia to work on the Russian- American Initiative on Land-Shelf Environments sponsored by the National Science Foundation. The goal of the project is to estimate the flux of nutrients from Eurasia to the Arctic Ocean. Nearby on Martha's Vineyard, scientists from The Ecosystems Center are working on a pilot ecosystem restoration program. Assistant Scientists Chris Neill and Mathew Williams have begun collaborating with The Nature Conservancy on a 10-year study of how a forest functions. The project involves large-scale cutting, burning, and restoration on a track of land on the Vineyard. Pre-treatment monitoring will be conducted in 2000; cutting and burning treatments are scheduled to begin in 2001. This is a great opportunity to see how a forest works and to determine how a prior, less forested landscape functioned. Center scientists will concentrate on understanding how such treatments influence water balance, soil nitrogen cycling, and the retention and movement of nitrogen to adjacent coastal ponds. The project will also provide The Nature Conservancy with practical information on restoration design and techniques, as well as provide a place to bring people who are interested in biodiversity preservation and its connection with ecosystem functions. Center scientists see this as a pilot project for how they might eventually restore larger areas of fire-adapted ecosystems in the Vineyard State Forest and at the Massachusetts Military Reservation. Because of the need to identify and confront real and present worldwide threats to the environment, it is not surprising that The Ecosystems Center has grown so dramatically in size, scope, and reputation. The staff has increased six-fold and the budget has more than doubled in recent years, making the Center's office and laboratory space inadequate, and forcing researchers to work at scattered locations around the MBL campus. Soon, Ecosystems Center scientists will be in the Environmental Sciences Building on Albatross Street. This new building will have a cutting-edge geographic information systems facility, state-of-the-art laboratories for plant and soil sample analysis, a stable isotope laboratory, offices, teaching facilities, a classroom/conference room for the Semester in Environmental Science Program, ample storage areas for all equipment, and field staging areas. To that end, the MBL has received an important challenge grant of $500.000 from The Kresge Foundation. Report of the Director and CEO R3 Payment of that grant is conditional upon the MBL raising an additional $2. 1 million for the project. With further fundraising success, we hope to break ground for the $8 million building this spring. What a fitting way to celebrate The Ecosystem Center's 25th anniversary. Josephine Bay Paul Center for Comparative Molecular Biology and Evolution At the Bay Paul Center, 35 scientists and support staff continue to explore a number of aspects of molecular evolution and comparative molecular biology. Their efforts to sequence the genome of Giardia, a water-borne human pathogen that attacks the intestinal tract, is now more than 50 percent complete. Bay Paul Center Director Mitchell Sogin is the principal investigator on that study, which is sponsored by a major grant from the National Institutes of Health. In 1998 the Center welcomed Dr. Michael Cummings to the scientific staff. He is currently investigating ways to accurately predict drug-resistant strains of tuberculosis by examining specific gene sequences. With a recent grant from the Alfred P. Sloan Foundation, he is also developing novel computer-based analytical procedures to study color vision. In January 2000, Dr. Jennifer Wernegreen joined the staff as an Assistant Scientist. Dr. Wernegreen comes to the MBL from the University of Arizona where she was an NIH postdoctoral fellow in the Department of Ecology and Evolutionary Biology. Wernegreen uses comparative approaches to explore the molecular evolution of certain species of bacteria that live symbiotically with specific insects. These bacteria are thought to supplement nutritionally unbalanced diets of their hosts by providing essential amino acids, vitamins, and other nutrients. Other Research Initiatives Elsewhere at the MBL, scientists are working on a variety of biological and biomedical problems. Dr. David Keefe has developed a new method of non-invasively imaging the meiotic spindle of eggs during human in vitro fertilization at his clinic at Women and Infants Hospital in Rhode Island. This technique was developed at the MBL using the polscope designed by Dr. Rudolf Oldenbourg. Application of this exciting technology has doubled the pregnancy rates during intracytoplasmic sperm injection, and improved clinicians' ability to predict fertilization. Dr. Keefe has also been working with Dr. Peter Smith, Director of the BioCurrents Research Program at the MBL. They have developed a novel approach to measuring oxygen uptake by individual mammalian embryos using a non-invasive, self- referencing oxygen sensor. This work was recognized as one of the 50 most important biotechnology breakthroughs at a special program at the National Institutes of Health. It was also a semi-finalist for the Christopher Columbus Science Innovation Award sponsored by Discover magazine. MBL Distinguished Scientist Shinya Inoue was awarded two patents in 1999 for the Slit Scan Centrifuge Microscope and the Centrifuge Microscope Capable of Realizing Polarized Light Observation. These instruments were developed in collaboration with Olympus Optical and Hamamatsu Photonics Co. Dr. Inoue has been using these and other microscopes that he has developed over the years to study several unexplored attributes of living cells. Over the past year he has taken the first measurements of crawling forces of a cell, shown how mammalian cells can be separated into fractions that only contain certain types of organelles, and recorded thin R4 Annual Report optical slices of rapid molecular changes in living cells hitherto unseen. He's been able to trace the assembly of protein filaments that move chromosomes in yeast cells and track the movement of individual protein molecules within those filaments — something thought impossible only a few years ago. Two new resident scientists joined the MBL in 1999. Dr. Paul Colinvaux, an Adjunct Scientist, reconstructs Pleistocene climatic and environmental histories of the continents from the sediments of ancient lakes, particularly those found in the Amazon basin. Research has shown that the lowland Amazon forests persisted through glacial cycles, with some re-assortment of species as temperatures fluctuated from the last glacial maximum to the present. Although Colinvaux's research now is concentrated in the Neotropics, he maintains sites in Alaska and Russia for continued study of the paleoecology of the Arctic. Dr. Ayse Dosemeci came to the MBL in October from the National Institutes of Health as an Adjunct Scientist. She is a neurobiologist who studies synaptic plasticity, a biological phenomenon that may be involved in learning and memory and other central nervous system functions. Summer Research Last summer — as has been the case for more than a century — investigators from around the world came to the Marine Biological Laboratory to do research. As always, the excitement of study, scientific exchange, and discinery was great. The 126 principal investigators came from 12 countries and represented more than 100 institution-- One of the many highlights from last summer's research was the ivf-nt publication of a paper in Nature magazine by Drs. Miuuel Holmgren, Jonathan Wagg. Francisco Bezanilla, Robert Rakowski, Paul De Weer, and David Gadsby. In that paper they describe their latest findings about how a specialized cellular machine, the sodium/potassium exchange pump, works. Because this pump is essential to the health of virtually every cell in all animals, including humans, scientists at the MBL have spent years studying the molecular mechanisms by which this pump transports sodium and potassium ions across cellular membranes. They use the giant nerve cell of the Woods Hole squid as a model system for their research. These investigators already knew that this pump, which is a single protein molecule, transports three sodium ions across the cell membrane at once. In the Nature paper they showed that three separate changes in the shape of the pump protein release the three sodium ions from the pump one at a time, in a fixed sequence. This new information will help scientists understand in greater detail how these, and other, essential ion pumps perform the crucial work that keeps all our cells alive. Another important cellular pump, the sarcoplasmic reticulum-Ca~+ pump, is being studied by Dr. Larry Rome and his colleagues from the University of Pennsylvania. They are interested in learning more about how muscle design influences an organism's behavior. This past summer at the MBL they developed a new way to measure, in real time, calcium pumping and sarcoplasmic reticulum function in muscle fibers — a necessary first step in understanding the biological basis of behavior. As their model, they used the swimbladder muscle of the toadfish, the fastest known of all vertebrate fast muscle tissues. The muscles that envelop the swimbladder contract and relax at a remarkable 200 times per second, creating the animal's distinctive "boatwhistle" mating call. These muscles operate almost 100 times faster than the fish's locomotory muscles, which function just adequately to get the rather sluggish creature where it needs to go. Among the principal summer investigators at the Laboratory last summer, 1 2 were awarded Grass Fellowships in neurobiology and 19 were awarded other named MBL Fellowships to conduct research on a variety of biological topics at the MBL. These scientists come from around the country and the world to work in Woods Hole for the summer. For example. Pavel Balaban of the Russian Academy of Sciences used the mollusc Helix to study putative command neurons that modulate Report of the Director and CEO R5 withdrawal behavior and the activities of neurons underlying this behavior. Elizabeth Jonas of Yale University School of Medicine measured ionic currents on membranes of mitochondria during neurotransmission in squid. Anthony DePass of Long Island University used sea urchins to study how calcium enters heart and nerve cells when a cell is stimulated. And David Ogden of the National Institute for Medical Research in London studied how the skate senses small electric potentials in surrounding seawater to locate prey. Joining the annual gathering of scientists were 19 print and broadcast journalists who had been awarded MBL Science Writing Fellowships. This program offers writers the chance to step into the shoes of people they cover, to study basic biomedical and environmental science and — for some — to spend additional time doing course work in Woods Hole or research at Ecosystems Center field sites in Alaska and Brazil. Education It is my great pleasure to tell you that in 1999 the Howard Hughes Medical Institute awarded a new grant of $2.2 million to the MBL. The four-year award will support many of our advanced laboratory courses for graduate students, postdoctoral fellows, and university faculty members. The MBL has received $8.2 million in HHMI grants since 1988 for which we are extremely grateful. In that time, more than 4900 students have participated in courses taught by the best faculty in the world. Last summer, the MBL offered 20 courses, involving 579 faculty and guest lecturers and 427 students. Molecular Biology of Aging and a second session of Medical Informatics were added to the course list in 1999. Also in 1999 we welcomed David Garbers (HHMI, University of Texas Southwestern Medical Center) and Randall Reed (HHMI, Johns Hopkins University School of Medicine) as new directors of the Physiology course. This past summer was also the final year in the tenures of the directors of the Neurobiology, Neural Systems & Behavior, and Microbial Diversity courses. My special thanks to retiring directors Gary Banker and Dan Madison, Janis Weeks and Harold Zakon, and Ed Leadbetter and Abigail Salyers. Last fall, the Semester in Environmental Science Program was held at the MBL for the third time, and results again were impressive. In 1999 four new schools — Beloit College, Lawrence University, Southwestern University, and Trinity University — joined the consortium of institutions that participate in the program, bringing the total number of colleges and universities to 37. Undergraduates from a number of these small liberal arts colleges and universities around the country were immersed in a 15-week program of lectures, laboratory and fieldwork and independent research, all of this under the sponsorship of the MBL's Ecosystems Center. Students explored how human activity, such as deforestation, fisheries exploitation, changes in biodiversity, eutrophication and fossil fuel combustion alter ecological processes and ecosystem structure locally, regionally, and globally. Trustees The Board of Trustees elected three new members at November's meeting. Nobel Laureate Dr. Torsten I. Wiesel is President Emeritus and Vincent and Brooke Astor Professor Emeritus of The Rockefeller University. Dr. George M. Langford is the Ernest Everett Just Professor of Natural Sciences and Professor of Biological Sciences at Dartmouth College and Adjunct Professor of Physiology at Dartmouth Medical School. M. Howard Jacobson has been a Senior Advisor at Bankers Trust Private Bank since 1991. Current Board members G. William Miller, Frank Press, and Christopher M. Weld were re-appointed to the Board as members of the class of 2004. Longtime Trustee and stalwart supporter of the MBL Mary Ellen Cunningham was appointed an honorary member of the board. The Board also recognized the efforts of retiring members Alexander W. Clowes. Story C. Landis, and Irwin B. Levitan. The Discovery Campaign The Discovery Campaign, now in its final year, was remarkably successful in 1999, thanks to the tireless efforts of our volunteers and the generosity of donors. More than $9.9 million in private support was raised — the most ever in a single year. Our Annual Fund surpassed R6 Annual Report the half-million dollar mark last year as well, raising nearly 12 percent more than the year before. This record support pushed the Discovery Campaign past its $25 million goal in August of 1999, an astonishing 16 months ahead of schedule. By January 1. 2000, we had raised $30 million in support of research, education, and facilities at the MBL. As I write this report, some key objectives still remain to be funded before the Campaign ends in December 2000. I am confident that we will meet, if not exceed, these important goals. With many important gifts and the efforts of the Board's Investment Committee, the Laboratory's endowment has also grown dramatically — from $ 1 6 million when I came in 1992 to $47 million at the end of 1999. I thank all Campaign donors and volunteers for making 1999 such a successful year. Your strong support is a testimony to your belief in the special mission of the Marine Biological Laboratory. /// Conclusion As was noted many years ago in Life Science in Woods Hole, science is more than the accumulation of facts and findings and more than their interpretation. Science is, most of all, a grand collective curiosity. At the MBL, that collective curiosity is alive and well, and everyone who is touched by this place, who investigates, studies, learns and wonders is looking into the new century with hope and great expectations. I know that this place has touched me, and I will always be grateful for having been a part of it. I will miss this special institution and the many friends that I have made here. Thank you for making my tenure at the MBL such a pleasure. — John E. Burris Report of the Treasurer During 1999 the Marine Biological Laboratory had an outstanding year. This was the result of favorable growth in all the areas of operating support and auspicious investment results that further strengthened the balance sheet. All six areas of Operating Support grew by at least 6%, with double-digit growth in Government Grants (11.9%), Private Contracts ( 1 1.7%), and Fees for Conferences and Services (10.2%). The biggest change was from Contributions, which increased $3.8 million (78.8%) from 1998 levels as the Discovery Campaign hit full stride. These increases in Operating Support combined with restraining expense growth to 8% resulted in an almost sevenfold increase in the Change in Net Assets before Non-operating Activity. Also note that this included an underlying positive change in unrestricted net assets from operations of $138 thousand, the first surplus since 1994. This is particularly favorable considering these results include coverage of $1.56 million in depreciation. Reviewing our Non-operating Activities further demonstrates positive trends. New to our presentation this year is a breakout of the Contributions to Plant, which almost tripled. We have taken this step in light of our extensive capital improvement plans for the next few years. By pulling this out of the ongoing operations we have a much more informative display that gives a better indication of how we are doing in both areas. Total Investment Income and Earnings increased sixfold and enabled us to reinvest over $4.6 million after using roughly one-fifth for Operations. As a result, we reported for the fifth year in a row a positive Total Change in Net Assets. The increase of $11.2 million represents a very healthy 16.7% return on average net assets. A review of the 1 999 balance sheet further demonstrates the positive trends and our continued strong liquidity. The increase in Pledges and Other Receivables reflects $3 million in increased pledge receivables. The Endowment and Similar Investments increased 21.4%. Also Property. Plant and Equipment (net) increased (3%) for the first time in five years. Total Net Assets increased 18.3% during the year, with Unrestricted Net Assets increasing 7.8%. A subsequent event, which will have a major impact on the balance sheet, occurred on March 8, 2000, when the Massachusetts Development Finance Agency issued on behalf of the Laboratory $10.2 million in variable rate revenue bonds. A portion was used to refinance the Laboratory's long-term debt at a lower interest cost. The balance of the proceeds will be used to finance capital improvements of the Laboratory's educational, research, and administrative facilities, including the Environmental Sciences Building. The leverage ratio (unrestricted & temporarily restricted net assets-to-debt) on a pro forma basis is an acceptable 521% and represents suitable leverage of the financial strength of the Laboratory. In summary, the Laboratory had a very successful year of operations, fundraising, and investment performance that has greatly enhanced the financial strength of the Laboratory. The success of the Discovery Campaign and the bond financing will facilitate the upgrading of the MBL's physical plant and the continued expansion of our research and educational activities in the new millennium. — Mary B. Conrad R7 Financial Statements PricewaterhonseCoopers LLP One International Place Boston MA 02 110 Telephone (hi 7) 478 5000 f.ii •.mule ((,17) 478 3900 REPORT OF INDEPENDENT ACCOUNTANTS To the Board of Trustees of Marine Biological Laboratory: In our opinion, the accompanying balance sheet of Marine Biological Laboratory (the "Laboratory") as of December 31, 1999 and the related statements of activities and of cash flows for the year then ended present fairly, in all material respects, the financial position of the Laboratory as of December 31, 1999, and the changes in its net assets and its cash flows for the year then ended in conformity with accounting principles generally accepted in the United States. These financial statements are the responsibility of the Laboratory's management; our responsibility is to express an opinion on these financial statements based on our audit. We conducted our audit in accordance with auditing standards generally accepted in the United States. Those standards require that we plan and perform the audit to obtain reasonable assurance about whether the financial statements are free of material misstatement. An audit includes examining, on a test basis, evidence supporting the amounts and disclosures in the financial statements. An audit also includes assessing the accounting principles used and significant estimates made by management, as well evaluating the overall financial statement presentation. We believe that our audit provides a reasonable basis for the opinion expressed above. Our audit was conducted for the purpose of forming an opinion on the basic financial statements taken as a whole. The supplemental schedule of functional expenses as of December 31, 1999 is presented for the purpose of additional analysis and is not a required part of the basic financial statements. Such information has been subjected to the auditing procedures applied in the audit of the basic financial statements and. in our opinion, is fairly stated, in all material respects, in relation to the basic financial statements taken as a whole. April 7, 2000 R8 MARINE BIOLOGICAL LABORATORY BALANCE SHEET As of December 31. 1999 (with comparative totals as of December 31, 1998) ASSETS Cash and cash equivalents Short-term investments, at market (Note 3) Accounts receivable, net of allowance for doubtful accounts of $59,978 in 1999 and $34.195 in 1998 Current portion of pledges receivable (Note 8) Receivables due for costs incurred on grants and contracts Other assets Total current assets Long-term investments, at market (Notes 3 and 4) Pledges receivable, net of current portion (Note 8) Plant assets, net (Notes 2. 5 and 6) Total long-term assets Total assets 1999 $ 1.942,285 3.182.537 1.158,073 3.974.385 1,380,766 306.518 I | .944.564 45.001,493 3,498.787 20.118.725 68.619.005 $80.563.569 1998 $ 1.187,954 3.56 1. 544 1,242.530 1.607.664 1.531.083 557,908 9.688.683 37,054.120 2.855.352 19.536.171 59.445.643 $69,134.326 LIABILITIES AND NET ASSETS Current portion of long-term debt (Note 5) Accounts payable and accrued expenses Deferred income and advances on contracts Total current liabilities Annuities and unitrusts payable Long-term debt, net of current portion (Note 5) Advances on contracts Total long-term liabilities Total liabilities Commitments and contingencies (Notes 5, 7, 9 and 10) 267,404 1.957,508 656.745 2.881,657 1.460.94X 2,056.692 1.574.758 5.0C)2.39S 7.974,055 243.274 2,057.741 462.873 2.763.888 1.412.200 2,324.096 1.272.390 5.008.686 7.772.574 Net assets: Unrestricted Temporarily restricted Permanently restricted Total net assets (Note 2] Total liabilities and net assets 19.887.437 33,349,244 19.352.833 72,589.514 $80,563.569 1 8.45 1 ,865 25,635,237 17.274.650 61.361.752 $69,134.326 The accompanying notes are an integral part of the financial \tiiicnifiii.\. R9 MARINE BIOLOGICAL LABORATORY STATEMENT OF ACTIVITIES for the year ended December 31, 1999 (with comparative totals for the year ended December 31, 1998) Operating support and revenues: Government grants Private contracts Laboratory rental income Tuition Fees for conferences and services Contributions Investment income Miscellaneous revenue Present value adjustment to annuities Net assets released from restrictions Total operating support and revenues Expenses: Research Instruction Conferences and services Other programs (Note 2) Total expenses Change in net assets before nonoperating activity Nonoperating revenue: Contributions to plant: Private Government Release from restriction Invested in plant Total investment income and earnings Less: investment earnings used for operations Reinvested (utilized) investment earnings Total change in net assets Net assets, beginning of year Net assets, end of year Temporarily Permanently 1999 1998 Unrestricted Restricted Restricted Total Total SI 2.248.442 $ — $ — $12.248.442 $10,943.239 1,819,240 — — 1.819.240 1,629,283 1.548.168 — — 1,548.168 1.470.372 537.835 — — 537,835 489,726 3.765,039 — — 3,765.039 3,415,519 1,781.643 4.604.501 2.234.375 8.620.519 4.822,227 705,85 1 1.354.627 2,060.478 1,955,735 466.903 — — 466.903 405,633 — (22.680) (7,853) (30,533) (76,702) 3.705,796 (3.705.796) — — 26,578,917 2,230,652 2.226,522 31,036,091 25,055.032 14.147.645 14.147.645 12,666,746 4,742,287 — — 4.742.287 4,433.789 2.252.842 — — 2,252,842 1.999.433 5.297,773 — — 5.297.773 5,365,530 26,440,547 26.440.547 24.465.498 138.370 2.230.652 2,226,522 4.595.544 589.534 1,507,319 250,000 1,757.319 515,775 198.443 — — 198,443 — 912,046 (912,046) — 1.1 10.489 595.273 250,000 1,955.762 515,775 220.372 6.116.443 (398.339) 5,938.476 819,705 (33.659) (1.228.361) — (1.262.020) (1,246,913) 186.713 4,888.082 (398.339) 4.676,456 (427,208) 1.435.572 7,714.007 2.078,183 11.227,762 678.101 18,451.865 25.635.237 17,274.650 61,361.752 60.683.651 $19,887,437 $33.349.244 $19.352.833 $72.589.514 $61.361.752 The accompanying notes tire cm integral port of the financial sttttetnents. RIO MARINE BIOLOGICAL LABORATORY STATEMENT OF CASH FLOWS for the year ended December 31. 1999 (with comparative totals for the year ended December 31, 1998) 1999 Cash flows from operating activities: Change in net assets Adjustments to reconcile change in net assets to net cash provided hy (used in) operating activities: Depreciation Unrealized (gain) loss on investments Realized gain on investments Present value adjustment to annuities and unitrusts payable Contributions restricted for long-term investment and annuities Provision for bad debt Provision for uncollectible pledges Change in certain balance sheet accounts: Accounts receivable Pledges receivable Grants and contracts receivable Other assets Accounts payable and accrued expenses Deferred income and advances on contracts Annuities and unitrusts payable Advances on contracts Net cash provided by operating activities Cash flows from investing activities: Purchase of property and equipment Proceeds from sale of investments Purchase of investments Net cash used in investing activities Cash flows from financing activities: Payments on annuities and unitrusts payable Receipt of permanently restricted gifts Annuity and unitrusts donations received Payments on long-term debt Net cash provided by financing activities Net increase in cash and cash equivalents Cash and cash equivalents at beginning of year Cash and cash equivalents at end of year $ 11.227,762 1,562.487 (3,544.380) (1,639.795) 30.533 (2,485,624) 36,968 47.489 (3,010.156) 150,317 25 1 .390 (100,233) 193.872 68,112 302.368 3,091.110 (2,145.041) 63.101.047 (65.485.238) (4,529. 232 1 (49.897) 2,438,148 47,476 (243.274) 2.192.453 754.33 1 1.187.954 $ 1.942,285 1998 $ 678,1 1,505,696 2,755.079 (2,805.560) 76,702 (682,817) 15.771 250.000 (36,520) (255.134) (373.918) 2.361 562.793 78,615 163.700 (160.818) 1,774,051 (1.015.287) 18,935.050 (19.478.036) (1.558,273) (41,785) 653.152 29,665 (229.657) 411.375 627,153 560.801 $ 1,187.954 The accompanying notes are an integral part of the financial statements. Rll R12 Annual Report Marine Biological Laboratory Notes to Financial Statements 1. Background: The Marine Biological Laboratory (the "Laboratory") is a private, independent not-for-profit research and educational institution dedicated to establishing and maintaining a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural history. The Laboratory was founded in 1888 and is located in Woods Hole. Massachusetts. 2. Significant Accounting Policies: Basis of Presentation The accompanying financial statements have been prepared on the accrual basis of accounting and in accordance with the principles outlined in the American Institute of Certified Public Accountants' Audit Guide, "Not-For-Profit Organizations." The financial statements include certain prior-year summarized comparative information in total but not by net asset class. Such information does not include sufficient detail to constitute a presentation in conformity with generally accepted accounting principles. Accordingly, such information should be read in conjunction with the Laboratory's financial statements for the year ended December 31, 1998, from which the summarized information was derived. The Laboratory classifies net assets, revenues, and realized and unrealized gains and losses based on the existence or absence ol donor-imposed restrictions and legal restrictions imposed under Massachusetts State law. Accordingly, net assets and changes therein are classified as follows: Unrestricted Unrestricted net assets are not subject to donor-imposed restrictions of a more specific nature than the furtherance of the Laboratory's mission Revenues from sources other than contributions are generally reported as increases in unrestricted net assets. Expenses are reported as decreases in unrestricted net assets. Gains and losses on investments and other assets or liabilities are reported as increases or decreases in unrestricted net asset-, unless their use is restricted by explicit donor stipulations or law. Expirations of temporary restrictions on net assets, that is, the donor-imposed stipulated purpose has been accomplished and or the stipulated time period has elapsed, are reported as reclassirications between the applicable classes of net assets and titled "Net assets released from restrictions." Temporarily Restricted Temporarily restricted net assets are subject to legal or donor-imposed stipulations that will be satisfied either by the actions of the Laboratory, the passage of time, or both. These assets include contributions for which the specific, donor-imposed restrictions have not been met and pledges, annuities, and unitrusts for which the ultimate purpose of the proceeds is not permanently restricted. As the restrictions are met, the assets are released to unrestricted net assets. Also, rcali/cd/unrealized gains/losses associated with permanently restricted gifts which are not required to be added to principal by the donor are classified as temporarily restricted but maintain the donor requirements for expenditure. Permanently Restricted Permanently restricted net assets are subject to donor-imposed stipulations that they be invested to provide a permanent source of income to the Laboratory. These assets include contributions, pledges and trusts which require that the corpus be invested in perpetuity and only the income be made available for program operations in accordance with donor restrictions. Nonoperating revenues include realized and unrealized gains on investments during the year as well as investment income on the master pooled investments and revenues that are specifically for the acquisition or construction of plant assets. Investment income from short-tenn investments and investments held in trust by others are included in operating support and revenues. To the extent that nonoperating investment income and gains are used for operations as determined by the Laboratory's total return utilization policy (see below), they are reclassilied from nonoperating as "Investment earnings used for operations" to operating as "Investment income" on the statement of activities. All other activity is classified as operating revenue. The Laboratory recorded net realized gains of $1.639.795. net unrealized gains of $3.544.380 and dividend and interest income of $1.533.579 in 1999. Cash anil Cash Equivalents Cash equivalents consist of resources invested in overnight repurchase agreements and other highly liquid investments with original maturities of three months or less. Financial instruments which potentially subject the Laboratory to concentrations of risk consist primarily of cash and investments. The Laboratory maintains cash accounts with one hankini; institution. Investments purchased by the Laboratory are carried at market value. Donated investments are recorded at fair market value at the date of the gift. For closely held non-publicly traded investments, management determines the fair value, based upon the most recent information available from the Limited Partnership. For determination of gain or loss upon disposal of investments, cost is determined based on the first-in, first-out method. Investments with an original maturity of three months to one year, or those that are available for operations within the next fiscal year, are classified as short-term. All other investments are considered long-term. Investments are maintained primarily with three institutions. In 1424, the Laboratory became the beneficiary of certain investments, included in permanently restricted net assets, which are held in trust by others. The Laboratory has the continuing rights to the income produced by these funds in perpetuity, subject to the contractual restrictions on the use of such Financial Statements R13 funds. Accordingly, the trust has established a process to conduct a review every ten years by an independent committee to ensure the Laboratory continues to perform valuable services in biological research in accordance with the restrictions placed on the funds by the agreement. The committee met in 1994 and determined that the Laboratory has continued to meet the contractual requirements. The market values of such investments are $7,275,488 and $7,673,828 at December 31, 1999 and 1998. respectively. The dividend and interest income on these investments, included in unrestricted support and revenues, totaled $221.882 and $260.80? in 1999 and 1998. respectively. Investment Income ami Distribution For me master pooled investments, the Laboratory employs a total return utilization policy that establishes the amount of the investment return made available for spending each year. The Finance Committee of the Board of Trustees has approved a standing policy that the withdrawal will be based on a percentage of the 12 quarter average ending market values of the funds. The market value includes the principal plus reinvested income, realized and unrealized gains and losses. Spending rates in excess of 5%, but not exceeding 7%. can be utilized if approved in advance by the Finance Committee of the Board of Trustees. For fiscal 1999 and 1998, the Laboratory obtained approval to expend 6% of the latest 12 quarter average ending market values of the investments. The net appreciation on permanently and temporarily restricted net assets is reported together with temporarily restricted net assets until such time as all or a portion of the appreciation is distributed for spending in accordance with the total return utilization policy and applicable state law. Investment income on the pooled investment account is allocated to the participating funds using the market value unit method (Note 4). Plum Assets Buildings and equipment are recorded at cost. Donated facility assets are recorded at fair market value at the date of the gift. Depreciation is computed using the straight-line method over the asset's estimated useful life. Estimated useful lives are generally three to five years for equipment and 20 to 40 years for buildings and improvements. Depreciation is not recorded for those assets classified as construction-in-process as they have not yet been placed into service. Depreciation expense for the years ended December 31. 1999 and 1998 amounted to $1.562.487 and $1,505.696. respectively, and has been recorded in the statement of activities in the appropriate functionalized categories. When assets are sold or retired, the cost and accumulated depreciation are removed from the accounts and any resulting gain or loss is included in unrestricted income for the period. Annuities and Unitrnsts Payable Amounts due to donors in connection with gift annuities and unitrusts are determined based on remainder value calculations, with varied assumptions of rates of return and payout terms. Deferred Income and Advances on Contracts Deferred income includes prepayments received on Laboratory publications and advances on contracts to be spent within the next year. Advances on contracts includes funding received for grants and contracts before it is earned. Long-term advances are invested in the master pooled account until they are expended. Revenue Recognition Sources of revenue include grant payments from governmental agencies, contracts from private organizations, and income from the rental of laboratories and classrooms for research and educational programs. The laboratory recognizes revenue associated with grants and contracts at the time the related direct costs are incurred or expanded. Recovery of related indirect costs is recorded at predetermined fixed rates negotiated with the government. Revenue related to conferences and services is recognized at the time the service is provided, while tuition revenue is recognized as classes are offered. The tuition income is net of student financial aid of $527.258 and $523,190 in 1999 and 1998. respectively. Fees for conferences and other services include the following activities: housing, dining, library, scientific journals, aquatic resources and research services. Contributions Contribution revenue includes gifts and pledges. Gifts are recognized as revenue upon receipt. Pledges are recognized as temporarily or permanently restricted revenue in the year pledged and are recorded at the present value of expected future cash flows, net of allowance for unfulfilled pledges. Gifts and pledges, other than cash, are recorded at fair market value at the date of contribution. Expenses Expenses are recognized when incurred and charged to the functions to which they are directly related. Expenses that relate to more than one function are allocated among functions based upon either modified total direct cost or square footage allocations. Other programs expense consists primarily of fundraising, year-round labs and library room rentals, costs associated with aquatic resource sales and scientific journals. Total fundraising expense for 1999 and 1998 is $1.008,920 and $1.037.495. respectively. Use of Estimates The preparation of financial statements in conformity with generally accepted accounting principles requires management to make estimates and assumptions that affect the reported amounts of assets and liabilities and disclosure of contingent assets and liabilities at the date of financial statements and the reported amounts of revenues and expenses during the reporting period. Actual results could differ from those estimates. Tax-Exempt Status The Laboratory is exempt from federal income tax under Section 501(c)(3) of the Internal Revenue Code. Reclassification Certain prior year balances have been reclassified to conform with the current year presentation. R14 Annual Report 3. Investments: value of investments at December 31, 1999 and 1998: Market Cost The following is a summary of the cost and market Certificates of deposit Money market securities U.S. Government securities Corporate fixed income Common stocks Mutual funds Limited partnerships Total investments Investment portfolios for the years ended December Short-Term Investments 1999 $ 40.000 1,781,128 69, 1 25 2,364,068 15,665.205 26,664,204 1 ,600,300 1998 1999 $ 40,000 1,781,128 69,951 2,536,808 10.608,588 23.851,004 958,982 7998 $ 40.000 1.052,276 1.136.219 2,472.653 4,290.581 26.225,214 958.982 $ 40,00(1 1,052,276 1,397,686 2,504,507 5,033,704 29,548,891 1,038,600 $48, 1 84.030 $40,615.664 $39,846,461 $36,175,925 31, 1999 and 1998 are as follows: Market Cost 1999 7998 $ 40,000 559,314 2,955,989 6,241 1999 $ 40,000 233,938 2,965.273 33, 1 1 9 1998 $ 40,000 233,938 2,875,480 33,119 $ 40.000 559,314 2,940.929 6,241 Certificates of deposit Money market 1 784 Fund Mutal funds Common stocks in transit Total investments Lang-Term Investments 3,182.537 3,561,544 3,272,330 3,546,484 $35,354,938 5.717,108 1.558.380 2,371.067 $27,057,909 6,038.153 1,635,675 2.322,383 $27,514,505 5,335,721 1 ,448,569 2.275,336 $23,723,343 5,433,574 Pooled investments: Master pooled investments Separately invested: General Chase Trust Library Chase Trust Annuity and unitrust investments Total Total investments 1,477,462 1,995.062 45.001.493 37,054.120 36.574,131 32,629,441 $48,184,030 $40.615,664 $39,846.461 $36,175,925 Financial Statements R15 4. Accounting for Pooled Investments: Certain net assets are pooled for investment purposes. Investment income from the pooled investment account is allocated on the market value unit basis, and each fund subscribes to or disposes of units on the basis of the market value per unit at the beginning of the calendar quarter within which the transaction takes place. The unit participation of the funds at December 31, 1999 and 1998 is as follows: Unrestricted Temporarily restricted Permanently restricted Advances on contracts 1999 8.573 42.351 65.789 5.557 122.270 4.001 44.455 65.016 d.437 119,909 Pooled investment activity on a per-unit basis was as follows: Unit value at beginning of year Unit value at end of year Total return on pooled investments 1999 $ 225.51 2X3.37 $ 57.86 1998 $ 220.30 225.51 $ 5.21 5. Long-Term Debt: Long-term debt consisted of the following at December 31: Variable rate (6.3% at December 31, 1999) Massachusetts Industrial Finance Authority Series 1992A Bonds payable in annual installments through 2012 6.63% Massachusetts Industrial Finance Authority Series 1992B Bonds. payable in annual installments through 2012 5.8% The University Financing Foundation. Inc.. payable in monthly installments through 2000 5.8% The University Financing Foundation. Inc., payable in monthly installments through 2002 1999 $ 890.1 1 , 1 75,000 120,929 138.167 $2,324.096 /99.S' $ 925,000 1,230,000 226,024 186,346 Subsequent to year-end, all existing debt was extinguished and new debt was issued (Note 10). In 1992, the Laboratory issued $1,100,000 Massachusetts Industrial Finance Authority (MIFA) Series 1992A Bonds with a variable interest rate and $1,500,000 MIFA Series 1992B with an interest rate of 6.63%'. Interest expense totaled $142,545 for the year ended December 31. 1999. The Series 1992 A and B Bonds mature on December 1. 2012 and are collateralized by a first mortgage on certain Laboratory property. On March 17. 1998. the Laboratory entered into a ten-year interest rate swap contract in connection with the Series 1992 A Bonds. This contract effectively fixes the interest rate at 6.30% through December 17, 2008. This contract was canceled as part of the extinguishment of debt and new debt issuance. The agreements related to these bonds subject the Laboratory to certain covenants and restrictions. Under the most restrictive covenant of this debt, the Laboratory's operating surplus, exclusive of interest expense and depreciation expense, must he greater than or equal to 1.2 times all debt service payments, as defined by the agreement. In 1996. the Laboratory borrowed $500.000 with an interest rate of 5.8% per annum from the University Financing Foundation, Inc. The interest expense for the year ended December 31, 1999 was $10.345. The loan matures in 2000 and is collateralized by 50,000 shares of a fixed income fund with a fair value of $576,000 at December 31, 1999. In 1997, the MBL borrowed $2.50,000 with an interest rate of 5.8%^ per annum from the University Financing Foundation, Inc. The interest expense for the year ended December 3 1 , 1999 was $9,541. This loan matures in 2002 and is collateralized by 19,440 shares of a fixed income mutual fund with a fair value of $223,949 at December 31, 1999. The Laboratory has a line of credit agreement with a commercial bank from which it may draw up to $1,000,000. This line of credit has an interest rate of prime plus V? percent. The line has no expiration date but is reviewed periodically by the hank for renewal. No amounts were outstanding under this agreement as of December 31, 1999 and 1998. R16 Annual Report 6. Plant Assets: Plant assets consist of the following at December 3 1 : Land Buildings Equipment Construction in process Total Less: Accumulated depreciation Plant assets, net 7999 $ 702,908 33,702,485 4,667,026 1.510.821 40,583,240 (20.464,515) $20.118.725 /99.S' $ 702,908 33,082.164 4.401.1X4 251.943 38,438,199 (18.902.028) $19,536.171 7. Retirement Pliin: The Laboratory participates in the defined contribution pension plan of TIAA-CREF (the "Plan"). The Plan is available to permanent employees who have completed two years of service. Under the Plan, the Laboratory contributes 10% of total compensation for each participant. Contributions amounted to $785,509 and $737,156 for the years ended December 31, 1999 and 1998, respectively. 8. Pledges: Unconditional promises to give are included in the financial statements as pledges receivable and the related revenue is recorded in the appropriate net asset category. Unconditional promises to give are expected to be realized in the following periods: In one year or less Between one year and five years After five years 1999 $3.974,385 3.632,683 202.948 J99.S $1,607,664 3.110.354 146.586 Total 7.810.016 4.864.604 Less: discount of $236,844 in 1999 and $301,588 in 1998 and allowance of $100.000 in 1999 and $100,000 in 1998 (336.844) (401.588) $7,473.172 $4.463.016 Financial Statements R17 9. Postretiremen! Benefits: The Laboratory accounts for its postretiremen! benefits under Statement No. 106. "Employers' Accounting for Postretiremen! Benefits Other than Pensions," which requires employers to accrue, during the years that the employee renders the necessary service, the expected cost of benefits to be provided during retirement As permitted, the Laboratory has elected to amortize the transition obligation over 20 years commencing on January I. 1994. The Laboratory's policy is that all current retirees and certain eligible employees who retired prior to June 1. 1994 will continue to receive postretiremen! health benefits. The remaining current employees will receive benefits; however, those benefits will be limited as defined by the Plan. Employees hired on or after January 1, 1995 will not be eligible to participate in the postretiremen! medical benefit plan. The following tables set forth the Plan's funded status as of December 31: Benefit obligation at December 31 Fair value of plan assets at December 3 1 Funded status Accrued benefit cost Weighted-average assumptions as of December 31: Discount rate Expected return on plan assets Compensation increase rate Benefit cost Employer contribution Benefits paid $ 2,091,057 935.257 $(1.155.8(10) $ (26.654) 6.75% 7.25% N/A 209,430 190.090 129,589 799* $ 2.171.1 19 820,645 S( 1.350.474) $ (2h.h54i 6.75% 7.25% N/A 210.339 192,082 109.404 For measurement purposes a 6.75% annual rate of increase in the per capita cost of covered health care benefits was assumed for 2000. The rate was assumed to decrease by half of 1.00% per year to 4.25% in 2006 and remain at that level thereafter. Pension plan assets consist of investment in a money market fund. 10. Subsequent Event: On March 8. 2000, the Massachusetts Development Finance Agency issued on behalf of the Laboratory a series of Variable Rate Revenue Bonds (the "Bonds") in the amount of $10.200,000. The initial interest rate on the issue was 3.65% and the interest rate will be reset weekly. The bonds are scheduled to mature on February 1 , 2030. The Laboratory is required to make interest payments only for the first five years. The first principal payment is due February 1 , 2006 with incremental increases through maturity. The proceeds of these bonds are to be used to finance the capital improvements of the Laboratory's educational, research and administrative facilities, specifically the construction and equipping of the Environmental Sciences building. A portion of the proceeds were used to extinguish all of the Laboratory's capital obligations (Note 51. As collateral for the bonds, the Laboratory has entered into a Letter of Credit Reimbursement Agreement which is set to expire on March 15. 2007. The Letter of Credit is in an amount sufficient to pay the aggregate principal amount of the bonds and up to forty-six days' interest. -f rj OS m 2 Z - c - so 3 oo (N sO O sO sO OO ri r- oo so — r«-j r Z ri Tf 1 S i ;/; oo 3 ; r' rn - U~! O C in S -T oo' ri n* ri — -" r i -t r i — r*~, oo v~, rj m w~. o r- sD ir, ^t O Os rl ri rl — oo O VO r-. r-- — — 00 r^i ri r*-, O r- r-- ri Os so in CO m" o ~ra Wj C CO 6 S ^c '"° u J ^ y z £ S u "i fN OS sO -t 3 sO* j= D- Si D g T3 g- U w; w H D O o 3 2 "• OS -r)- — 00 -1- r ] — *n tr -t oo Os r { rt >-, z Z 1J b^ ~~ m' in oj rt 5 i Ui •^3 •5 o | U-l a, a. "*" 1 8 5J r~- r~- Os c Os O u~( r oq «n — - is Os OO — sO " OO Os Os sO — -t ri r- CO O^ \O rn in r- IT. r- ri ri -t C cfl « _J 2 c^ 0 S * m u", ^o' r rn \O m, ~ I rn in oo' to — r — r i m' o — Os fft oo § Os r[ a S E CJ Q 1 — ^_* ri rt o r" C « t/5 *rt '*- •^ .11 . — - .c -t ri — r*-| vO OO r- m oo ri os •* os so co co os r- so -f — os -t rn 8 P i/~i r O sD -t o ri OO OO r) 11 g O' rn ••* ri-j r^"j — - r^. ri "t r^, t rn O O OO r- r- ri OS ri (N QJ W t; -r — r*-, r*~j OS O r^ [~~- C £ •S ^ m* — 5 E 8 ° U n-, o r- c r\ m <— « cxa in — ' OO Os -t __ m <*, •£ f> O ' — ' s, 2 — m so m — sO oo -t jj rt os in u i CN ^t r- oo o OS Os ri sO ^ "O 1 ~- -^ o " sO OO rn C t -^ os m — — • Is so rn SO t "3- OO -rf ^t -t ri n r*-| -t 1 r-' r-' -t C 3 8.1 <^ Tf — ' Os' -f ^ " '£ £? o r3 ^ , ' .Si — « U •S -P • 118 C i; ^5SSr QJ - E2 HO KI8 Report of the Library Director The Library has been on the move since the fall of 1999. thanks to a long-overdue and welcome project to install air conditioning in the Library offices, reading rooms, and stacks. During the construction, each volume in the front stack was moved, alphabetized, and cleaned. When Library staff and patrons felt the first cool breezes of air conditioning in the stacks in early March, we quickly forgot about the inconveniences we encountered during this complex and time-consuming project. The construction was well worth the effort, and I am pleased to report that this summer Library patrons will find a clean, organized, and cool environment, which will be a comfort to them as well as a benefit to the collection. The Traditional Library At the brink of a new millennium we are focusing on the Library's traditional mission: acquiring, preserving, conserving, and distributing volumes in our collection. An article published recently in the Library Journal titled "Farewell to Alexandria: Not Yet!" reports on the number of publications that flow from an individual institution in relation to its library holdings, including citation productivity. The latter provides a rough measure of the scholarship quality of an individual institution. The statistics support the conclusion that the size of library holdings and institutional scholarly productivity go hand- in-hand. In this new era we need to continue to support acquisitions and preservation and conservation efforts as well as provide leadership in the creation of the new, technology-driven, scholarly environment. Books and journals continue to be printed in numbers inconceivable even a half century ago. While the MBL/ WHOI Library is providing new services and creating greater access to digital collections worldwide, we have not outlived print. Therefore, we must store it, preserve it, and make it available to our patrons. Special Collections The Library completed the inventory of several Special Collections in 1999. These include Charles Wilkes and his U.S. Exploring Expedition, Memoirs of the Museum of Comparative Zoology (Harvard) from 1865 to 1899, and the Harriman Alaska Expedition, the re-creation of which will become the basis of a public television special. Science historian Dr. Garland Allen has also recently provided us with the final installment of a much-needed survey of valuable journals currently stored on the open shelves of the stacks that require preservation and secure storage. The Journals The Library solicited bids for a new serials vendor in 1999 and awarded the contract to RoweCom/Faxon. Our electronic resources were improved with the purchase of Science Direct from Elsevier. This product provides full- text access to the 107 titles that the Library subscribes to plus a transactional allowance for staff-limited access to all 1 100 titles provided by Elsevier. Combined with the R19 R20 Annual Report addition of 400 electronic journals published by Springer- Verlag, our digital library has increased substantially. We have also recently purchased The Procatyotes. an on-line version of the book series; Marine Mammal Science, volumes 1 to 13 on CD-ROM; and Cambridge Scientific Abstracts Biological Sciences, an interdisciplinary database offering abstracts and citations to a wide range of research in biomedicine, biotechnology, zoology, ecology, and some aspects of agriculture and veterinary science. Harvard Depositorv By early 1999, the space remaining to accommodate future growth of the journal collection in the stacks had been exhausted. This problem was further exacerbated by the installation of air conditioning ductwork in the area. Therefore, we needed to find remote storage for approximately 8000 journal volumes. Because room for collection growth was needed in the active stacks, it was decided that volumes of cancelled series in that section were prime candidates for storage. Holding records of the selected series were created for display in the on-line catalog, and an in-house inventory of each series and volume was created. By the end of the year, 5038 volumes had been prepared and shipped to the Harvard Depository for storage. An additional 3000 volumes, consisting of series for which we have purchased an online counteipart, were subsequently sent to the Depository. Although these volumes are no longer in Woods Hole, they may be retrieved within 24 hours from the Harvard Depository. Document Delivery A major accomplishment in Document Delivery was the creation and implementation of the web-based Inter- Library Loan (ILL) request form. Members of the Woods Hole scientific community may now request ILLs with this form rather than using the traditional paper form. Also, with the addition of full text electronic journals, desktop delivery of information is now a reality. Cooperating Libraries The Boston Library Consortium has received funding for a virtual catalog and interlibrary loan direct distance- borrowing project. Our Library is an early participant in the project, which will eventually make it possible for patrons to easily ascertain which of the 16 BLC Libraries has the desired material and then order it directly from that Library. The National Library of Medicine's Medical Informatics course, sponsored by the Library, has expanded to two sessions, one in June and the other in October. The course continues to be very popular and successful with a focus on medical database design. Internet interfacing, and web page design. The MBLAVHOI Library hosted the 25th Anniversary Conference of IAMSLIC (International Association of Aquatic and Marine Science Libraries and Information Centers) in October. The group was organized and held its first meetings in Woods Hole in 1975. Over the years it has grown from the original 25 East Coast Marine Science Librarians to the international organization of 295 members it is today. The Library signed a new five-year contract with NOAA for the continuing operation and support of the NMFS Library at the Northeast Fisheries Center in Woods Hole and support of their serial and monograph collection held at the MBL. Volunteers Once again we thank Carol Winn and Millie and Bob Huettner for their tireless help and support with Rare Books and Special Collections. During 1999, more than 150 volumes were sent out for preservation under the Huettner' s tutelage. Carol has provided cataloging support for esoteric material in languages from Old German to 19"' century Swedish. — Catherine Norton Educational Programs Summer Courses Biology of Parasitism: Modern Approaches (June 10-August 13) Directors Pearce, Edward. Cornell University Tsehudi, Christian. Yale University School of Medicine Faculty Phillips, Meg, University of Texas Southwest, Dallas Russell. David, Washington University Medical School Scott, Phillip. University of Pennsylvania Selkirk. Murray. Imperial College of Science, Technology and Medicine, United Kingdom Sibley, David. Washington University Medical School Ullu, Elisabetta. Yale University School of Medicine Waters. Andrew P., University of Leiden. The Netherlands Teaching Assistants Appleby. Todd. Cornell University Beatty, Wandy, Washington University Medical School Giddings, Olivia, Washington University Medical School Hussein. Ayman. Imperial College of Science, Technology and Medicine. United Kingdom Kinch, Lisa, University of Texas Southwest La Flamme, Anne Camille. Cornell University Mair, Gunnar, Yale University School of Medicine Mordue, Dana, Washington University Medical School van der Wei. Annemarie, Biomedical Primate Research Centre, The Netherlands Zaph. Colby, University of Pennsylvania Lecturers Andrews, Norma, Yale University School of Medicine Bangs. James, University of Wisconsin, Madison Beckers. Cornelis, University of Alabama. Birmingham Beverley, Stephen, Washington University Medical School Carucci, Daniel. Naval Medical Research Institute Clark, Theodore. Cornell University Cully, Dons, Merck & Co. Day, Karen. Oxford University, United Kingdom Dell, Anne. Imperial College of Science, Technology and Medicine, United Kingdom Doolan, Denise, Naval Medical Research Institute Englund, Paul, Johns Hopkins University School of Medicine Finkelman, Fred, Veterans Administration Medical Center Frevert, Ute, New York University Medical Center Goldberg, Daniel, Washington University Medical School Grencis, Richard K., University of Manchester. United Kingdom Gull. Keith. University of Manchester, United Kingdom Hajduk, Steve, University of Alabama, Birmingham Hedstrom, Liz, Brandeis University Hunter, Christopher, University of Pennsylvania Johnson, Patricia. University of California, Los Angeles Komuniecki. Richard, University of Toledo Kopf, Manfred, Basel Institute for Immunology. Switzerland Langhorne. Jean. Imperial College of Science, Technology and Medicine, United Kingdom Long, Carol, National Institutes of Health Matthews, Keith. University of Manchester, United Kingdom Mottram. Jeremy. University of Glasgow. United Kingdom Pearlman. Eric, Case Western Reserve University Rathod, Pradip, Catholic University of America Roos, David, University of Pennsylvania Sacks, David, National Institutes of Health Scherf, Artur. Institut Pasteur, France Sher, Alan, National Institutes of Health Sollner-Webb, Barbara, Johns Hopkins University School of Medicine Tarlelon, Rick, University of Georgia Turco, Sam, University of Kentucky Medical Center Ullman, Buddy, Oregon Health Sciences University Wirth, Dyann. Harvard School of Public Health Workshop Coordinators Cooper, Peter, National Institutes of Health Ealich, Steve. Cornell University Lo Verde, Philip. State University of New York, Buffalo Course Assistants Chappie. Taylor, Boston University Chipperfield, Caitlin Nadine, Cornell University Students Angeli, Ve'ronique. Pasteur Institute, France Aviles, Hernan, Indiana State University Barragan. Antonio, Karolinska Institute. Sweden Batchelor, Adrian, Walter and Eliza Hall Institute, Australia Bishop, Joseph, University of Alabama. Birmingham Djimde, Abdoulaye, University of Maryland Dobbin. Caroline, University of Technology, Sydney, Australia Falcone, Franco, University of Edinburgh. United Kingdom R21 R22 Annual Report Gavrilescu. Cristina, Cornell University Jones, Stacy, University of Virginia Montgomery, Jacqui, Walter and Eliza Hall Institute, Australia Santori, Isabel, University of Buenos Aires, Argentina Sodre, Catia, Universidade Federal do Rio de Janeiro, Brazil Stem, Leah, University of California, San Francisco Toe, Laurent, World Health Organization, West Africa Wang. Zefeng, Johns Hopkins University Lartillot. Nicholas, Universite Paris-Sud, France Maduro. Morris, University of California. Santa Barbara Muriani, Francesca, University of California. Berkeley Micchelli, Craig. University of Wisconsin, Madison Ober. Hike. Max-Planck-Institute. Germany Pepicelli, Carmen. Harvard University Pizette. Sandrine, Memorial Sloan-Kettering Cancer Center Trainor, Paul, Medical Research Council, United Kingdom Wallingford. John, University of California, Berkeley Walsh, Emily. University of California. San Francisco Wilson. Valerie, University of Edinburgh, United Kingdom Lecturers Davidson. Eric. California Institute of Technology Heasman-Wylie. Janet, University of Minnesota School of Medicine Holland. Linda, University of California. San Diego Hopkins, Nancy, Massachusetts Institute of Technology Levine, Michael, University of California, Berkeley Rosenthal, Nadia, Massachusetts General Hospital-East Rothenberg, Ellen, California Institute of Technology Soriano. Philippe, Fred Hutchinson Cancer Research Center Stem. Claudio. Columbia University Tabin, Clifford, Harvard University Medical School Wylie, Christopher C., University of Minnesota Medical School Embryology: Concepts and Techniques in Modern Developmental Biology (June 13-July 24) Directors Bronner-Fraser. Marianne, California Institute of Technology Fraser, Scott, California Institute of Technology Faculty Adoutte, Andre. University of Paris-Sud, France Blair, Seth S., University of Wisconsin, Madison Carroll, Sean, University of Wisconsin, Madison Collazo, Andres. House Ear Institute Eltensohn. Charles. Carnegie Mellon University Harland, Richard, University of California, Berkeley Hartenstein. Volker, University of California. Los Angeles Henry, Jonathan. University of Illinois Krumlauf, Robb, National Institute for Medical Research, United Kingdom Martindale, Mark. Kewalo Marine Laboratory Niswander, Lee, Memorial Sloan-Kettering Cancer Center Rothman, Joel. University of California. Santa Barbara Saunders. John Jr.. Marine Biological Laboratory Shankland. Martin, University of Texas, Austin Wray, Gregory. State University of New York, Stony Brook Zeller, Robert, University of California, San Diego Teaching Assistant.* Baker, Clare. California Institute of Technology Baker, Julie. University of California. Berkeley Georgopoulos, Katia. Harvard University Hartenstein. Amelia, University of California. Berkeley Kourakis, Matlhew. University of Chicago Kuhlman, Julie, Memorial Sloan-Kettering Cancer Center Lane. Mary Ellen. University of Massachusetts Medical Center Course Assistant.* Stringer. Kristen. Marine Biological Laboratory Wylie, Matthew, Marine Biological Laboratory Lab Assistant Wylie, Sara, Marine Biological Laboratory Students Basch. Martin, California Institute of Technology Casanueva, Olivia. University of Chicago Clements, Wilson, University of Washington Corson, Laura, Ludwig Institute for Cancer Research Ewald. Andrew. California Institute of Technology Freistadt, Marion, Louisiana State University Medical Center Glavic, Alvaro, University of Chile. Chile Gould, Thomas. Wake Forest University Medical School Junghlut, Benno, University of Tubingen, Germany Li, Dongling. University of Texas. Austin Lwigale. Peter, Kansas State University Meyers, Jason, University of Virginia Mui, Stina, University of California, San Diego Nance. Jeremy. University of Arizona Panopoulou. Georgia, Max-Planck-Inslitute, Germany Paul, Angelika, University of Otago, New Zealand Pfeiffer, Sven, National Institute for Medical Research. United Kingdom Pizer, Margaret. State University of New York, Stony Brook Ragusa, Maria, Alberto Monroy Foundation. Italy Robertson. Christie. University of Washington Saiide, Leonor, National Institute for Medical Research. United Kingdom Spengler. Tatjana, Universite Paris. France Sumanas. Saulius, University of Minnesota Vukovich. Wolfgang, Max-Planck-Institute, Germany Zigler. Kirk. Duke University Educational Programs R23 Microbial Diversity (June 13-july 29) Directors Leadbetter, Edward, University of Connecticut Salyers, Abigail, University of Illinois, Urbana Faculty Dawson, Scott, University of California. Berkeley Hanselmann. Kurt, University of Zurich. Switzerland Holmes, Dawn, University of Massachusetts, Amherst Kenyon, Sarah, Forsyth Dental Center Klappenhach. Joel. Michigan State University Plugge. Caroline M.. Wageningen Agricultural University, The Netherlands Schauder, Rolf, Frankfurt. Germany Lecturers Blake. Ruth, Yale University Emerson. David. ATCC Farrand. Stephen, University of Illinois, Urbana Fouke, Bruce, University of Illinois. Urbana Hayes, John, Woods Hole Oceanographic Institution Leadbetter, Jared. University of Iowa Lovely, Derek, University of Massachusetts Metcalf, William, University of Illinois Newman. Dianne. Harvard University O'Neill, Scott, Yale University Paster, Bruce, Forsyth Dental Center Ruby. Ned. University of Hawaii Runimel, John, NASA Schmidt. Thomas. Michigan State University Shoemaker, Nadja, University of Illinois, Urbana Sogin. Mitchell. Marine Biological Laboratory Stein, Jeffrey, Quorum Pharmaceuticals Teske, Andreas. Woods Hole Oceanographic Institute Visscher, Pieter, University of Connecticut. Avery Point Waterbury, John, Woods Hole Oceanographic Institute Whitman, William. University of Georgia Young. Lily, Rutgers University Course Assistants Ament, Nell, Marine Biological Laboratory White. Kalina, University of Connecticut Students Aislabie, Jacqueline. Landcare Research, New Zealand Bedard, Donna, General Electric Corporate Research Center Casillas. Lilliam. Autonomous University of the State of Puebla. Mexico Christner, Brent, Ohio State University Chyba. Christopher. SETI Institute Dollhopf, Sherry, Michigan State University Gaidos. Eric. California Institute of Technology Gillor. Osnat, The Hebrew University, Israel Gregory. Kelvin, University of Iowa Niggemyer. Allison. University of Idaho Norris, Tracy, University of Oregon Nyholm, Spencer, University of Hawaii Pomper, Barbara, Max-Planck-Institute. Germany Rukayadi, Yaya, Bogor Agricultural University, Indonesia Salmassi. Tina, California Institute of Technology Shipman. Joseph, University of Illinois, Urbana Tuit. Caroline, Massachusetts Institute of Technology Van Lith. Yvonne. Swiss Federal Institute of Technology, Switzerland Warren, Lesley, McMaster University, Canada Zopfi. Jakob, Max-Planck-Institute. Germany Neural Systems & Behavior (June 13-August 6) Directors Weeks, Janis, University of Oregon Zakon, Harold, University of Texas, Austin Faculty Barnes, Carol, University of Arizona, Tucson Calabrese, Ronald L.. Emory University Carr, Catherine, University of Maryland French, Kathleen, University of California, San Diego Glanzman, David, University of California, Los Angeles Hooper, Scott, Ohio University Hyson, Richard, Florida State University Kristan. William, University of California. San Diego Levine. Richard, University of Arizona. Tucson McNaughton. Bruce, University of Arizona, Tucson Muir, Gillian, University of Saskatchewan, Canada Nadim, Farzan, Rutgers University Nusbaum, Michael, University of Pennsylvania School of Medicine Prusky, Glen, University of Lethbridge, Canada Roberts. William, University of Oregon Wenning-Erxleben. Angela, Universitat Konstanz, Germany Wood, Emma, University of Edinburgh, Scotland Teaching Assistants Armstrong. Cecilia, University of Oregon Blitz, Dawn Marie, University of Chicago Bower, Mark, University of Arizona. Tucson Chitwood, Raymond. University of Texas. San Antonio Few. Preston. University of Texas, Austin Gamkrelidze. Georgi, Lucent Technology Gerrard, Jason, University of Arizona, Tucson Golowasch. Jorge, Brandeis University Hill, Andrew, Emory University Lenzi, David, University of Oregon McAnclly, Lynne, University of Texas Melville, Johnathan. Oregon State University Murphy, Geoffrey. Lmiversity of California. Los Angeles R24 Annual Report I Sandstrom, David, University of Arizona, Tucson Shaw, Brian, The Neurosciences Institute Villareal. Greg, University of California, Los Angeles Yong. Rocio, University of California, Los Angeles Zee, Michelle. University of Oregon Zirpel. Lance. University of Utah School of Medicine Lecturers Augustine, George, Duke University Medical Center Barlow, Robert, State University of New York Health Science Center Beer, Randall, Case Western Reserve University Bodznick, David, Wesleyan University Cohen, Avis, University of Maryland Davis, Graeme, University of California, San Francisco Katz, Paul, Georgia State University Scholar-ill -Resilience Abbott, Lawrence. Brandeis University Nishikawa, Kiisa C., Northern Arizona University Wilson. Martin, University of California, Davis Lab Technician Stengel. Keith. Neuralynx Inc. Course Assistants Aimers, Lucy, Marine Biological Laboratory Stell. Brandon. Marine Biological Laboratory Students Baca, Serapio, University of California, San Diego Beenhakker. Mark. University of Pennsylvania Cain. Shaun, University of North Carolina. Chapel Hill Chance. Frances. Brandeis University Coddington, Emma, Oregon State University Crisp, Kevin, University of Minnesota Franks. Kevin, University of California. San Diego Greenwood, Anna, Stanford University Hausrath, Cassandra, University of Virginia Kao, Mimi, University of California, San Francisco Knittel, Laura, Oregon Health Sciences University Kricger. Patrik. Karolinska Institute!, Sweden Maravall. Miguel. Cold Spring Harbor Laboratory Maruska, Karen, Florida Institute of Technology Paradis. Suzanne. University of California, San Francisco Rao, Shankaranar. National Centre for Biological Sciences, India Rut, Jason. Boston University Suadicani, Sylvia, Albert Einstein College of Medicine Wainger. Brian, Columbia University Wissman, Anne Marie. University of Washington Neurobiology (June 13-Angust 14) Directors Banker, Gary, Oregon Health Sciences University Madison, Daniel. Stanford University Medical Center Section Directors Greenberg. Michael, Children's Hospital Smith. Stephen, Stanford University School of Medicine Faculty Delaney, Kerry, Simon Fraser University. Canada Edmonds, Brian, Universily of California. Los Angeles Feller, Maria, National Institutes of Health Ginty. David, Johns Hopkins University School of Medicine Griffith, Leslie. Brandeis University Hanson, Phyllis, Washington University School of Medicine Hart, Anne, Massachusetts General Hospital Haydon, Philip, Iowa State University Khodakhah, Kamran, University of Colorado School of Medicine Reese, Thomas, National Institutes of Health Schweizer. Felix, University of California, Los Angeles Shamah, Steven, Children's Hospital Smith, Carolyn. National Institutes of Health Terasaki. Mark, University of Connecticut Health Center Thompson, Stuart. Stanford University Van Vactor, David, Harvard University Medical School Teaching Assistants Boies. Sarah, Brandeis University Brinkhaus, Heike. Friedrich Miescher Institute, Switzerland Imani, Farzin, University of Colorado School of Medicine McQuiston, Rory. Duke University Medical Center Pereda, Alberto. Allegheny University of the Health Sciences Winters, Christine. National Institutes of Health Lecturers Barres, Ben. Stanford University School of Medicine Birren, Bruce. Massachusetts Institute of Technology Burden. Steven, New York University Ehrlich, Barbara. Yale University School of Medicine Ellisman. Mark. University of California. San Diego Faber, Donald, Allegheny University of the Health Sciences Flanagan, John, Harvard University Medical School Greene. Lloyd, Columbia University College of Physicians and Surgeons Hanson, Roland, Arizona State University Heuser, John. Washington University Medical School Lipscombe. Diane, Brown University Llinas, Rudolfo, New York University Nicoll, Roger. University of California, San Francisco Ogden, David, National Institute tor Medical Research. LInited Kingdom Rosenberg, Robert, LIniversity of North Carolina. Chapel Hill Li-Huei, Tsai. Harvard University Medical School Ziff, Edward, New York University Medical Center Zimmerberg. Joshua, National Institutes of Health Zimmerman, Anita, Brown University Educational Programs R25 Course Assistants Baughman, Kenneth, Boston University Chiu, Delia. Stanford University Students Abenavoli, Allesandra, Scientific Institute San Raffael. Italy Diana, Marco, Max-Planck-Institute, Germany Haapasalo, Annakaisa, A.I. Virtanen Institute. Finland Hrahetova, Sahina, New York University Medical Center Matsui. Ko, University of Tokyo, Japan Samuel. Aravinthan, Harvard University Schmolesky, Matthew, University of Utah Smith, Gregory, Princeton University Spotts, James, Children's Hospital Vollrath, Melissa. Baylor College of Medicine Yoon. Miri. Northwestern University Yu. Xiang. Medical Research Council. United Kingdom Physiology: The Biochemical and Molecular Basis of Cell Signaling (June 13-July 24) Directors Garbers. David, University of Texas Southwestern Medical Center Reed, Randall, Johns Hopkins University School of Medicine Faculty Beuve, Annie, University of Texas Southwestern Medical Center Munger. Steven. Johns Hopkins University School of Medicine Prasad, Brinda. Johns Hopkins University School of Medicine Quill. Timothy A., University of Texas Southwestern Medical Center Robinson, Susan W.. University of Texas Southwestern Medical Center Wang, Song S., Johns Hopkins University School of Medicine Wedel, Barbara, University of Texas Southwestern Medical Center Zhao, Haiqing, Johns Hopkins University School of Medicine Zielinski, Raymond. University of Illinois. Urbana Lecturers Brady, Scott, University of Texas Southwestern Medical Center Buck, Linda, Harvard University Medical School Clapham. David. Harvard University Medical School Corey. David, University of Texas Southwestern Medical Center Devreotes, Peter, Johns Hopkins University School of Medicine Dixon, Jack. University of Michigan Medical School Flanagan, John, Harvard University Medical School Furlow. John. University of California. Davis Ginty. David. Johns Hopkins University School of Medicine Huganir, Richard. Johns Hopkins University School of Medicine Hurley, James, National Institutes of Health Kirschner, Marc. Harvard University Medical School Li. Min, Johns Hopkins University School of Medicine Ranganathan. Rama, University of Texas Southwestern Medical Center Yanagisawa, Masashi. University of Texas Southwestern Medical Center Course Coordinator Rossi. Kristen. University of Texas Southwestern Medical Center Course Assistant Kirby, Melissa, Marine Biological Laboratory Students Chen, Lihong, University of North Carolina, Chapel Hill D'Souza, Jacinta. Tata Institute of Fundamental Research. India van Drogen. Frank, Swiss Institute for Experimental Cancer Research, Switzerland Duncan. Tod, Imperial Cancer Research Fund, United Kingdom Fort, Alfredo, Albert Einstein College of Medicine Franco, Peter, Harvard University Medical School Ganguly, Anindita. University of Utah Han, Qin. University of California, San Francisco Holdaway-Clarke. Terena, University of Massachusetts. Amherst Horn. Erik, University of California. San Francisco Jessani, Nadim. Scripps Research Institute Kimbell, Jennifer, University of Hawaii Macias. Chanda. Howard University March. Tony, University of Idaho Mazzatenta, Andrea, University of Pisa, Italy Narayan. Sujatha, Bryn Mawr College Nzambi. Eduardo, Howard University O'Neill, Forest, University of California, Santa Barbara Purves, Dianne. California State University. Sacramento Rao. Anita. University of Maryland Sawai, Satoshi. Tohoku University, Japan Sutton, Timothy, Indiana University Tefft, Denise. University of Southern California Tidwell, Judy, Wake Forest University Varshney, Anurag, National Centre for Biological Sciences, India Welman, Arkadiusz. Friedrich Miescher Institute, Switzerland Wen. Ying, University of North Carolina, Chapel Hill Woo, Caroline, Albert Einstein College of Medicine Special Topics Courses Analytical and Quantitative Light Microscopy (May 6 -May 14) Directors Sluder, Greenfield, University of Massachusetts Medical School Wolf. David. University of Massachusetts Medical School Faculty Amos, William B., Medical Research Council. United Kingdom Cardullo, Richard, University of California. Riverside Chaisson, Eric, Tufts University R26 Annual Report Gelles, Jeff, Brandeis University Hinchcliffe, Edward. University of Massachusetts Medical School Inoue. Shinya, Marine Biological Laboratory Lippincott-Schwartz. Jennifer. National Institutes of Health Oldenbourg. Rudolf, Marine Biological Laboratory Silver, Randi, Cornell University Medical College Spring. Kenneth. National Institutes of Health Swedlow, Jason. University of Dundee, Scotland Tuft, Richard, University of Massachusetts Medical School Teaching Assistant Thompson, Christine, University of Massachusetts Medical School Course Coordinator Miller, Frederick, University of Massachusetts Medical School Students Bearman, Gregory. Jet Propulsion Laboratory Botvinick. Elliot. University of California, San Diego Bowden. Emma. Georgetown University Brooks. John. Bio-Rad Microscience Bulseco. Dylan. University of Massachusetts Medical School Carrero. Jenny. Unilever Research U.S., Inc. Danuser, Gaudenz. Swiss Federal Institute of Technology, Switzerland Faulkner, Nicole, University of Massachusetts Medical School Heynen, Susanne, University of California, San Diego Hochegger, Helfrid, Imperial Cancer Research Fund, United Kingdom Holbrook, Pamela, National Institutes of Health Holz. Ronald. University of Michigan Hughes Fulford, Millie, University of California, San Francisco Keating, Christine, Penn State University Koehler. Julia. Whitehead Institute Kreitzer. Geri. Cornell University Medical College Kwan. Kristen, Harvard University Medical School Levin. Max, Wallenberg Laboratory for Cardiovascular Research, Sweden Lmdberg, Seth. Procter & Gamble Co. McDonald, John. Mayo Clinic Scoltsdale Novoradovskaya, Natalia. Stratagenc Pfister. Kevin. University of Virginia Reichelt. Stefanie, University of London. United Kingdom Roberts, Theresa, National Institutes of Health Rohatgi, Rajat, Harvard University Medical School Shirani. Jamshid, Albert Einstein College of Medicine Shonn, Marion, University of California, San Francisco Tanphaichitr. Nongnuj, Loeb Health Research Institute, Canada Tse. William, Children's Hospital Van Dover, Cindy Lee, College of William and Mary Yarovoi, Serge, University of Massachusetts Medical School Frontiers in Reproduction: Molecular and Cellular Concepts and Applications (May 24-July 4) Directors Hunt, Joan, University of Kansas Medical Center Mayo, Kelly, Northwestern University Schatten, Gerald, Oregon Health Sciences University Faculty Ascoli. Mario, University of Iowa Bowen, Jeffery A.. University of Kansas Medical Center Camper. Sally. University of Michigan Medical School Croy, Barbara Anne, University of Guelph. Canada Handel, Mary Ann, University of Tennessee Herr. John C., University of Virginia School of Medicine Hunt, Patricia A.. Case Western Reserve University Jaffe, Laurinda, University of Connecticut Health Center Petroff, Margaret. University of Kansas Medical Center Shupnik, Margaret. University of Virginia Health Sciences Center Simerly. Calvin, Oregon Regional Primate Research Center Terasaki, Mark, University of Connecticut Health Center Tnmarchi, James, Marine Biological Laboratory Weigel, Nancy. Baylor College of Medicine Teaching Assistants Aldrich. Carrie, University of Chicago Berard, Mark, University of Michigan Cunningham. Meghan, Georgetown University Diekman. Alan, University of Virginia Health Sciences Center Giusti, Andrew. University of Connecticut Health Sciences Center Greenwood. Janice, University of Guelph, Canada Hinkle, Beth Anne, University of Connecticut Health Sciences Center Hodees, Craig, Case Western Reserve University Mukherjee, Abir. Northwestern University Nakamura, Kazuto, University of Iowa Medical School Phillips. Teresa. University of Kansas Medical Center Resnick. Eileen, University of Virginia Health Sciences Center Rowan. Brian. Baylor College of Medicine Runt't. Linda, University of Connecticut Health Center Westbrook. Anne. University of Virginia Health Sciences Center Lecturers Behringer. Richard. University of Texas Campbell. Keith. PPL Therapeutics, Scotland Carroll. David. University of California. Santa Barbara Crowley. William. Massachusetts General Hospital Dominko, Tanja. Oregon Regional Primate Research Center Fazleabas. Asgi, University of Illinois Handyside. Alan, St. Thomas' Hospital, United Kingdom Hennighausen, Lothar, National Institutes of Health Hewitson. Laura. Oregon Regional Primate Research Center Johnson. Peter M.. University of Liverpool Medical School, United Kingdom Keefe, David, Marine Biological Laboratory Kopf, Greg, University of Pennsylvania Medical Center Ober. Carole, University of Chicago Orth, Joanne, Temple University School of Medicine Pederson. Roger, University of California, San Francisco Pollard, Jeffrey W., Albert Einstein College of Medicine Richards. JoAnne, Baylor College of Medicine Ruderman. Joan. Harvard University Medical School Shenker. Andrew. Children's Memorial Hospital Tilly, Jonathan L., Massachusetts General Hospital Wessel, Gary, Brown University Woodruff. Teresa. Northwestern University Course Administrator Emme. Michelle, Oregon Health Sciences University Educational Programs R27 Chief Course Coordinator Payne, Christopher, Oregon Health Sciences University Course Coordinators Daggett, Melissa, University of Kansas Medical Center McMullen. Michelle. Northwestern University Students Akhmedkhanov, Arslan, New York University School of Medicine Belts, Dean, University of Guelph. Canada Bos-Mikich, Adriana, Fundaijao Universitaria de Endocrinologia e Fcrtilidade, Brazil Buhimschi, Irina, University of Maryland El Guiziry. Dalai, Alexandria University, Egypt Johanputra, Vaidehi, All India Institute of Medical Sciences, India Johnson, Quinton, University of the Western Cape Lue, Yanhe, Harbor-University of California, Los Angeles, Medical Center Man'n Bivens, Carrie, University of Massachusetts, Amherst Mendeluk, Gabriela, University of Buenos Aires. Argentina Natesampillai, Sekar, University of Virginia Ollero, Mario, Harvard University Medical School Paidas. Michael, New York University School of Medicine Pritts. Elizabeth, Yale University School of Medicine Sprague, David, Texas A&M University Witlin, Andrea, University of Texas Medical Branch. Galveston Medical Informatics (May 30-june 5) Director Masys. Daniel, University of California, San Diego Students Adams. Martha. Duke University Babbitt, Patricia, University of California, San Francisco Barclay. Donald, Houston Academy of Medicine Bernhard, Jeffrey, University of Massachusetts Medical School Finley, Allen, Dalhousie University. Canada Goldstein, Cynthia, Tulane University Medical Library Lin. Chen-Tan, University of Colorado Health Sciences Center Lindherg, Don. Regenstrief Institute Lyons, Amy, University of Buffalo Health Sciences Library Mahoney, Diane, Hebrew Rehabilitation Center for Aged Markovitz, Barry, Washington University McGrath, St. John, Tufts University School of Medicine Meyers, Arlen, University of Colorado Health Sciences Center Mulluly-Quijas, Peggy, University of Missouri, Kansas City Pelok, Scott, University of Michigan Pifer, Eric, University of Pennsylvania Health System Robinson, Judith, Eastern Virginia Medical School Rosnian. Alan, Bronx Veteran's Administration Medical Center Sack, Jean, Johns Hopkins University Sarchet, Patricia, University of Buffalo Health Sciences Library Seago, Brenda, Virginia Commonwealth University Siblcy. Deborah. University of Massachusetts Medical School Stroman, Rosalie. National Institutes of Health Library Swanson. Sandra. Cook Institute of Research and Education Thompson, Laurie, State University of New York Health Science Center. Syracuse Tomlinson, Louise, Morehouse School of Medicine Travers. Robin, Boston University School of Medicine Tunnan, Lynne, Virginia Commonwealth University Volpp, Bryan, Veterans Affairs Medical Center Warlick, Becky, Duke University Medical Center Medical Informatics (October 3-October 9) Director Cimino, James, Columbia University Faculty Bakken, Suzanne, Columbia University Canese, Kathi, National Library of Medicine Cimino, Chris, Albert Einstein College of Medicine Friedman. Charles. University of Pittsburgh Hightower, Allen, Centers for Disease Control and Prevention Jenders, Robert, Columbia University Lindberg, Donald, National Library of Medicine Masys. Daniel, University of California, San Diego Safran, Charles. Center for Clinical Computing Starren, Justin, Columbia University Wheeler, David. National Library of Medicine Faculty Cimino. James. Columbia University Friedman, Charles, University of Pittsburgh Hightower, Allen, Centers for Disease Control and Prevention Hripcsak, George, Columbia-Presbyterian Medical Center Kingsland, Lawrence, National Library of Medicine Landsman, David, National Library of Medicine Lindberg, Donald D.A.B., National Library of Medicine Safran, Charles. Center for Clinical Computing Sengupta. Soumitra, Columbia University Starren, Justin, Columbia University Students Beidas, Sary, Prince George's Hospital Center Boyle, Marian, University of Florida Calarco. Pascal, Virginia Commonwealth University Chong. Lisa, Science magazine Cohen, Arlene, University of Guam Coster, Trinka, US Army Medical Research Institute Delia. Catherine, George Washington University Dimitroff, Alexandra. University of Wisconsin-Milwaukee Doyle. Jacqueline, Samaritan Health Systems, Phoenix Eaton, Elizabeth, Tufts University Health Sciences Library R28 Annual Report Feldman, Marc, University of Alabama. Birmingham Francis, Marcia. Idaho Slate University Fuller. Howard. University of California. San Francisco Hogan, Linda, University of Pittsburgh Hornby. Kathryn. University of British Columbia. Canada Knight, Barbara, University of North Dakota Kufreja. Neera. Cook County Hospital Linton. Anne. George Washington University Livingston, Jill, University of Connecticut Health Center Massanari. Mike. Wayne State University Miller. Stephen, Massachusetts General Hospital/Martha's Vineyard Hospital Parada. Jorge. Cook County Hospital Reilly. James, State University of New York Health Science Center, Brooklyn Strassner. Howard, Rush-Presbyterian-St. Luke's Medical Center Swanton, James, Harlem Hospital Center Swiatek-Kelley. Janice. Bridgeport Hospital Teal. Janis, University of New Mexico Walker. James. Penn State College of Medicine Wu, Carol, New York University School of Medicine Wulff, Judith, University of Louisville Yue, Cheung. MetroHealth Medical Center Methods in Computational Neuroscience (August 1 -August 28) Directors Bialek, William. NEC Research Institute van Steveninck. Rob de Ruyter. NEC Research Institute Faculty Abbott. Lawrence. Brandeis University Colby. Carol, University of Pittsburgh Dan, Yang, University of California. Berkeley Delaney. Kerry. Simon Fraser University. Canada Doupe, Allison, University of California, San Francisco Ermentrout, Bard, University ol Pittsburgh Hoplield, John, Princeton University Johnston. Daniel, Baylor College of Medicine Kelley, Darcy, Columbia University Klemleld, David, University of California, San Diego Kopell. Nancy. Boston University Marder. Eve, Brandeis University Meister. Markus. Harvard University Miller. K. D.. University of California. San Francisco Mitra. Partha. AT&T Bell Laboratories Rieke. Fred, University of Washington Seung, H. Sebastian, Massachusetts Institute of Technology Sigvardt, Karen, University of California, Davis Solla, Sara A.. Northwestern University Sompi'linsk\ . Haim. Hebrew University of Jerusalem. Israel Tank, David. AT&T Bell Laboratories Tishby. Nattali. Hebrew University of Jerusalem. Israel Zucker. Sie\ni. " il>- University Teaching Asv/.vMm.s Aguera y Areas. B . r,i!u.;-u>ii 1 'niversity Jensen. Roderick. \Visk-\.,n I HUCIM!) Koberle. Roland. Universidade ih Sac Paulo, Brazil Lewen. Geoffrey David. NEC Research Institute Nemenman. I., Princeton University White. John. Boston University Lecturers Baylor. Denis. Stanford University Medical Center Laughlin. Simon Barry, University of Cambridge, United Kingdom Logothetis, Nikos. Max-Planck-Institute for Biological Cybernetics. Germany Srinivasan, Mandyam V.. Australian National University, Australia Course Coordinator Stogryn. Krista, Marine Biological Laboratory Students Borisyuk. Alia, New York University Buss, Robert. McGill University, Canada Gang, Jianhua, University of Virginia Chechik. Gal. Hebrew University of Jerusalem, Israel Cowen, Stephen, University of Arizona Dumont. Sophie, Princeton University Garcia de Polavieja. Gonzulo. University of Cambridge, United Kingdom Jacobson, Gilad, Hebrew University of Jerusalem, Israel Karbowski. Jan. Boston University Kefalov. Vladimir, Boston University School of Medicine Kozloski, James. Columbia University Laing, Carlo, University of Pittsburgh Mayraz. Guy. University College London. United Kingdom Mazurek, Mark. University of Washington Naylor, David, University of California, Los Angeles Petersen, Ras. International School of Advanced Studies, Italy Prescott, Steven, McGill University, Canada Rubin. Jonathan. Ohio State University Spence. Andrew, Cornell University Still, Susanne, University of Zurich, Switzerland Wittenberg. Gayle. Princeton University Zeddies, David. Northwestern University Microinjection Techniques in Cell Biology (May 18-May 25) Director Silver. Robert B.. Marine Biological Laboratory Faculty Cousins, Susan, Cornell University Klaessig. Suzanne, Cornell University Kline, Douglas, Kent State University Mehlmann. Lisa. University of Connecticut Health Center Shelden. Eric. University of Michigan Teaching Assistant Warnke. Honey, University of Maine Students Araujo, Loraine, State University of Rio de Janeiro, Brazil Cohen. David, Oregon Health Sciences University Dabrowski, Konrad. Ohio State University James. Marianne. Massachusetts General Hospital Koulen. Peter, Yale University Kozek, Wieslaw, University of Puerto Rico Kuan, Chia-Yi, Yale University Lahti. Jill. St. Jude Children's Research Hospital Larkin. Janet. Barnard College Educational Programs R29 Li. Bin, University of California. San Francisco McGowan. Francis. Harvard University Medical School Nemoto, Yasuo, Yale University Nusser. Kevin. Oregon Regional Primate Research Center Pai. Vinay, Florida Slate University Rueda. Angelica, Centre de Investigation y de Estudios Avanzados del I.P.N.. Mexico Wentz-Hunter, Kelly, University of Illinois, Chicago Yu, Han-Gang, State University of New York. Stony Brook Molecular Biology of Aging (August 10 -August 27) Directors Guarente, Leonard P.. Massachusetts Institute of Technology Wallace, Douglas, Emory University School of Medicine Faculty Aiken, Judd M., University of Wisconsin-Madison Austad. Steven. University of Idaho Bohr, Vilhelm A.. National Institutes of Health Campisi, Judith, Berkeley National Laboratory Finch. Celeb. University of Southern California Grossman. Lawrence, Johns Hopkins University Harley. Calvin, Geron Corporation Hekimi. Siegfried, McGill University. Canada Johnson, Thomas, University of Colorado Jones. Dean P.. Emory University Kenyon. Cynthia, University of California. San Francisco Kim. Stuart. Stanford University of Medicine Kirkwood. Tomas, University of Manchester. United Kingdom de Lange. Titia, The Rockefeller University Lithgow, Gordon J.. University of Manchester, United Kingdom Longo. Valter, University of Southern California Martin, George, University of Washington School of Medicine Melov. Simon. Buck Center for Research in Aging Richardson, Arlan, University of Texas Health Science Center Ruvkun, Gary. Massachusetts General Hospital Sohal, Rajindar. Southern Methodist University Tanzi, Rudolph E., Harvard University Medical School Tower. John, University of Southern California Van Voorhies. Wayne. University of Arizona. Tucson Wright. Woodring E.. University of Texas Southwestern Medical Center Teaching Assistants Bilger. Johannes, Emory University Pinar, Elif. Emory University Cottrell, Barbara. Emory University Esposito, Luke. Emory University Jegalian. Beatrice, Massachusetts Institute of Technology Johnson, Brad. Massachusetts Institute of Technology Kokoszka. Jason. Emory University Levy. Shawn, Emory LIniversity McNabb. David. Massachusetts Institute of Technology Murdock. Deborah, Emory University Course Coordinator Burke, Rhonda E.. Emory University School of Medicine Course Assistant Abisla, Richard. University of Chicago Students Ayala-Torres. Sylvette. University of Texas Medical Branch Brown. Jeremy. Roslin Institute, Scotland Brown-Borg, Holly, University of North Dakota Chen. Yaohui. Yale University Medical School Chung. Namjm, Duke University Medical Center Crawford. Douglas, University of California, San Francisco de Lacalle, Sonsoles. Beth Israel Deaconess Medical Center Eshoo, Mark. Buck Center for Research in Aging Ford. Carolyn. Northwestern University Goto, Joy, University of California. Los Angeles Henning. Karla, National Institutes of Health Kennell, John, Southern Methodist University Kukull. Walter, University of Washington Martin, Kareen, Biological Gerontology Group. Manchester, LInited Kingdom McChesney, Patricia, University of Texas Southwestern Medical Center Merker. Robert, New York University Medical Center Squier. Thomas. University of Kansas Torres-Ramos. Carlos. University of Texas Medical Branch Molecular Mycology: Current Approaches to Fungal Pathogenesis (August 8-August 27) Directors Edwards, John Jr., Harbor-UCLA Medical Center Magee, Paul T.. University of Minnesota Mitchell. Aaron P.. Columbia University Faculty Casadevall. Arturo, Albert Einstein College of Medicine Cole, Gary T., Medical College of Ohio Davidson. Robert, Duke University Medical Center Davis, Dana, Columbia University Filler. Scott. Harbor-UCLA Medical Center Fonzi. William, Georgetown University Medical Center Heitman, Joseph. Duke University Medical Center Keath, Elizabeth, St. Louis University School of Medicine Klein. Bruce. University of Wisconsin-Madison Kurtz, Myra. Merck Research Lab Kwon-Chung, June, National Institutes of Health Lodge. Jennifer. St. Louis University School of Medicine Murphy. Juneann, University of Oklahoma Oliver, Brian. 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Louisiana State University Medical Center Neural Development and Genetics of Zebrafish (August 15-Augiist 27) Directors Dowling, John E., Harvard University Hopkins, Nancy, Massachusetts Institute of Technology Faculty Baker, Robert, New York University Medical Center Collazo, Andres, House Ear Institute Eisen, Judith S., University of Oregon Fetcho. Joseph, State University of New York, Stony Brook Fricke, Cornelia, University of Utah Medical Center Hanlon, Roger, Marine Biological Laboratory Kimmel, Charles, University of Oregon Lin. Shuo. Medical College of Georgia Neuhauss, Stephan, Max-PIanck-Institut fur Entwicklungsbiologie, Germany Talhot. William S., Stanford University Wilson. Stephen, University College London, United Kingdom Teaching Assi 'ants Clarke, .lon.iih.m, ' niversity College London. United Kingdom Fadool. James Hi l.i State University Granato. Michiu ' , of Pennsylvania Kainz, Pamela, Harvard i 'Diversity Link, Brian, Harvard Uimvrsity Lorent, Kristin, Um\cisii\ i,l Pennsylvania School of Medicine Moens, Cecilia, Fred Hulchmson Cancer Research Center Mullins, Mary, University of Pennsylvania Sirotkin, Howard. New York Universiiv School of Medicine Walker-Durchanck, Charline, Universiiv of Oregon Lecturers Astrosfky. Keith, Massachusetts Institute of Technology Fraser, Scott. California Institute of Technology Laboratory Technicians Linnon, Beth, Marine Biological Laboratory Mazanec, April, LIniversity of Oregon Course Coordinator Schmitt. Ellen. Harvard University Course Assistant Sweeney, Neal, Marine Biological Laboratory Students Ashworth. Rachel. University College London, United Kingdom Belletroid, Enc. Universite Libre de Bruxelles. Belgium Bishop, Charles, Baylor College of Dentistry Chan. Joanne. Dana-Farber Cancer Institute Endres. James, University of California. San Diego Glanzman, David, University of California, Los Angeles Levandoski, Mark. Brown University Lightfoot. Kurt, University of the Witwatersrand, South Africa Lunde, Karen, University of California, San Diego Poznanski, Ann, Midwestern University Rmkwitz, Silke. New York University Medical Center Tong, Betty. Whitehead Institute Vlachakis, Nikolaos. University of Massachusetts Medical Center Waterbury. Julie. University of Pennsylvania Wiemelt, Anthony, University of Pennsylvania Williams, Fred, University of Toledo Optical Microscopy and Imaging in the Biomedical Sciences (October 6-October 14) Director Iz/ard, Colin. State University of New York, Albany Faculty DePasquale, Joseph. New York Stale Department of Health Dunn, Kenneth, Indiana University Medical Center 1 laid, Robert, State University of New York, Buffalo Herman, Brian, University of Texas Health Science Center Murray, John, University of Pennsylvania School of Medicine Piston, David M., Vanderbilt University Snyder, Kenneth, State University of New York, Buffalo Spring, Kenneth, National Institutes of Health Swedlow. Jason. University of Dundee, Scotland Teaching Assistants Pierini, Lynda, Cornell University Medical College Sigurdson, Wade, State University of New York, Buffalo Lecturers Hinsch, Jan, Leica, Inc. Inoue, Shinya. Marine Biological Laboratory Keller, H. Ernst, Zeiss Optical Systems Educational Programs R31 Students Bhalla, Needhi, University of California, San Francisco Biggins, Sue, University of California. San Francisco Brotz, Tilmann. National Cancer Institute Chien, Edward, University of Chicago Combs, Christopher, National Institutes of Health Cromey. Douglas. University of Arizona Duca, Karen, University of Wisconsin Fletcher, Tara, Albany Medical College Gustashaw, Karen. Case Western Reserve University Hoja, Mary-Rose. Karolinska Institute Stockholm, Sweden Holt. Matthew. Medical Research Council. United Kingdom Hudson, Emma, University of Dundee, Scotland Kaplan, David, Food and Drug Administration Lin, Keng-hui, University of Pennsylvania Love, Dona, National Institutes of Health Martini, Lene, University of Copenhagen, Denmark Murnion. Mairead, University of Dundee. Scotland Reilly, Thomas, Johns Hopkins LIniversity School of Medicine Saslowsky. David. Virginia Tech Shestopalov, Valery, Washington University Silverman. Michael, Oregon Health Sciences University Sossick, Alex, University of Cambridge, United Kingdom Tang, Cha-Mei, Creative MicroTech. Inc. Wallace, Wes. Brown University Pathogenesis of Neuroiinmiinologic Diseases (August 16-August 27) Directors Brosnan, Celia F., Albert Einstein College of Medicine Rosenbluth, Jack. New York University School of Medicine Faculty Etty. Benveniste, University of Alabama, Birmingham Berman. Joan, Albert Einstein College of Medicine Brightman, Milton W., National Institutes of Health Burden, Steven, New York University School of Medicine Coyle, Patricia, State University of New York, Stony Brook Darnell, Robert, Rockefeller University Drachman. Daniel, Johns Hopkins University School of Medicine Felten, David, Loma Linda University School of Medicine Gould, Robert M., New York State Institute of Basic Research Griffin. Diane, Johns Hopkins University Griffin, John, Johns Hopkins University Hickey, William. Dartmouth-Hitchcock Medical Center Itescu, Silviu, Columbia/Presbyterian Medical Center Kaplan. Gilla. Rockefeller University Knopf. Paul, Brown University Kocsis. Jeffery D.. Yale University School of Medicine Kuchroo. Vijay, Brigham and Women's Hospital Lipton, Stuart, The Burnham Institute Martiney, James, Picower Institute for Medical Research McKinnon, Randall D., R. W. Johnson Medical School Popko. Brian, University of North Carolina, Chapel Hill Price. Donald L.. Johns Hopkins University School of Medicine Ransohoff. Richard. Cleveland Clinic Foundation Ransom. Bruce, University of Washington School of Medicine Reder, Anthony, University of Chicago Salzer, James, New York University Medical Center Saper, Clifford, Beth Israel Hospital Shin, Moon, University of Maryland School of Medicine Shrager, Peter. University of Rochester Medical Center Solimena. Michele, Yale University Sontheimer, Harald, University of Alabama, Birmingham Steinberg. Esther. National Institutes of Health Waksman, Byron. Foundation for Microbiology Weiner. Howard, Harvard University Medical School Course Coordinator Stogryn. Krista, Marine Biological Laboratory Students Andjelkovic. Anuska, University of Connecticut Brundula. Veronika, University of Calgary, Canada D'Aversa, Teresa, Albert Einstein College of Medicine DeFeo, Anthony. Mercy College Dzenko. Kirk, University of Connecticut Health Center Fischer. Falko. Harvard University Medical School Hillert. Jan, Karolinska Institute, Sweden Hjelmstrom, Peter, Yale University Janson, Christopher, Thomas Jefferson University Kuljis. Rodrigo, University of Miami Lu, Weiquan, State University of New York, Stony Brook Luedtke, Robert. University of North Texas O'Brien, Niklci. Australian National University, Australia Odyniec, Artur, Medical Academy of Lodz, Poland Regardsoe. Emma, University of Oxford, United Kingdom Reis, Donald, Cornell Medical College Robichaud, Lillian. Parke-Davis Research Salzberg, Heather, Rutgers University Sivakumar, M. R.. Apollo Hospitals. India Troncoso. Juan, Johns Hopkins University Vari, Gabor. Brown University Vijayan, Shrijay. City University of New York Woodman, Scott. Albert Einstein College of Medicine Wu, Dona. Alberl Einstein College of Medicine Yates, Jennifer. University of North Carolina, Chapel Hill Workshop on Molecular Evolution (August 1-Aiigust 13) Directors Davison, Daniel B.. Bristol-Myers Squibb PRI Sogin, Mitchell, Marine Biological Laboratory R32 Annual Report Faculn Cummings, Michael, Marine Biological Laboratory Eddy, Sean, Washington University Edwards, Scott, University of Washington Eisen. Jonathan, Institute for Genomic Research Felsenstein, Joseph. University of Washington Fitch. David H.A., New York University Fraser, Claire M.. Institute for Genomic Research Kuhner. Mary, University of Washington Maddison. David, University of Arizona, Tucson Miyamoto, Michael, University of Florida Muse, Spencer, North Carolina State University Olsen, Gary, University of Illinois, Urbana Pace, Norman, University of Colorado, Boulder Pearson, William, University of Virginia Health Sciences Center Rice. Ken. SmithKline Beecham Pharmaceuticals Riley, Margaret. Yale University Swofford, David, Smithsonian Institution Teaching Assistants Edgcomb. Virginia, Marine Biological Laboratory Mac-Arthur, Andrew, Marine Biological Laboratory Thompson, Steven, Florida State University Laboratoiy Technician Holder. Michael. University of Houston Course Coordinator Hams, Marian. Marine Biological Laboratory Students Ariey. Frederic, Inslitut Pasteur, France Babm. Josephine. Louisiana State University Baumgarlner. Manuela. University Regensburg. Germany Beati. Lorenza. Centers for Diseases Control and Prevention Becker, Jennifer. Lehigh University Best, Aaron, University of Illinois Blatter. Robert, University of Basel, Switzerland Bond, Philip, University of Wisconsin Bouchet. Valerie. Boston University School of Medicine Bouzat, Juan, University of Illinois Brazeau, Dan, University of Florida Brinkmann, Anna, University of Wisconsin-Milwaukee Bnones. Marcelo. Universidade Federal de Sao Paulo. Brazil Carrigan. Matthew. University of Florida Chaturvedi, Vishnu, New York State Department of Health Chyba, Christopher, SETI Institute Clark. Ann Marie. University of Florida Craven, Kelly, University of Kentucky Dacks, Joel, Dalhousie University, Canada Dennis, Paige, University of Massachusetts, Boston Di Meo, Carol, University of Delaware Dimsoski, Pero, United Slates Environmental Protection Agency Fitzpatrick, Jennifer, Tufts University School of Medicine Fleming. Melissa. University of Alaska Museum Franck, Jens. Occidental College Freire. Nicole. University of Florida Gasparich. Gail. Towson University Gaudier, Eric. University of Florida Gribaldo, Simonetta. Universita "La Sapienza," Italy Gueneau-Novoa, Pulchene, Instituto Venezolano Investigaciones Cientih'cas, Venezuela Hansen, Jan, Technical University of Denmark, Denmark Harbmski, Fred, Harvard University Ho, Hoi Yan, Chinese University of Hong Kong. Hong Kong Hurtado, Luis. Rutgers University Inagaki. Yuji. Dalhousie University. Canada Klingbeil. Michele. Johns Hopkins School of Medicine Lawrence. Carolyn, University of Georgia Liebert, Cynthia. University of Georgia Maiwald. Matthias, Stanford University McGraw, Beth, Yale University Mead, Louise, University of Massachusetts Moore, Jon. National Marine Fisheries Service Pilcher, Carl. NASA Headquarters Pineda, Augustin, Florida Slate University Posada, David. Brigham Young University Pntham. Ellen, Universily of Massachusetls, Boston Reed. David. Louisiana State Universin Richardson. Susan, Yale University/Woods Hole Oceanographic Institution Rinke De Wit, Tobias. Ethiopian Health and Nutrition Institute. The Netherlands Sabo, Aniko, Purdue University Salamin, Nicolas. Universite de Lausanne, Switzerland Schreiber, Edgar. PE Applied Biosystems Seffemick. Jennifer. University of Minnesota Sellers. Holly. United States Department of Agriculture Sinclair, Elizabeth. Brigham Young University Skirnisdottir, Sigurlaug. IceTherm Inc.. Iceland Stockley. Bruce. Southampton Oceanography Centre. United Kingdom Worapong, Jeerapun, Montana State University Zmasek. Christian, Washington University Medical School Other Programs Marine Models in Biological Research Undergraduate Program (June 8-August 6, 1999) Directors Browne. Carole L.. Wake Forest University Tytell. Michael. Wake Forest University School of Medicine Course Assistant Begley. Gail. Marine Biological Laboratory Faculty Allen. Nina S., North Carolina State University Borst, David, Illinois State University Furie, Barbara. Harvard University Furie, Bruce. Harvard University Hanlon. Roger. Marine Biological Laboratory Jonas, Elizabeth. Yale University Laufer. Hans, University of Connecticut Malchow, R. Paul, University of Illinois Mensinger, Allen. Washington University Wainwright, Norman. Marine Biological Laboratory Seminar Speakers Frank. Tammy. Harbor Branch Oceanographic Institution Inoue, Shinya, Marine Biological Laboratory Kuzirian, Alan. Marine Biological Laboratory Reinisch, Carol. Marine Biological Laboratory Silver. Robert. Marine Biological Laboratory Kducutional Programs R33 Students Baliga, Meghan. Wake Forest University Clifton. Christine. Mount Holyoke College Harris!. Alexia. Yale University Helm, Jessica, Washington and Lee University Lassen, Kara. Wake Forest University Mitchell, Michael. Wake Forest University Peck, Raphaela. Reed College Price. Nichole, Connecticut College Ramsey, David, Harvard University Rankin, Ellen, Colgate University Tang, Kathleen, Washington University Taylor, Kevin, Wake Forest University Vasse, Aimee, Williams College NASA Planetary Biology Internship (June-September 1999) Directors Margulis, Lynn, University of Massachusetts Dolan, Michael F., University of Massachusetts Interns Caylor, Kelly, University of Virginia Chacon, Elizabeth. Universidad Nacional Autonoma de Mexico Franklin. Rima, University of Virginia French, Jason, University of Alberta. Canada Gauci. Vincent, The Open University, United Kingdom Marx, Joseph G., International Space University, France Omelon, Christopher, McGill University, Canada de Peyer, Oliver, University of Reading, United Kingdom Popa. Radu, University of Cincinnati de Vera Gomez. Alvin. University of the Philippines Wilson, Cindy, University of Montana Sponsors Cady. Sherry L., Portland State University Des Marais, David, NASA Ames Research Center Joyce, Gerald, Scripps Research Institute Knoll, Andrew, Harvard University Mancmelli. Roco, NASA Ames Research Center Matthews. Elaine, Goddard Institute for Space Studies Nealson, Ken. Jet Propulsion Laboratory Privette, Jeff, NASA Goddard Space Flight Center Rothschild, Lynn. NASA Ames Research Center Teske, Andreas, Woods Hole Oceanographic Institution Wheeler, Raymond, NASA Kennedy Space Center Semester in Environmental Science (Septembers-December 17, 1999) Administration Hobbie, John E., Director Foreman, Kenneth H., Associate Director Moniz, Polly C., Administrative Assistant Faculty Deegan, Linda A. Giblin, Anne E. Hopkinson. Charles S. Jr. Hughes, Jeffrey Liles, George Nadelhoffer, Knute J. Neill, Christopher Peterson. Bruce J. Rastetter, Edward B. Shaver. Gams R. Vallino, Joseph J. Williams, Mathew 7999 Research and Teaching Assistants Bahr, Michelle Kelsey, Sam Kwiatkowski. Bonnie Micks. Patricia Parker, Sophie Tholke. Kris 7999 SES Students Arling, Jeremy. Bowdoin College Avery, Jennifer. Brandeis University Butman, David. Connecticut College Glueck, Lara. Claremont McKenna College Greenbaum, Adena, Wellesley College Hinckley, Eve-Lyn, Middlebury College Horowitz. Julie, Hampshire College Kirkby, Ryan, Harvey Mudd College Mathrani, Vandana, Scripps College Mathrani. Varsha, Scripps College Mifflin. Amanda. Wellesley College Morrisseau, Sarah, Connecticut College Peterson, G. Gregory. Wesleyan University Romagnano. Joseph. Worcester Polytechnic Sohm, Jill, Harvey Mudd College Spivak, Amanda, Bryn Mawr College Williams, Samantha. Mount Holyoke College Ziemann, Tori. Beloit College SPINES — Summer Program in Neuroscience, Ethics and Survival (June 12-July 10) Directors Martinez, Joe L. Jr. Townsel, James Fellows Herne. Moss, Boston University School of Medicine Hubbard. Aida, University of Texas, San Antonio McCrery. Karen, Texas Women's University Meadows, Adimika, Boston University Mohamed, Somaia, University of Iowa Nelson, Rhonda, Meharry Medical College Orfila, James, University of Texas, San Antonio Simples. James. University of Pittsburgh Villarreal. Julissa, University of Texas. San Antonio Zayas, Ricardo, Tufts University Teachers' Workshop: Living in the Microbial World (August 15-21) Course Directors Olendzenski, Lorraine, University of Connecticut, Storrs Dugas, Jeff, University of Connecticut, Storrs Curriculum Specialist Dorritie, Barbara, Cambridge Rindge and Latin School, Cambridge, MA R34 Annual Report Course Assistant Wier, Andrew. University of Massachusetts, Amherst Presenters Margulis, Lynn. University of Massachusetts. Amherst Guerrero. Ricardo, University of Barcelona. Spain Knoll. Andrew, Harvard University Edgcomb. Virginia. Marine Biological Laboratory Cast. Rebecca. Woods Hole Oceanographic Institution Runimel. John. National Aeronautics and Space Administration Teacher Participants Molyneaux, Leslie, Hanover Middle School, Hanover, MA Buckley, Kalhryn. Mashpee High School, Mashpee, MA Henderson, Forest, Bellingham Jr./Sr. High School, Bellinghain, MA Muscatell. Gina, Bellingham Jr./Sr. High School, Bellingham. MA Bennett. Tara, Norwell High School, Norwell, MA Webber. Alan. Norwell High School. Norwell. MA Yuhas, Joseph. Kennebunk High School. Kennebunk. ME Johnston, Ross B.. Nauset Regional High School. N. Eastham, MA Albright, Lori, Nauset Regional High School. N. Eastham, MA Carotenuto, Sheila, Quashnet River School. Mashpee. MA Rocio, Zamaria. Horace Mann Middle School. San Diego. CA Conn, Kathleen. West Chester Area School District, West Chester, PA Carty, Susan, West Chester Area School District, West Chester, PA Rutland. Susan, West Chester Area School District. West Chester. PA Settertield. Elena. King Ethelbert School, Kent, England Scales, Sacha, King Ethelbert School, Kent, England Cronin, Maureen. Nonington C.E.P. School, Kent. England Scott. Nyree. Nonington C.E.P. School. Kent, England Summer Research Programs Principal Investigators Adamo, Shelley, Dalhousie University, Canada Armstrong, Clay, University of Pennsylvania Armstrong, Peter B.. University of California, Davis Augustine, George J., Duke University Medical Center Balaban, Pavel. Russian Academy of Sciences, Russia Barlow. Robert B. Jr.. State University of New York Health Science Center Beauge, Luis, Institute de Investigation Medica "Mercedes y Martin Ferreyra," Argentina Beckman, Matthew, University of Alabama. Birmingham Ben-Jonathan, Nira, University of Cincinnati Bennett. Michael V. L., Albert Einstein College of Medicine Bodznick. David, Wesleyan University Boron, Walter, Yale University Medical School Borst, David, Illinois State University Boyer, Barbara, Union College Boyle. Richard. Oregon Health Sciences University Brady. Scott T. The University of Texas Southwestern Medical Center. Dallas Brock, Matthew, Stanford University Browne, Carole, Wake Forest University School of Medicine Burger, Max M.. Friedrich Miescher Institut, Switzerland Cardullo, Richard, University of California, Riverside Carvan, Michael, University of Cincinnati Chappell. Richard L., Hunter College, City University of New York Cohen, Lawrence B., Yale University School of Medicine Cohen, William D., Hunter College, City University of New York Crespi. Marco, Scientific Institute S. Raffaele, Italy De Weer, Paul, University of Pennsylvania School of Medicine DePass. Anthony, Long Island University, Brooklyn DePina, Ana S., Dartmouth College DiPolo, Reinaldo. Instituto Venezolano Investigaciones Cientificas, Venezula Dodge, Frederick, State University of New York Health Science Center Doussau, Frederic, Duke University Medical Center Edds-Walton, Peggy. Parmly Hearing Institute Ehrlich, Barbara, Yale University School of Medicine Fay, Richard, Loyola University of Chicago Field, Christine, Harvard University Medical School Fishman, Harvey M., University of Texas Medical Branch, Galveston Flamarique, Inigo Novales, University of Victoria. Canada Gadsby, David. Rockefeller University Gerhart, John, University of California. Berkeley Giuditta. Antonio, University of Naples, Italy Goldman, Robert D., Northwestern University Medical School Gould, Robert, New York State Institute for Basic Research Groden. Joanna, University of Cincinnati Haimo, Leah, University of California, Riverside Han. Yi. Baylor College of Medicine Heck. Diane. Rutgers University Hershko, Avram, Technion-Israel Institute of Technology, Israel Highstein, Steven M., Washington University School of Medicine Hill, Susan Douglas. Michigan State University Hines, Michael, Yale University School of Medicine Hoskin. Francis, US Army Natick RD&E Center Innocenti. Barbara. Iowa State University Johnston. Daniel, Baylor College of Medicine Jonas, Elizabeth, Yale University School of Medicine Jones, Teresa, National Institutes of Health Joye, Samantha, University of Georgia Kaczmarek, Leonard, Yale University School of Medicine Kaplan, Barry, National Institutes of Mental Health Kaplan, Ilene M., Union College Kier, William. University of North Carolina, Chapel Hill Kirschner, Marc, Harvard University Medical School Koulen, Peter, Yale University School of Medicine Kuhns, William, The Hospital for Sick Children, Canada Later. Eileen M., University of Texas Health Science Center Landowne, David, University of Miami School of Medicine Langford, George, Dartmouth College Laskin, Jeffrey, University of Medicine and Dentistry of New Jersey Laufer, Hans. University of Connecticut LaVail. Jennifer, University of California, San Francisco Lipicky, Raymond J.. Food and Drug Administration Llinas, Rodolfo R., New York University Medical Center Magee, Jeff, Louisiana State University Medical Center Major, Guy, Lucent Technologies Malgaroli, Antonio. University of Milan. Italy Martinez. Joe, University of Texas. San Antonio McAllister. A. Kimberly, Salk Institute of Biological Studies McNeil. Paul, Medical College of Georgia Mensinger, Allen. Washington University School of Medicine Metuzals. Janis, University of Ottawa Faculty of Medicine, Canada Mitchison, Timothy, Harvard University Medical School Miyakawa. Hiroyoshi, Tokyo University of Pharmacy and Life Science, Japan Moore, John W., Duke University Medical Center Mooseker, Mark, Yale University R35 R36 Annual Report Nasi, Ennco. Boston University School of Medicine Ogden, David. National Institute for Medical Research Ogunseitan. Oladele A.. University of California, Irvine Palazzo, Robert, University of Kansas Pant, Harish, National Institutes of Health Parysek. Linda, University of Cincinnati Paydarfar, David, University of Massachusetts Medical School Quigley, James P., State University of New York, Stony Brook Rabhitt, Richard, University of Utah Rakowski. Robert F.. Finch University of Health Sciences/The Chicago Medical School Ramus. Seth. Boston University Ratner. Nancy, University of Cincinnati Reese, Thomas S., National Institutes of Health Rieder, Conly, Wadsworth Center Ripps, Harris, University of Illinois College of Medicine Rome, Larry, University of Pennsylvania Russell. John M., Hahnemann University Salmon, Edward, University of North Carolina, Chapel Hill Siwicki. Kathleen. Swarthmore College Sloboda. Roger D.. Dartmouth College Spiegel, Evelyn, Dartmouth College Spiegel. Melvin, Dartmouth College Srinivas, Miduturu. Albert Einstein College of Medicine Standart, Nancy, University of Cambridge. United Kingdom Steinacker, Antoinette, University of Puerto Rico Sugimon, Mutsuyuki, New York University Medical Center Suszkjw, Janusz, University of Cincinnati Telzer, Bruce, Pomona College Tilney, Lewis, University of Pennsylvania Trinkaus. John P.. Yale University Troll, Walter, New York University Medical Center Tytell, Michael. Wake Forest University School of Medicine Walters. Edgar, University of Texas. Houston Weidner, Earl. Louisiana State University Yamaguchi, Ayako. Columbia University Yamoah, Ebenezer, University of Cincinnati College of Medicine Zecevic, Dejan P.. Yale University School of Medicine Zimmerberg, Joshua, National Institutes of Health Zito, Karen, University of California, Berkeley Zochowski, Michal. Yale University School of Medicine Zottoli. Steven, Williams College Zukin. R. Suzanne. Albert Einstein College of Medicine Other Research Personnel Adams, Curt, University of California, Riverside Akanki. Feyisara, Williams College Allen. Nina. North Carolina State University Antic, Srdjan Henry, Yale University School of Medicine Anton. Roberto, Hunter College April, liana, Connecticut College Armstrong, Clara. University of Pennsylvania Asokan. R., Universitv of California, Davis Baliga, Meghna. Wake Forest University Banini, Bubu. Swarthmore College Bashi, Esther. Yale University Bearer, Elaine, Brown University Benjamins. Steven, Groningen University. The Netherlands Bergamaschi, Andrea, Fondazione Centra San Raffaele del Moute Tabor. Italy Berger-Sweeney. Joanne. Wellesley College Bertetto. Lisa, Wesleyan University Bezanilla, Francisco. University of California, Los Angeles Billack. Blase. Rutgers University Bingham. Eula. University of Cincinnati Medical School Bonacci. Lisa, Hunter College Bronner-Fraser, Marianne. California Institute of Technology Brown. Joel, Albert Einstein College of Medicine Bucior, Inona, Friedrich Miescher Institute, Switzerland Burris, Jennifer, Northwestern University Medical School Chan, Sena. Long Island University Cho, Myoung-Soon. National Institutes of Health Clarkson, Melissa, University of Kansas Clifton. Christine. Mount Holyoke College Crawford, Karen, St. Mary's College of Maryland Davis, Bruce. Yale University Debowy, Owen, New York University School of Medicine Desai. Arshad. European Molecular Biology Laboratory, Germany Detrait. Eric. University of Texas Medical Branch Devlin, Leah. Penn State University Doherty. Ann, Connecticut College Dou. Hongwei, University of Cincinnati Dumollard, Remi, Station Zoologique de Ville-Franche Sur Mer. France Eddleman. Christopher. University of Texas Medical Branch. Austin Escalona de Motta. Gladys. University of Puerto Rico Eyman, Maria. University of Naples, Italy Fakhrzadeh, Ladan. Rutgers University Felke. Erin, University of Illinois Femandez-Busguets. Xavier. Friedrich Miescher Institute. Switzerland Fraser, Scott. California Institute of Technology Fukui. Yoshio. Northwestern University Medical School Gainer. Harold. National Institutes of Health Galanis, Jennifer. National Institutes of Health Galbraith. James A., National Institutes of Health Gallant, Paul E.. National Institutes of Health Summer Research R37 Gallo. Michael, University of Medicine and Dentistry of New Jersey Gerosa-Erni, Daniela, Friedrich Miescher Institute. Switzerland Gioio, Anthony, National Institutes of Mental Health Gleeson. Richard, University of Florida Goldman, Anne E., Northwestern University Medical School Gomez, Maria del Pilar, Boston University School of Medicine Gonzalez-Lima, Francisco, University of Texas, Austin Grant, Philip, National Institutes of Health Grassi, Daniel, Food and Drug Administration Gyoeva, Fatima K., Institute of Protein Research. Russia Hagar, Robert, Yale University School of Medicine Harrington, John, University of South Alabama, Mobile Harrist, Alexia, Yale University Harrow, Faith, Hunter College Harwood, Claire, University of Pennsylvania Helfand, Brian, Northwestern University Medical School Helm. Jessica, Washington and Lee University Hernandez, Carlos, New York University School of Medicine Herrick, Scott, University of California, Riverside Hitt, James, State University of New York Health Science Center Hiza, Nicholas, Williams College Ho-Sang. Dwight, Williams College Hogan. Emilia, Yale University Medical School Holford, Kenneth, Illinois State University Holmgren, Miguel, Harvard University Medical School Hoof, Laura, University of Chicago Nguyen, Michael P., University of Texas Medical Branch Oegema. Karen, European Molecular Biology Laboratory. Germany Ogan, Jeff, Illinois State University Orfila, James, University of Texas, San Antonio Peck, Raphaela, Reed College Petersen, Jennifer. National Institutes of Health Powers, Maureen. Vanderbilt University Prahcad, Veena, Northwestern University Medical School Prasad, Kondury. University of Texas Health Science Center Price, Nichole, Connecticut College Quinn, Kerry, Yale University School of Medicine Ingrassia. Rosaria, University of Milan, Italy Inoue, Masashi, Tokyo University of Pharmacy and Life Science, Japan Intravaiu. Anthony, NASA Ames Research Center Janowitz, Tobias, University Hannover, Germany Ramsey, David, Harvard University Rankin, Ellen, Colgate University Reinsch, Sigrid, NASA Ames Research Center Rhodes, Paul, New York University Medical School Ring, Sabine, University of Frankfurt. Germany Rosenstein. Fred, Hunter College Ruta, Vanessa, Hunter College Kamino, Kohtaro, Tokyo University School of Medical and Dental, Japan Kannenberg, Kai. University of Milan, Italy Kapoor, Turun, Harvard University Medical School Kifaieh, Nidal. Long Island University King. Alison Jane, Dalhousie University, Canada Klimov, Andrei, University of Pennsylvania Koroleva, Zoya, Hunter College Kuner, Thomas, Duke University Medical Center Lassen, Kara G., Wake Forest University Lee, Kyeng Gea, Hunter College Lee, Rosalynn, University of Georgia Lesher, Sarah. University of Maryland Leznik, Elena, New York University School of Medicine Loboda. Andrey, University of Pennsylvania Lowe. Christopher, University of California, Berkeley Schuette. Etha, Hunter College Schwartz. William, University of Massachusetts Medical School Simpson, Tracy, University of Hartford Steffen, Walter. University of Rostock, Germany Steinacker, Antoinette. University of Puerto Rico Stockbridge, Norman, Food and Drug Administration Szalisznyo, Krisztina, Hungarian Academy of Science. Hungary Tamse, Catherine, University of Rhode Island Tan, Xiao, Williams College Tang, Kathleen. Washington University Taylor, Kevin, Wake Forest University Thorn, George, University of Cambridge. United Kingdom Tokumaru. Hiroshi. Duke University Medical Center Tokumaru. Keiko, Duke University Medical Center Townsel, James G.. Meharry Medical College Tran. Phong. Columbia University Twersky. Laura, Saint Peter's College Maddox, Paul. University of North Carolina, Chapel Hill Malchow. Robert Paul, University of Illinois, Chicago Melishchuk. Alexey. University of Pennsylvania Mitchell, Michael, Wake Forest University Miyake, Katsuya, Fukushima Medical College, Japan Mohan, Nishal, Hunter College Moir, Robert. Northwestern University Motta. Melissa, Williams College Vasse. Aimee, Williams College Villa-Komaro. Lydia. Northwestern University Wachowiak, Matt, Yale University School of Medicine Ward, Rita. National Institutes of Health Watts. Kisha, Williams College Wen, Huajie, National Institutes of Mental Health Wisniewski, Rachel. University of Georgia R38 Annual Report Woellert, Torsten, University of Rostock, Germany Wu, Samuel, Baylor College of Medicine Yancey. Sadiga, Mass Bay Community College Yang, Stacy. University of California, Irvine Young, Iain, University of Pennsylvania Zakevicius, Jane M., University of Illinois College of Medicine Zavilowitz, Joe, Yale University Library Readers Abbott, Jayne, Marine Research, Inc. Adelberg. Edward, Yale University Ahmadjian. Vernon, Clark University Allen. Garland, Washington University Allen, Nina, North Carolina State University Alliegro, Mark, Louisiana State University Alsup, Peggy, Tennessee Department of Health Anderson. Everett. Harvard Medical School Barrett, Dennis, University of Denver Barry. Susan, Mount Holyoke College Bedard, Andre, York University Benjamin, Thomas, Harvard Medical School Bcrnhard, Jeffery, University of Massachusetts Medical School Bernheimer, Alan, New York University School of Medicine Borgese, Thomas, Lehman College-CUNY Boyer. John, Union College Campos, Ana, McMaster University Candelas. Graciela. University of Puerto Rico Chang. Donald, Hong Kong University Child, Frank. Woods Hole. MA Clark, Douglas, John Hopkins University Clarkson, Kenneth, Lucent Technologies Cobb, Jewel, California State University Cohen, Seymour. Woods Hole, MA Colinvaux, Paul. Marine Biological Laboratory Collier, Marjorie, University of Louisiana Cooperstein. Sherwin, University of Connecticut Health Center Copeland, Eugene, Woods Hole, MA Corwin, Jeffery, University of Virginia Couch, Ernest, Texas Christian University Cowling, Vincent, University of Florida Duncan, Thomas K., Nichols College Epstein, Herman. Brandeis University Farmant'armaian, A. Verdi, Rutgers University Fee, Michale, Bell Laboratories Frenkel. Krystyna. New York University School of Medicine Gabriel, Mordecai, Brooklyn College Galatzer-Levy. Robert. University of Chicago German, James, Cornell University Ginsberg, Harold, National Institutes of Health Goldstein, Moise, Johns Hopkins University Grossman, Albert, New York University School of Medicine Gruner, John, Cephalon, Inc. Guttenplan. Joseph, New York University Dental and Medical School Haimo. Leah, University of California Harrington, John, University of South Alabama Hays, Thomas, University of Minnesota Hernandez, Mari-Luz, University of Nice Herskovits. Theodore, Fordham University Hunter. Robert, Gartnaval Royal Hospital Inoue, Sadayuki, McGill University Jacobson, Allan, University of Massachusetts Josephson, Robert, University of California Kaltenbach, Jane, Mount Holyoke College Kamino, Kohtaro, Tokyo Medical and Dental School of Medicine Karlin, Arthur, Columbia University King, Kenneth, Falmouth, MA Klein, Donald, Colorado State University Kornberg. Hans, Boston University Krane, Stephen M., Harvard Medical School Laster, Leonard, University of Massachusetts Medical Center Lee, John, City College of CUNY Levy, Arthur, St. Vincents Hospital Lorand, Laszlo, Northwestern University Medical School Luckenbill, Louise, Ohio University MacNichol. Edward, Boston University School of Medicine Masland. Richard, Massachusetts General Hospital Mauzerall, David, Rockefeller University Mitchell. Ralph, Harvard University/DEAS Mizell, Merle, Tulane University Nagel, Ronald. Albert Einstein College of Medicine Narahashi, Toshio, Northwestern University Medical School Naugle, John. National Aeronautics and Space Administration Nicaise, Ghislain, University of Nice Nickerson. Peter. State University of New York. Buffalo Olds, James, George Mason University Pappas. George, University of Illinois, Chicago Pollen, Dan, University of Massachusetts Medical Center Porter, Mary, University of Minnesota Schippers, Jay, The HNG Foundation Schuel, Herbert, University of Buffalo, SUNY Shepro. David, Boston University Shriftman, Molly, Woods Hole, MA Spector, Abraham, Columbia University Spotte, Stephen, University of Connecticut Sundquist, Eric, U.S. Geological Survey Sweet, Frederick, Washington University School of Medicine Trager, William, Rockefeller University Tweedell, Kenyon, University of Notre Dame Tykocinski. Mark. University of Pennsylvania Van Holde, Kensal, Oregon State University Walton. Alan, University of Cambridge Warren, Leonard, Wistar Institute Wennger, Elora, Pfizer Central Research 1999 Library Room Readers Dan Alkon National Institute of Health Lucio Cariello Stazione Zoologica A. Dohm Giuseppe D'Alessio University of Naples Robert Goldman Northwestern University Medical School Roberto GonzalezPalaza Northwest Indian College Harlyn Halvorson Marine Biological Laboratory Michael Hines Yale Univ. School of Medicine Andres Kanner Rush University Alex Keynan Israel Academy of Science Kamino Kohtaro Tokyo Medical and Dental John W. Moore Duke University Medical Center Leyla Morrel Rush University Michael Rabinowitz Marine Biological Laboratory George Reynolds Princeton University Ann Stuart UNC Chapel Hill Gerald Weissmann NYU School of Medicine Summer Research Whillaker, J. Richard, University of New Brunswick Wolken, Jerome J., University of Pittsburgh Yevick, George, Stevens Institute of Technology Domestic Institutions Represented Acorda Therapeutics Alabama. University of. Birmingham Alaska Museum, University of Albany Medical College Albert Einstein College of Medicine Allegheny University of the Health Sciences Arizona State University Arizona, University of, Tucson Barnard College Baylor College of Dentistry Baylor College of Medicine Bell Laboratories Berkeley National Laboratory Beth Israel Hospital Beth Israel-Deaconess Medical Center Boston University Boston University School of Medicine Brandeis University Bridgeport Hospital Brigham and Women's Hospital Brigham Young University Bristol-Myers Squibb PRI Bronx Veterans Administration Medical Center Brown University Bryn Mawr College Buck Center for Research in Aging Buffalo, University of Burnham Institute California Institute of Technology California State University. Sacramento California, University of, Berkeley California, University of, Davis California, University of, Irvine California, University of, Los Angeles California, University of. Riverside California, University of, San Diego California, University of, San Francisco California, University of, Santa Barbara Carl Zeiss, Inc. Carnegie Mellon University Case Western Reserve University Catholic University of America Center for Clinical Computing Centers for Disease Control and Prevention Chicago, University of Children's Hospital, Boston Cincinnati, University of Cleveland Clinic Foundation Cold Spring Harbor Laboratory Colgate University Colorado Health Science Center, University of Colorado School of Medicine, University of Colorado, University of. Boulder Columbia University Columbia-Presbyterian Medical Center Connecticut College Connecticut Health Center, University of Connecticut, University of Cook County Hospital Cook Institute for Research and Education Cornell University Cornell University Medical Center Cornell University Medical College Creative Micro Tech. Inc. Dana-Farber Cancer Institute Dartmouth College Dartmouth-Hitchcock Medical Center Delaware, University of Duke University Duke University Medical Center Eastern Virginia Medical School Emory University Emory University School of Medicine Finch University of Health Sciences Florida Institute of Technology Florida State University Florida, University of Food and Drug Administration Forsyth Dental Center Foundation of Microbiology Fred Hutchinson Cancer Research Center General Electric Corporate Research Center George Washington University Georgetown University Georgetown University Medical Center Georgia State University Georgia, University of Geron Corporation Guam. University of Hahnemann University Harbor-UCLA Medical Center Harlem Hospital Center Hartford, University of Harvard University Medical School Harvard School of Public Health Harvard University Hawaii, University of Hebrew Rehabilitation Center for Aged House Ear Institute Houston Academy of Medicine Houston, University of Howard University Hunter College Idaho State University Idaho, University of Illinois State University Illinois. University of, Chicago Illinois, University of, Urbana-Champaign Indiana State University Indiana University Indiana University School of Medicine Institute for Genomic Research R40 Annual Report Iowa State University Iowa, University of Jet Propulsion Laboratory Johns Hopkins University School of Medicine Kansas Medical Center. University of Kansas, University of Kent State University Kentucky. University of Kewalo Marine Laboratory Lehigh University Leica. Inc. Loma Linda University School of Medicine Long Island University Louisiana State University Louisiana State University Medical Center Louisville. University of Loyola University of Chicago Lucent Technologies Ludwig Institute for Cancer Research. San Diego Marine Biological Laboratory Maryland School of Medicine, University of Maryland. University of Mass Bay Community College Massachusetts General Hospital Massachusetts Institute of Technology Massachusetts Medical School, University of Massachusetts. University of Mayo Clinic Scottsdale Medical College of Georgia Meharry Medical College Memorial Sloan-Kettermg Cancer Center Merck & Co. Merck Research Laboratory Mercy College MetroHealth Medical Center Miami. University of Michigan Medical School, University of Michigan State University Michigan. University of Midwestern University Minnesota School of Medicine, University of Minnesota, University of Missouri, University of Montana State University Morehouse School of Medicine Mount Holyoke College NASA Ames Research Center National Cancer Institute National Institutes of Health National Institutes of Health Library National Institutes of Mental Health National Library of Medicine National Marine Fisheries Service Naval Medical Research Institute Nebraska. University of Neuralynx Inc. Neurosciences Institute New Jersey. University of Medicine and Dentistry New Mexico. University of New York and Presbyterian Hospital New York Health Science Center. State University of New York State Department of Health New York State Institute for Basic Research New York University Medical Center New York University School of Medicine New York, City University of New York, State University of, Albany New York, State University of. Buffalo New York, State University of. Stony Brook North Carolina State University North Carolina, University of, Chapel Hill North Dakota. University of North Texas, University of Northern Arizona University Northwest Indian College Northwestern LIniversity Northwestern University Medical School Occidental College Ohio State University Ohio University Ohio, Medical College of Oklahoma, University of Oregon Health Science University Oregon Regional Primate Research Center Oregon State University Oregon, University of Parke-Davis Research Parmly Hearing Institute PE Applied Biosystems Penn State University Pennsylvania Health System, University of Pennsylvania LIniversity School of Medicine Pennsylvania. University of Pharmacia & Upjohn Pittsburgh, University of Pomona College Prince George's Hospital Center Princeton LIniversity Procter & Gamble Puerto Rico, University of Purdue University Quorum Pharmaceuticals Reed College Rhode Island, University of Rochester Medical Center, University of Rockefeller University Rush-Presbyterian-St. Luke's Medical Center Rutgers University Saint Peter's College Samaritan Health System. Phoenix Scnpps Institution of Oceanography Seattle Biomedical Research Institute SETI Institute SmithKlme Beecham Pharmaceuticals Smithsonian Institution South Alabama, University of. Mobile Southern California, University of Southern Methodist University Summer Research R-ll St. Jude Children's Research Hospital St. Louis University School of Medicine St. Mary's College of Maryland Stanford University Stanford University Medical Center Stratagene Swarthmore College Temple University School of Medicine Tennessee. University of Texas A&M University Texas Health Science Center. University of Texas Medical Branch. University of Texas Southwestern Medical Center, University of Texas Southwestern, University of Texas, University of Thomas Jefferson University Toledo, University of Towson University Tufts University Tufts University Health Sciences Library Tufts University School of Medicine Tulane University Medical Library Unilever Research Union College United States Army Medical Research Institute United States Army Natick RD&E Center United States Department of Agriculture United States Environmental Protection Agency Utah Medical Center, University of Utah, University of Vanderbilt University Veterans Administration Medical Center Virginia Commonwealth University Virginia Health Sciences Center. University of Virginia School of Medicine. University of Virginia Tech Virginia. University of Wadsworth Center for Labs and Research Wake Forest University Wake Forest University School of Medicine Washington and Lee University Washington University School of Medicine Washington, University of Wayne State University Wellesley College Wesleyan University Western Cape, University of the Whitehead Institute for Biomedical Research William and Mary, College of Williams College Wisconsin, University of, Madison Wisconsin. University of, Milwaukee Woods Hole Oceanographic Institution Yale University Foreign Institutions Represented Alberto Monroy Foundation Palermo, Italy Alexandria University, Egypt All India Institute of Medical Sciences, India Apollo Hospitals, India Australian National University. Australia Autonomous University of the State of Puebla, Mexico Basel Institute for Immunology. Switzerland Basel, University of. Switzerland Bio-Rad Microscience. United Kingdom Bogor Agricultural University, Indonesia British Columbia, University of, Canada Bruxelles, Universite Libre de, Belgium Buenos Aires, University of, Argentina Calgary, University of. Canada Cambridge, University of. United Kingdom Centre de Investigation y de Estudios Avanzados, Mexico Chile, University of. Chile Copenhagen, University of. Denmark Dalhousie University. Canada Denmark. Technical University of. Denmark Dundee. University of. Scotland Edinburgh. University of. Scotland Ethiopian Health and Nutrition Institute. The Netherlands European Molecular Biology Laboratory. Germany Fondazione Centre San Raffaele del Moute Tabor, Italy Frankfurt. University of, Germany Friedrich Meischer Institute. Switzerland Fukushima Medical College. Japan Fundacao Universitaria de Endocrinologia e Fertilidade, Brazil Glasgow, University of. United Kingdom Gromngen University, The Netherlands Guelph, University of. Canada Hannover. University of, Germany Hebrew University of Jerusalem, Israel Hong Kong, The Chinese University of. Hong Kong Hospital for Sick Children. Canada Hungarian Academy of Science. Hungary IceTherm Inc., Iceland Imperial Cancer Research Fund, United Kingdom Imperial College of Science, Technology and Medicine, United Kingdom Institute of Protein Research. Russia Instituto de Investigacion Medica "Mercedes y Martin Ferreyra.' Argentina Instituto Venezolano Investigaciones Cientificas, Venezuela International School of Advanced Studies. Italy Karolmska Institute Stockholm. Sweden Konstanz, University of. Germany Kuopio. University of, Finland Landcare Research, New Zealand Lausanne. University of, Switzerland Leiden, University of. The Netherlands Lethhridge, University of, Canada Liverpool Medical School, University of. United Kingdom Lodz, Medical Academy of. Poland R42 Annual Report Loeh Health Research Institute. Canada London, University of. United Kingdom Manchester, University of. United Kingdom Max-Planck-Institut, Germany McGill University, Canada McMaster University, Canada Medical Research Council. United Kingdom Melbourne. University of, Australia Milan, University of, Italy Naples. University of, Italy National Centre for Biological Sciences, India National Institute for Medical Research. United Kingdom National Research Council, Canada Otago, University of. New Zealand Ottawa, University of, Canada Oxford, University of. United Kingdom Paris, University of, France Pasteur Institute-Lille, France Palerson Institute for Cancer Research, United Kingdom Pisa. University of, Italy PPL Therapeutics, Scotland Regensburg, University of, Germany Rio de Janeiro, Federal University of, Brazil Rio de Janeiro, State University of, Brazil Roslin Institute, Scotland Rostock. University of, Germany Russian Academy of Sciences. Russia Sao Paulo, University of. Brazil Saskatchewan, University of. Canada Scientific Institute San Raffaele. Italy Simon Fraser University, Canada Southampton Oceanography Centre, United Kingdom St. Thomas' Hospital London. United Kingdom Station Zoologique de Ville-Franche Sur Mer. France Swiss Federal Institute of Technology, Switzerland Swiss Institute for Experimental Cancer Research. Switzerland Sydney, University of Technology, Australia Sydney, University of. Australia Tata Institute of Fundamental Research, India Technion-Israel Institute of Technology, Israel Tohoku University, Japan Tokyo University of Pharmacy and Life Science, Japan Tokyo University School of Medical and Dental. Japan Tokyo. University of, Japan Toronto, University of, Canada Tubingen, University of. Germany Universidad Nacional Autonoma de Mexico, Mexico Universita "La Sapienza," Italy Universite Paris-Sud, France University College London. United Kingdom Wageningen Agricultural University, The Netherlands Wallenberg Laboratory for Cardio Research, Sweden Walter and Eliza Hall Institute, Australia Witwatersrand, University of the. South Africa World Health Organization. West Africa Zurich, University of, Switzerland Year-Round Research Programs Architectural Dynamics in Living Cells Program The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution Established in 1992, this program focuses on architectural dynamics in living cells — the timely and coordinated assembly and disassembly of macromolecular structures essential for the proper functioning, division, motility. and differentiation of cells; the spatial and temporal organization of these structures; and their physiological and genetic control. The program is also devoted to the development and application of powerful new imaging and manipulation devices that permit such studies directly in living cells and functional cell-free extracts. The Architectural Dynamics in Living Cells Program promotes interdisciplinary research carried out by resident core and visiting investigators. Resident Core Investigators Danuser, Gaudenz, Postdoctoral Fellow Inoue, Shinya. Distinguished Scientist Katoh, Kaoru. Postdoctoral Scientist Oldenbourg, Rudolf. Associate Scientist Staff Geer, Thomas. Research Assistant Knudson, Robert. Instrumental Development Engineer Baraby. Diane. Laboratory Assistant MacNeil. Jane. Executive Assistant Staff Arimoto. Rieko, Washington University School of Medicine Biggs. David, AutoQuant Imaging Inc. Desai. Arshad. EMBL. Heidelburg. Germany Fukui, Yoshio, Northwestern University Medical School Goda, Makoto. Kyoto University. Japan Inoue. Theodore D., Universal Imaging Corporation Keefe, David, Rhode Island Women and Infants Hospital Liu. Lin. Rhode Island Women and Infants Hospital Maddox, Paul, University of North Carolina-Chapel Hill Matsurnoto, Brian. University of California-Santa Barbara Milchison, Timothy J.. Harvard Medical School Murray. John M., University of Pennsylvania Salmon, Edward D., University of North Carolina-Chapel Hill Tran. Phong, Columbia University Major emphasis in the Josephine Bay Paul Center in Comparative Molecular Biology and Evolution is placed upon comparative/ phylogenetic studies of genes and genomes, molecular microbial ecology/biodiversity and evolution of host defense mechanisms in marine invertebrates. The Center encourages studies of genotypic diversity across all phyla and promotes the use of modern molecular genetics and phylogeny to gain insights into the evolution of molecular structure and function. The Josephine Bay Paul Center is a member of NASA's Virtual Institute for Astrobiology. Other major research programs include Mitchell Sogin's studies of molecular evolution in eukaryotes and studies of genome sequences from parasitic microorganisms, Monica Riley's metabolic database and evolutionary studies of protein sequences, Neal Cornell's comparative molecular studies of genes critical to heme biosynthesis, and Michael Cummings' studies of evolution of pathogenetic microorganisms. Other collaborative projects include studies of P450 evolution (M. Sogin and John Stegeman's laboratory at Woods Hole Oceanographic Institution [WHOI] ). a molecular ecology component of the Long Term Ecological Research project (M. Sogin's laboratory and John Hobbie of The Ecosystems Center), and studies of molecular diversity among marine protists and bacteria (with marine microbiologists at WHOI). Future recruiting efforts will focus upon molecular evolution in developmental biology and genome sciences. The Center has excellent resources for studies of molecular evolution: automated DNA sequencing, well-equipped research laboratories, and powerful computational facilities. In addition to participating in the Parasitology and Microbial Diversity courses, the Center sponsors the Workshop in Molecular Evolution at the MBL, which has gained an international reputation for excellence. This Workshop offers 60 students a series of lectures and minisymposia that are complemented by a state- of-the-art computational facility. The Josephine Bay Paul Center in Comparative Molecular Biology and Evolution includes the laboratories of Neal Cornell. Michael Cummings. Monica Riley. and Mitchell Sogin. Resident Core Investigators Sogin. Mitchell, Director and Senior Scientist Cornell, Neal, Senior Scientist Cummings. Michael. Assistant Scientist Riley. Monica. Senior Scientist Wainwrieht, Norman, Senior Scientist R43 R44 Annual Report Adjunct Scientists Halanych. Ken, Woods Hole Oceanographic Institution Teske, Andreas, Woods Hole Oceanographic Institution Laboratory of Neal Cornell Research in this laboratory is concerned with the comparative molecular biology of genes that encode the enzymes for heme biosynthesis, with particular emphasis on 5-ammolevulinate synthase, the first enzyme in the pathway. Because the ability to produce heme from common metabolic materials is a near universal requirement for living organisms, these genes provide useful indicators of molecular aspects of evolution. For example, 5-aminolevulinate synthase in vertebrate animals and simple eukaryotes such as yeast and Plasmodium ftili'iparum have high sequence similarity to the enzyme from the alpha- purple subgroup of eubacteria. This supports the suggestion that alpha- purple bacteria are the closest contemporary relatives of the ancestor of eukaryotic mitochondria. The analysis also raises the possibility that plant and animal mitochondria had different origins. Aminolevulinate synthase genes in mitochondria-containing protists are currently being analyzed to obtain additional insight into endosymbiotic events. Also, genes of primitive chordates are being sequenced to gain information about the large-scale gene duplication that played a very important role in the evolution of higher vertebrates. Other studies in the laboratory have been concerned with the effects of environmental pollutants on heme biosynthesis in marine fish, and it has been shown that polychlorinated biphenyls (PCBs) enhance the expression of the gene for aminolevulinate synthase. Staff Cornell. Neal W., Senior Scientist Faggart. Maura A., Research Assistant Foster, Martin, Laboratory Assistant Frisbee, Cameran. Laboratory Assistant Visiting Scientist Fox, T.O., Harvard Medical School Laboratory of Michael P. Cummings The research is in the area of molecular evolutionary genetics and includes the study of the mechanisms of molecular genetic processes, and uses methods from molecular biology, statistics, computer science, molecular systematics, and population genetics. The basis for much of the research is comparative, across several levels of biological organization, and involves both computer-based and empirical studies. The major focus of research is using novel statistical methods to study relationships between genotype and phenotype. Current investigations in this area examine how gene sequence data can be used to understand and predict drug resistance in tuberculosis, variation in color vision, and basic immune system functions at the molecular level. For example, using drug resistance in Mycobacterium tuberculosis as a model system, we are investigating how well phenotype (level of drug resistance) can be predicted with genotype information (DNA sequence data). Drug resistance is a major problem in the treatment of infectious diseases. Understanding evolution of drug resistance, and developing accurate methods for its prediction using DNA sequence data can help in assessing potential resistance in a more timely fashion and circumvent the need for culturing bacteria, which takes several weeks in the case of tuberculosis. More generally, the relationship of genotype to phenotype is a fundamental problem in genetics, and through these investigations we hope to gain insight. The primary empirical work in the laboratory involves examination of opsins, proteins involved in color vision, from local species of Odonata (dragonflies and damselflies). Research on evolution of pathogenic bacteria also examines species within the genus Mycobacterium. Mycobacterium provides an excellent model system for studying evolution of pathogenicity and emergent pathogens; it is a large and widely distributed group that occupies a range of habitats (e.g., soil, water, skin), and exhibits a broad range of relationships with other organisms (e.g., free-living, commensal, parasitic). Importantly, the group contains a number of major human pathogens (e.g., those that cause tuberculosis and leprosy), including recently emerged pathogens. We are using phylogenetic analysis of DNA sequence data to study the evolutionary patterns of pathogenicity within Mycobacterium to discern patterns in the emergence of new pathogens. The goal of this work is to understand the origins of new pathogens and provide information that may aid in diagnosis and treatment efforts. Staff Cummings, Michael P., Assistant Scientist Mclnerney, Laura A., Research Assistant Visiting Scientist Neel, Maile C., University of California. Riverside Laboratory of Monica Ri/ey The genome of the bacterium Escheric/iia call contains all of the information required for a free-living chemoautotrophic organism to live, adapt, and multiply. The information content of the genome can be dissected from the point of view of understanding the role of each gene and gene product in achieving these ends. The many functions of E. coli have been organized in a hierarchical system representing the complex physiology and structure of the cell. In collaboration with Dr. Peter Karp of SRI International, an electronic encyclopedia of information is being constructed on the genes, enzymes, metabolism, transport Year-Round Research R45 processes, regulation, and cell structure of E. coli. The interactive EcoCyc program is now publicly available and has graphical hypertext displays, including literature citations, on nearly all of E. coli metabolism, all genes and their locations, a hierarchical system of cell functions and some regulation processes. This work is continuing. In addition, the E. coli genome contains valuable information on molecular evolution. We are analyzing the sequences of proteins of E. coli in terms of their evolutionary origins. By grouping like sequences and tracing back to their common ancestors, we learn not only about the paths of evolution for all contemporary E. coli proteins, but we extend even further back before E. coli, traversing millennia to the earliest evolutionary times when a relatively few ancestral proteins served as ancestors to all contemporary proteins of all living organisms. The complete genome sequence of E. coli and sophisticated sequence analysis programs permit us to identify evolutionary related protein families, determining ultimately what kinds of unique ancestral sequences generated all of present-day proteins. The data developed in the work has proven to be valuable to the community of scientists sequencing microbial genomes. E. coli data serve as needed reference points. Staff Riley. Monica, Senior Scientist Kerr. Alastair, Postdoctoral Scientist Liang, Ping, Postdoctoral Scientist MacGregor, Alicia, Laboratory Clerk Nalium, Laila, Postdoctoral Scientist Pelegrini-Toole, Alida, Research Assistant II Porterfield, Pamela, Laboratory Clerk Serres. Margerethe, Postdoctoral Scientist sudden evolutionary radiations that cannot be resolved by rRNA comparisons and will provide insights into the presence or absence of important biochemical properties in the earliest ancestors common to all eukaryotic species. More recently, we initiated a study of the complete genome of Giardia lamblia. Staff Sogin, Mitchell L., Director and Senior Scientist Amaral-Zettler, Linda, Postdoctoral Scientist Beaudoin, David, Research Assistant Bressoud, Scott, Laboratory Technician Eakin, Nora, Research Assistant Edgcomb, Virginia, Postdoctoral Scientist Fair. Rebecca, Research Assistant Gao, Lingqui, Research Assistant II Harris. Marian, Executive Assistant Holder, Greg, Research Assistant Kim, Ulandt, Research Assistant Kysela, David, Research Assistant Laan. Maris, Research Assistant II Lim. Pauline. Executive Assistant Luders, Bruce. Research Assistant McArthur, Andrew, Postdoctoral Scientist Medina, Monica, Postdoctoral Scientist Morrison, Hilary G., Postdoctoral Scientist Nixon, Julie. Postdoctoral Scientist Roger, Andrew, Postdoctoral Scientist Shakir, Muhhamed Afaq, Postdoctoral Scientist Silberman. Jeffrey. Postdoctoral Scientist Program in Comparative Molecular Biologv and Evolution: Laboratory of Mitchell L. Sogin This laboratory in molecular evolution employs comparative phylogenetic studies of genes and genomes to define patterns of evolution that gave rise to contemporary biodiversity on the planet Earth. The laboratory is especially interested in discerning how the eukaryotic cell was invented as well as the identity of microbial groups that were ancestral to animals, plants, and fungi. The lab takes advantage of the extraordinary conservation of ribosomal RNAs to define phylogenetic relationships that span the largest of evolutionary distances. These studies have overhauled traditional eukaryotic microbial classifications systems. The laboratory has discovered new evolutionary assemblages that are as genetically diverse and complex as plants, fungi, and animals. The nearly simultaneous separation of these eukaryotic groups (described as the eukaryotic "Crown") occurred approximately one billion years ago and was preceded by a succession of earlier diverging protist lineages, some as ancient as the separation of the prokaryotic domains. At the same time, this data base provides a powerful tool for the newly emerging discipline of molecular ecology. Using the ribosomal RNA data base and nucleic acid-based probe technology, it is possible to detect and monitor microorganisms, including those that cannot be cultivated in the laboratory. This strategy has uncovered new habitats and major revelations about geographical distribution of microorganisms. The laboratory has initiated a program to sample genomic diversity from eukaryotic microorganisms that do not have mitochondria. The lab previously demonstrated that these taxa represent some of the earliest diverging lineages in the evolutionary history of eukaryotes. The objective is to develop a set of additional molecular markers for studying molecular evolution. These will be invaluable in unraveling Visiting Investigators Bahr, Michele, The Ecosystems Center Campbell, Robert, Serono Laboratories, Inc. Crump. Byron. The Ecosystems Center Weil. Jennifer. Joslin Diabetes Center Adjunct Scientists Halanych. Kenneth, Woods Hole Oceanographic Institution Teske, Andreas, Woods Hole Oceanographic Institution R46 Annual Report BioCurrents Research Center The Biocurrents Research Center (BRCl. one of the NIH National Centers for Research Resources, pioneers methods in the study of transmembrane currents and hosts numerous research pursuits. The Center provides visiting investigators access to a variety of unique technologies as well as new approaches to experimentation in the hiomedical sciences. Four systems are available at the BRC. All these probe technologies are based on the principle of a self-referencing electrode, maximizing sensitivity by noise and drift reduction. All the probes are non-invasive and generally placed in close proximity to the membrane of cells or tissues, in some cases at sub-micron distances. The two older techniques are designed to measure the movement of ions across the membranes ot living tissues or cells with the minimum of disturbance. The current probe, developed in 1974, is still available for the study of external current densities resulting from the general net balance ot ion transport. Most use is made of the ion-selective probes (Sens), which measure and follow the transmembrane transport of specific ions such as calcium, potassium and protons. This system also can detect non-electrogenic transporters. Two newer techniques are also available: the BioKelvin probe and the non-invasive electrochemical or polarographic probe (Serp). The BioKelvin probe measures voltages around living tissues in air. A radically different approach is being taken to the measurements of biocurrents using the electrochemical microprobes. Presently applied to molecular oxygen, such a technique offers opportunity for the study of molecular transport by using the chemical redox potential. This probe has been applied to single neurons. |8-pancreatic cells, damaged neural tissues, developing embryos, and others. We are currently developing further applications of the Serp probes to measure nitric oxide, ascorbic acid, and insulin as well as the production of biosensors. A state-of-the-art system offers non-invasive ion probes coupled with current and voltage clamp (both single, two electrode, and patch) along with ratio imaging via a Zeiss Attofluor system, all of which are finding uses in the hosted biomedical studies, as well as BRC research and development. As in previous years, a wide variety of biological and biomedical subjects have been studied by BRC staff and visitors. In R&D we have continued developing the application of ion-selective and electrochemical microsensors. all applicable to single cells with square micron spatial resolution. We are currently exploring ways to combine these sensors with a variety of techniques known collectively as near field optical microscopy. In an experimental context we have advanced our technology into several fields, including reproductive physiology, diabetes research, neuroscience, development, gravitropic responses, ion transport, and homeostasis. Details of our research program and a list of publications can be found at . MBL year-round laboratories with which BRC is in active collaboration are the Laboratory of Rudolf Oldenbourg and the Laboratory of Reproductive Medicine, headed by David Keefe. Dr. Keefe and Dr. Peter Smith. BRC Director, are Co-Investigators on a project to support the development of new technology to assess the developmental potential of preimplantation embryos and to study the pathophysiology of oocyte dysfunction. Staff Smith. Peter J.S., Director and Senior Scientist Baikie. Iain D., Associate Scientist Danuser. Gaudenz M.. Postdoctoral Fellow Hammar. Katherine, Research Assistant 111 McLaughlin. Jane A., Research Assistant III Porterfield. D. Marshall, Staff Scientist I Sanger, Richard H., Research Assistant III Part Time and Temporary Staff' Jaffe. Lionel F.. Senior Scientist Moore. Laurel. Science Reference Librarian Pepperell. John R., Staff Scientist I Graduate Student Tamse, Catherine T.. University of Rhode Island Visiting Scientists and Publications This year the Research Center hosted 47 visitors. Scientific publications during the year numbered 25. Boston University Marine Program Faculty Atema, Jelle, Professor of Biology, Director Dionne, Vincent, Professor of Biology Golubic, Stjepko. Professor of Biology Humes, Arthur, Professor of Biology Emeritus Kaufman, Les, Associate Professor of Biology Lohel, Phillip. Associate Professor of Biology Voigt. Rainer, Research Associate Professor Ward, Nathalie, Lecturer Staff Decarie. Linette, Senior Staff Coordinator DiNunno. Paul, Research Assistant. Dionne Lab Hall. Sheri, Program Manager McCafferty. Michelle. Administrative Assistant Olson, Nancy, Program Assistant Tomasky. Gabrielle. Research Assistant, Valiela Lab Wheatlev, MaryJo, Information Officer Postdoctoral Investigators Basil. Jenny. Atema Laboratory Cehrian. Just. Valiela Laboratory Year-Round Research R47 Grasso, Frank, Atema Laboratory Trott, Thomas, Atema Laboratory Visiting Faculty and Investigators Hanlon, Roger, Marine Biological Laboratory Hecker. Barbara, Meeker Consulting Margulis, Lynn, University of Massachusetts, Amherst McFall-Ngai, Margaret, Kewalo Marine Laboratory Moore, Michael. Woods Hole Oceanographic Institution Nowacek, Douglas, Mote Marine Laboratory Ruby, Edward, Kewalo Marine Laboratory Simmons, Bill, Sandia National Laboratory Wainvvright. Norman, Marine Biological Laboratory Other Dolan, Mike. Visiting Teaching Assistant Weir. Andrew. Visiting Teaching Assistant Graduate Students PhD Students Existing Cole. Marci Dale. Jonathon Economakis. Alistair Hauxwell. Jennifer Herrold, Ruth Kroeger, Kevin Lindholm, James Ma, Diana Miller. Carolyn Oliver, Steven Sloan. Kevin Stieve, Erica Zettler, Erik Zhao, Jing New Dooley, Brad Tomasky, Gabrielle York. Joanna Masters Students Existing Allen, Christel Atkinson. Abby Barlas. Margaret Bentis, Christopher Bowen. Jennifer Cavanaugh, Joseph Chichester. Heather D'Ambrosio. Alison Evgenidou, Angeliki Ferland. Amy Fern. Sophie Fredland, Inga Griffin, Martin Homkow, Laura Keith, Lucy Koenig, Eduardo Konkle, Anne Lamb. Amy Lawrence. David Levine, Michael McKenna. Ian Neviackas. Justin Ramon. Marina Smith, Spence Watson. Elise Wright. Dana New Casper, Brandon Errigo, Michael Frenz, Christopher Grable, Melissa Grebner, Dawn Kollaros, Maria Lever, Mark Malley, Vanessa Martel, David Oweke, Ojwang William Perez, Edmundo Pugh, Tracy Ripley, Jennifer Roycroft, Karen Stueckle, Todd Sweeny. Melissa Tuohy-Sheen, Elizabeth Weiss, Erica Undergraduate Students Spring 99 Champagne, Jaimie Preto, Luca Watkins, Cari Weisbaum. Dolores Fall 99 Burgess, Robyn Gottlieb, Jennifer Griggs, Ryan Kwong, Grace Loewensteiner. David Matsumoto, Rae Muhlm. Jessica O'Connell. Timmy Peyton, Scott Pytel. Julie Sarno, Jillian Silverston, Jennifer von Kampen. Marie Walker. Andrew Williams. Jade Wingert, Sarah Woods. Pamela Summer 1999 Interns Berkey, Cristin Cantield, Susannah Cubbage, Andrea Hanna. John Komarow, Sharon McLaughlin, Leslie Mijos. Katnn R48 Annual Report Walters. Jennifer Wai M in, Amy Wolfe. Felisa Young. Talia Summer 1W9 Volunteers Hancock. Amy Qumn, Elizabeth Laboratory of J vile Atenui Many organisms and cellular processes use chemical signals as their main channel of information about the environment. All environments are noisy and require some form of filtering to detect important signals Chemical signals are transported by turbulent currents, viscous flow, and molecular diffusion. Receptor cells extract chemical signals from the environment through various filtering processes. In our laboratory, fish, marine snails, and Crustacea have been investigated for their ability to use chemical signals under water. Currently, we use the lobster and its exquisite senses of smell and taste as our major model to study the signal-filtering capabilities of the whole animal and its narrowly tuned chemoreceptor cells. Research in our laboratory focuses on amino acids, which represent important food signals for the lobster, and on the function and chemistry of pheromones used in lobster courtship. We examine animal behavior in the sea and in the lab. This includes social interactions and chemotaxis. To understand the role of chemical signals in the sea we use real lobsters and untelhered small robots. Our research includes measuring and computer modeling odor plumes and the water currents lobsters generate to send and receive chemical signals. Other research interests include neurophysiology of receptor cells and anatomical studies of receptor organs and pheromone glands. Liibomton' of Vincent Dionne How does the brain learn about an odor? This simple question frames a complex problem about how information is transferred into and within the brain. Odors are powerful stimuli. They can focus the attention, elicit behaviors, and resurrect forgotten memories. These actions depend on the initial transduction and encoding of odor signals by olfactory sensory neurons located deep in the nasal passages. Odor transduction involves a number of intracellular processes wherein odor receptors on the surfaces of olfactory receptor neurons are coupled to ion channels in the neuronal membrane through G proteins and other intracellular elements. Odors activate the transduction machinery, causing the neuron to fire a coded message carrying information that the brain is able to interpret. The information encoded after just one sniff of odor is actually earned by many olfactory neurons simultaneously, but each neuron appears to carry only part of the message. Thus encoding of odor information is a multicellular process, and different olfactory neurons can carry different pieces of the code. We are studying the cellular processes that underlie odor transduction and encoding in aquatic salamanders and in mice. Using electrophysiological, imaging, and pharmacological tools, our goal is to learn how these most fundamental actions work, for they represent an elegant and very ancient solution to a complex problem of neural function. Laboratory of Arthur G. Humes Research interests include systematics, development, host specificity, and geographical distribution of copepods associated with marine invertebrates. Current research is on taxonomic studies of copepods from invertebrates in the tropical Indo-Pacific area, and poecilostomatoid and siphonostomatoid copepods from deep-sea hydrothemial vents and cold seeps. Laboratory of Lex Kuufniuii Current research projects m the laboratory deal with speciation and extinction dynamics of haplochromine fishes in Lake Victoria. We are studying the systematics. evolution, and conservation genetics of a species flock encompassing approximately 700 very recently evolved taxa in the dynamic and heavily impacted landscape of northern East Africa. In the lab we are studying evolutionary morphology, behavior, and systematics of these small, brightly colored cichlid fishes. Another area of study is developmental and skeletal plasticity in fishes. We are studying the diversity of bone tissue types in fishes, differential response to mineral and mechanical challenge, and matrophic versus environmental effects in the development of coral reef fishes. We also study the biological basis for marine reserves in the New England fisheries. We are involved in collaborative research with NURC. NMFS, and others studying the relative impact on groundfish stocks of juvenile habitat destruction rtr.vii.v fishing pressure. Luhorator\ of Phi/lip Lobe/ Fishes are the most diverse vertebrate group and provide opportunities to study many aspects of behavior, ecology and evolution. We primarily study how fish are adapted to different habitats and behavioral ecology of species interactions. Current research focuses on fish acoustic communications. We are also conducting a long-term study of (he marine biology of Year-Round Research R49 Johnston Atoll, Central Pacific Ocean. Johnston Atoll has been occupied continuously by the military since the 1930s and proved a unique opportunity for assessing the biological impacts of island industrialization and its effects on reefs. Johnston Atoll is the site of the US Army's chemical weapons demilitarization facility. JACADS. Ongoing projects also include fish faunal studies in the African Congo, Belize Central America, and Wake Atoll, Pacific. Laboratory of Ivan Valiela A focus of our work is the link between land use on watersheds and consequences in the receiving estuarine ecosystems. The work examines how landscape use and urbanization increase nutrient loading to groundwater and streams. Nutrients in groundwater are transported to the sea, and, after biogeochemical transformation, enter coastal waters. There, increased nutrients bring about a series of changes on the ecological components. To understand the coupling of land use and consequences to receiving waters, we study the processes involved, assess ecological consequences, and define opportunities for coastal management. A second long-term research topic is the structure and function of salt marsh ecosystems, including the processes of predation, herbivory. decomposition, and nutrient cycles. Center for Advanced Studies in the Space Life Sciences In 1995, the NASA Life Sciences Division and the Marine Biological Laboratory established a cooperative agreement with the formation of the Center for Advanced Studies in the Space Life Sciences (CASSLS) at MBL. The Center's overall goals are to increase awareness of the NASA Life Sciences Program within the basic science community, and to examine and discuss potential uses of microgravity and other aspects of spaceflight as probes to provide new insights to fundamental processes of basic biology and medicine. Through symposia, workshops and seminars. CASSLS advises NASA and the biological science community on a wide variety of topics. Through fellowships. CASSLS supports summer research for investigators in areas pertinent to the aims of NASA life sciences. Since the Center began its operations in July 1995, more than 300 people have attended the seven CASSLS workshops. Typically these workshops last for two to four days and feature an international array of scientists and NASA/International space agency staff. In many cases, workshop chairs have a long-time association with the MBL. Workshop schedules incorporate many opportunities for interaction and discussion. A major outcome for workshops is the publication of proceedings in a peer-reviewed journal. Moreover, our meetings introduce outstanding biologists to research questions and to prominent scientists involved in gravitational biology and the NASA Life Sciences Program. The Center sponsored two workshops in 1999: Microgravity's Effects on Biological Systems and Behavior: An Integrative Approach, chaired by Richard Wassersug, Dalhousie University; and Cells in Spaceflight: Past, Present and Future, chaired by Dihp Kondepudi, Wake Forest University. The Center sponsored one Fellow during the summer of 1999: Dr. Paul McNeil of the Medical College of Georgia. Dr. McNeil used sea urchin eggs as a model system to study the subcellular and molecular basis of the cell's response to a temporary disruption in plasma membrane integrity. Staff Blazis. Diana E.J., Administrator Oldham. Pamela A., Administrative Assistant The Ecosystems Center The Center carries out research and education in ecosystems ecology. Terrestrial and aquatic scientists work in a wide variety of ecosystems ranging from the streams, lakes and tundra of the Alaskan Arctic (limits on plant primary production) to sediments of Massachusetts Bay (controls of nitrogen cycling), to forests in New England (effects of soil warming on carbon and nitrogen cycling), and South America (effects on greenhouse gas fluxes of conversion of rain forest to pasture) and to large estuaries in the Gulf of Maine (effects on plankton and benthos of nutrients and organic matter in stream runoff). Many projects, such as those dealing with carbon and nitrogen cycling in forests, streams, and estuaries, use the stable isotopes "C and 15N to investigate natural processes. A mass spectrometer facility is available. Data from field and laboratory research are used to construct mathematical models of whole- system responses to change. Some of these models are combined with geographically referenced data to produce estimates of how environmental changes affect key ecosystem indexes, such as net primary productivity and carbon storage, throughout the world's terrestrial biosphere. The results of the Center's research are applied, wherever possible, to the questions of the successful management of the natural resources of the earth. In addition, the ecological expertise of the staff is made available to public affairs groups and governmental agencies who deal with problems such as acid rain, coastal eutrophication, and possible carbon dioxide-caused climate change. The Semester in Environmental Science was offered again in Fall 1999. Eighteen students from 14 colleges participated in the program. There are opportunities for postdoctoral fellows. Administrative Staff Hobbie. John E., Co-Director Melillo, Jerry M., Co-Director Foreman, Kenneth H.. Associate Director. Semester in Environmental Studies Berthel, Dorothy J.. Administrative Assistant Donovan, Suzanne J., Executive Assistant Moniz, Priscilla C., Administrative Assistant. Semester in Environmental Studies Nunez, Guillermo, Research Administrator Seifert, Mary Ann, Administrative Assistant Scanlon, Deborah G.. Executive Assistant, LMER Coordination Office Scientific Staff Hobbie, John E.. Senior Scientist Melillo, Jerry M., Senior Scientist Deegan, Linda A.. Associate Scientist Giblin, Anne E., Associate Scientist Herbert. Darrell A., Staff Scientist Holmes, Robert M.. Staff Scientist Hopkinson, Charles S., Senior Scientist Hughes, Jeffrey E., Staff Scientist Nadelhoffer. Knute J., Associate Scientist Neill. Christopher. Assistant Scientist Peterson, Bruce J., Senior Scientist Rastetter, Edward B., Associate Scientist Shaver, Gaius R., Senior Scientist Steudler, Paul A., Senior Research Specialist Tian, Hanqin, Staff Scientist Vallino. Joseph J.. Assistant Scientist Williams, Mathew, Assistant Scientist R50 Annual Report Educational Staff Appointments Bovard. Brian. Postdoctoral Research Associate Buzby. Karen. Postdoctoral Research Associate Cieri. Matthew D.. Postdoctoral Research Associate Crump, Byron. Postdoctoral Research Associate Garcia-Montiel. Diana C.. Postdoctoral Research Associate Hartley, Anne E., Postdoctoral Research Associate Kappel-Schmidt, Inger, Postdoctoral Research Associate Nordin, Annika. Postdoctoral Research Associate Raymond. Peter. Postdoctoral Research Associate Tobias, Craig R., Postdoctoral Research Associate Technical Staff Ahrens, Toby. Research Assistant Bahr. Michele P., Research Assistant Bettez, Neil D., Research Assistant Byun, James P., Research Assistant Carpino, Elizabeth, Research Assistant Claessens, Lodevicus H. J. M., Research Assistant Colman, Ben, Research Assistant Downs, Martha R.. Research Assistant Fox, MaryKay, Research Assistant Garritt. Robert H., Senior Research Assistant Holland. Keri J.. Research Assistant Hrywna. Yarek. Research Assistant Jablonski. Sarah A., Research Assistant Jillson, Tracy A., Research Assistant Kelsey, Samuel, Research Assistant Kicklighter, David W., Senior Research Assistant Klcmhenz. Andrew, Research Assistant Kwiatkowski, Bonnie L., Research Assistant Laundre, James A., Senior Research Assistant Lux. Heidi, Research Assistant Micks, Patricia, Research Assistant Newkirk. Kathleen M., Research Assistant Nolin, Amy L., Research Assistant Nowicki. Genevieve, Research Assistant Pan, Shufen, Research Assistant Regan. Kathleen M., Research Assistant Ricca, Andrea, Research Assistant Schwamb. Carol, Research Assistant Slavik, Karie A., Research Assistant Thieler, Kama K., Research Assistant Tholke, Kristin S., Research Assistant Thomas, Suzanne M.. Research Assistant Tucker, Jane, Senior Research Assistant Vasiliou, David S., Research Assistant Weston, Nathaniel B., Research Assistant Wollheim. Wilfred M., Research Assistant Wright, Amos, Research Assistant Wyda, Jason C. Research Assistant Consultants Bowles, Francis P., Research Systems Consultant Bowles, Margaret C., Administrative Consultant Visiting Scientists and Scholars Banta, Gary. Roskilde University, Roskilde, Denmark Duncan, Thomas, Nichols College Fleischer. Dirk. Friedrich-Alexander Universitat Erlangen-Nurnberg. Germany Mondrup. Thomas. Roskilde University. Roskilde. Denmark Moore. Marianne, SES Faculty Fellow, Wellesley College Laboratory of Aquatic Biomedicine Work in this laboratory centers on comparative immunopathology and molecular biology using marine invertebrates as experimental models. Examples of current research include determining the prevalence of leukemia in M\a arenaria (the soft shell clam) in Massachusetts. Monoclonal antibodies developed by this laboratory are being used to diagnose clam leukemia, identify and characterize a tumor-specific protein, and differentiate other leukemias in bivalve molluscs. Development and chemically induced changes in gene expression and neuronal growth are also being studied in the surf clam. Spisula solidissima. Work in molecular biology is creating a clearer understanding of the comparative etiology and pathogenesis of tumors, particularly in environmentally impacted aquatic animals. Staff Reinisch, Carol L.. Senior Scientist Jessen-Eller, Kathryn, Postdoctoral Scientist Kreiling. Jill. Postdoctoral Scientist Visiting Scientists Stephens. Raymond, Boston University Walker, Charles, Professor of Zoology, University of New Hampshire Student Steiger. Daniel, Tufts University School of Veterinary Medicine Laboratory of Cell Communication Established in 1994, this laboratory is devoted to the study of intercellular communication. The research focuses on the cell-to-cell channel, a membrane channel built into the junctions between cells. This channel provides one of the most basic forms of intercellular communication in organs and tissues. The work is aimed at the molecular physiology of this channel, in particular, at the mechanisms that regulate the communication. The channel is the conduit of growth- regulating signals. It is instrumental in a basic feedback loop whereby cells in organs and tissues control their number; in a variety of cancer forms it is crippled. This laboratory has shown that transformed cells lacking communication channels lost the characteristics of cancer cells, such as unregulated growth and Himorigenicity, when their communication was restored by insertion of a gene that codes for the channel protein. Work is now in progress to track the channel protein within the cells from its point of synthesis, the endoplasmic reticulum. to its functional destination in the plasma membrane, the cell-to-cell junction, by expressing a fluorescent variant of the channel protein in the cells. Knowledge about the cellular regulation of this process will aid our understanding of what goes awry when a cell loses the ability to torm cell-to-cell channels and thus to communicate with its neighbors, thereby taking the path towards becoming cancerous. Another line of work is taking the first steps at applying information theory to the biology of cell communications. Here, the intercellular information spoor is tracked to its source: the macromolecular intracellular information core. The outlines of a coherent information network inside and between the cells are beginning to emerge. Year-Round Research R51 Staff Loewenstein, Werner, Senior Scientist Rose, Birgit, Senior Scientist Jillson, Tracy, Research Assistant Laboratory of Paul Colinvaux The research of this laboratory reconstructs Pleistocene climatic and environmental histories of the continents from the sediments of ancient lakes, particularly in the Amazon basin. The team has raised sediment cores from lakes in the lowland Amazon forests that span the last 30,000 years, including records of the last glacial maximum (LGM). Several of these sites were extremely remote. We use pollen analysis to reconstruct the history of vegetation around the lakes, an undertaking that required us to produce a pollen taxonomy for the diverse Amazon forests (published this year as an Amazon Pollen Manual and Alias). The research has shown that the lowland Amazon forests persisted through glacial cycles, with some reassortment of species as temperature fluctuated from the LGM to the present. These data are useful for the calibration of global climate models and in understanding how the great diversity of the Amazon biota is maintained. Because of the variety of analytical techniques other than pollen analysis used, we organize the research in collaboration with specialized laboratories at other institutions. Although our research is now concentrated in the Neotropics we also have a continued interest in the paleoecology of the arctic, with sites in Alaska and Russia. Staff Colinvaux, Paul, Adjunct Scientist Laboratory of Ayse Dosemeci The laboratory investigates molecular processes that underlie synaptic modification. The current project is aimed at clarifying how the frequency of activation at a synapse can determine whether the synapse will be potentiated (strengthened) or depressed (weakened) through the participation of an enzyme called CaM kinase II. Self-regulatory properties of this enzyme are investigated to prove that it can respond to the temporal pattern of calcium, the intracellular signal generated upon synaptic activation. Related projects in collaboration with Dr. Thomas Reese (NIH, NINDS) involve tracing changes in the distribution of CaMKII in cultured hippocampal neurons in response to sustained glutamate receptor activation and investigating the structural plasticity of the postsynaptic density under these conditions. primary laboratory is the synthesis and function of y-carboxyglutamic acid in blood clotting proteins and the role of vitamin K. Cone snails are obtained from the South Pacific and maintained in the Marine Resources Center. Until recently, the marine cone snail had been the sole invertebrate known to synthesize •y-carboxyglutamic acid (Gla). The venomous cone snail produces neurotoxic conopeptides. some rich in Gla, which it injects into its prey to immobilize it. To examine the biosynthetic pathway for Gla, we have studied the Conns carboxylase which converts glutamic acid to y-carboxyglutamic acid. This activity has an absolute requirement for vitamin K. The Conus carboxylase substrates contain a carboxylation recognition site on the conotoxin precursor. Given the functional similarity of mammalian vitamin In- dependent carboxylases and (he vitamin K-dependent carboxylase from Conus textile, we hypothesized that structurally conserved regions would identify sequences critical to this common functionality. Furthermore, we examined the diversity of animal species that maintain vitamin K- dependent carboxylation to generate y-carboxyglutamic acid. We have cloned carboxylase homologs in full length or partial form from the beluga whale (Delphinapterus leucas), toadfish (Opsanus tau), chicken (Callus gallus), hagfish (Myxine glutinosa), horseshoe crab (Limulus polyphemus) and cone snail (Conns textile} in order to compare these structures to the known bovine, human, rat and mouse cDNA sequences. Comparison of the predicted amino acid sequences identified a highly conserved 32-amino acid residue region in all of these putative carboxylases. In addition, this amino acid motif is also present in the Drosophila genome and identified a Drosophila homolog of the y- carboxylase. Assay of hagfish liver and Drosophila demonstrated carboxylase activity in these non-vertebrates. These results demonstrate the broad distribution of the vitamin K-dependent carboxylase gene, including a highly conserved motif that is likely critical for enzyme function. The vitamin K-dependent biosynthesis of -y-carboxyglutamic acid appears to be a highly conserved function in the animal kingdom. Novel y-carboxyglutamic acid-containing conopeptides have been isolated from the venom of Conus textile. The amino acid sequence, amino acid composition, and molecular weights of these peptides have been determined. For several peptides, the cDNA encoding the precursor conotoxin has been cloned. The three-dimensional structure of some of these Gla-containing conopeptides as well as conantokin G have been determined by 2D NMR spectroscopy. Complete resonance assignments were made from 2D 'H NMR spectra via identification of intraresidue spin systems using 'H-'H through-bond connectivities. NOESY spectra provided daN, dNN and dpN NOE connectivities and vicinal spin-spin coupling constants 3JHNa were used to calculate <$> torsion angles. Structure generation based on interproton distance restraints and torsion angle measurements yield convergent structures generated using distance geometry and simulated annealing methods. The goal of this project is to determine the structural role of y-carboxyglutamic acid in the Gla- containing conotoxins and other y-carboxyglutamic acid-containing proteins. Staff Dosemeci, Ayse, Adjunct Scientist Laboratory of Barbara Furie and Bruce Furie y-Carboxyglutamic acid is a calcium-binding amino acid that is found in the conopeptides of the predatory marine cone snail, Conus. This laboratory has been investigating the biosynthesis of this amino acid in Conus and the structural role of y-carboxyglutamic acid in the conopeptides. This satellite laboratory relates closely to the main laboratory, the Center for Hemostasis and Thrombosis Research, on the Harvard Medical School campus in Boston; the main focus of the Staff Furie. Barbara C. Adjunct Scientist Furie. Bruce, Adjunct Scientist Stenflo, Johan, Visiting Scientist Czerwiec, Eva, Postdoctoral Fellow Begley. Gail. Scientist I Rigby, Alan. Adjunct Scientist Laboratory of Roger Hanlon This laboratory investigates the behavior and neurobiology of cephalopods. Studies of various learning capabilities are currently being R52 Annual Report conducted, as are studies on reproductive strategies that include agonistic behavior, female mate choice, and sperm competition. The latter studies involve DNA fingerprinting to determine paternity and help assess alternative mating tactics. Currently we are studying sensory mechanisms and functions of polarization vision in cephalopods. Complementary field studies are conducted locally and on coral reefs. The functional morphology and neurobiology of the chromatophore system of cephalopods are also studied on a variety of cephalopod species, and image analysis techniques are being developed to study crypsis and the mechanisms that enable cryptic body patterns to be neurally regulated by visual input. Staff Hanlon, Roger, Senior Scientist Buresch, Kendra, Research Assistant Maxwell. Michael. Postdoctoral Scientist Rummel, John, Visiting Scientist Shashar, Nadav, Postdoctoral Scientist SusMnan. Raquel. Investigator Visiting Investigators Adamo, Shelley. Dalhousie University Baker, Robert, New York University Benjamins, Steven. Graduate Student, University of Groningen Boal, Jean, Adjunct Scientist Cavanaugh. Joseph. Graduate Student. Boston University Marine Program Fern. Sophie, Graduate Student. Boston University Marine Program Hatfield, Emma. Postdoctoral Fellow Kier. William, University of North Carolina King, Alison. Graduate Student. Dalhousie University Milbury, Coren, Research Assistant Ring. Sabine, Graduate Student, University of Frankfurt Saidel, William, Rutgers University Spotte, Stephen, University of Connecticut Laboratory of Shinya Inoue Scientists in this laboratory study the molecular mechanism and control of mitosis, cell division, cell motility, and cell morphogenesis, with emphasis on biophysical studies made directly on single living cells, especially developing eggs in marine invertebrates. Development of biophysical instrumentation and methodology, such as the centrifuge polarizing microscope, high-extinction polarization optical and video microscopy, digital image processing techniques including dynamic stereoscopic imaging, and exploration of their underlying optical theory are an integral part of the laboratory's efforts. Staff Inoue, Shinya, Distinguished Scientist Coda, Makoto. Visiting Scientist Baraby, Diane. Laboratory Assistant Knudson, Robert. Instrument Development Engineer MacNcil, Jane, Executive Assistant Laboratory of Alan M. Kuzirian Research in the laboratory explores the functional morphology and ultrastructurc of various organ systems in molluscs. The program includes mariculture of the nudibranch, Hennissenda crassicomis. with emphasis on developing reliable culture methods for rearing and maintaining the animal as a research resource. The process of metamorphic induction by natural and artificial inducers is being explored in an effort to understand the processes involved and as a means to increase the yield of cultured animals. Morphologic studies stress the ontogeny of neural and sensory structures associated with the photic and vcstibular systems which have been the focus of learning and memory studies, as well as the spatial and temporal occurrence of regulatory and transmitter neurochemicals. Concurrent studies detailing the toxic effects of lead on Hennis.iendti learning and memory, feeding, and the physiology of cultured neurons are also being conducted. New studies include cytochemical investigations of the Ca2+/GTP binding protein, calexcitin, and its modulation with learning and lead exposure. Collaborative research includes histochemical investigations on strontium's role in initiating calcification in molluscan embryos (shell and statoliths). immunocytochemical labelling of cell-surface antigens, neurosecretory products, second messenger proteins involved with learning and memory, as well as intracellular transport organelles using mono- and polyclonal antibodies on squid (Loligo pealei) giant axons and Herniixxcndu sensory and neurosecretory neurons. Additional collaborations involve studying neuronal development of myelin, myelination defects, as well as nerve regeneration and repair in phylogenetically conserved nervous systems. Additional collaborative research includes DNA fingerprinting using RAPD-PCR techniques in preparation for isogenic strain development of laboratory-reared Hermissenda and hatchery produced bay scallops (Argopectin irradians) with distinct phenotypic markers for the rapid field identification and stock assessments. Recently obtained funding will expand this research to perform population genetic analyses of currently designated yellowtail flounder (Limanda f'erruginea) stocks occurring in the Northeast Fisheries Region. Systematic and taxonomic studies of nudibranch molluscs, to include molecular phylogenetics, are also of interest. Scientific Stuff Ku/inan. Alan M.. Associate Scientist Visiting Investigators Chikarmane. Hemant, Investigator Clay, John R., NINDS/N1H Gould. Robert, NYS Institute of Basic Research Laboratory of Rudolf Oldenbourg The laboratory investigates the molecular architecture of living cells and of biological model systems using optical methods for imaging and manipulating these structures. For imaging cell architecture non- invasively and non-destructively, dynamically and at high resolution, we have developed a new polarized light microscope (Pol-Scope). The Pol- Scope combines microscope optics with new electro-optical components, video, and digital image processing for fast analysis of specimen birefringence over the entire viewing field. Examples of biological systems currently investigated with the Pol-Scope are microtubule-based structures (asters, mitotic spindles, single microtubules); actin-based structures (acrosomal process, stress fibers, nerve growth cones); zona pellucida of vertebrate oocytes; and biopolymer liquid crystals. Year-Round Research R53 Staff Oldenbourg, Rudolf, Associate Scientist Knudson, Robert, Instrument Development Engineer Baraby. Diane, Laboratory Assistant Laboratory of Michael Rabinowitz This laboratory investigates environmental geochemistry and epidemiology. Areas of recent activity include modeling lead bioavailability, writing a history of lead biokinetic models, performing a case control survey of tea drinking and oral cancer in Taiwan, quantifying the transport and fate of various sources of residential lead exposure, and serving on several advisory boards of Superfund research projects in Boston and New York. Current activity focuses on characterizing lead paints and pigments. Staff Rabinowitz, Michael, Associate Scientist Laboratory for Reproductive Medicine, Brown University and Women and Infants Hospital, Providence Work in this laboratory centers on investigating cellular mechanisms underlying female infertility. Particular emphasis is placed on the physiology of the oocyte and early embryo, with the aim of assessing developmental potential and mitochondria dysfunction arising from mtDNA deletions. The studies taking place at the MBL branch of the Brown Laboratory use some of the unique instrumentation available through the resident programs directed by Rudolf Oldenbourg and Peter J.S. Smith. Most particularly, non-invasive methods for oocyte and embryo study are being sought. Of several specific aims, one is to use the Pol-Scope to analyze the dynamic birefringence of meiotic spindles. An additional aim is to study transmembrane ion transport using non- invasive electro-physiological techniques available at the BioCurrents Research Center. The newly developed oxygen probe offers the possibility of looking directly at abnormalities in the mitochondria arising from accumulated mtDNA damage. Our laboratory has also focused on studying the mechanism underlying age-associated infertility in terms of oocyte quality and has attempted to rescue developmentally compromised oocytes or embryos through nuclear-cytoplasmic transfer technology. We have characterized oxidative stress-induced mitochondria! dysfunctions, developmental arrest and cell death in early embryos using animal models. Ultimately, this laboratory aims to produce clinical methods for assessing preimplantation embryo viability, an advance that will significantly contribute to the health of women and children. Staff Keefe, David, Director Liu, Lin, Research Scientist Trimarchi, James, Staff Scientist Laboratory of Osamu Shimomura Biochemical mechanisms involved in the biolurrunescence of various luminescent organisms are investigated. Based on the results obtained. various improved forms of bioluminescent and chemiluminesccnt probes are designed and produced for the measurements of intracellular free calcium and superoxide anion. Staff Shimomura, Osamu, Senior Scientist, MBL, and Boston University School of Medicine Shimomura, Akemi. Research Assistant Laboratory of Robert B. Silver The members of this laboratory study how living cells make decisions. The focus of the research, typically using marine models, is on two main areas: the role of calcium in the regulation of mitotic cell division (sea urchins, sand dollars, etc.) and structure and function relationships of hair cell stereociliary movements in vestibular physiology (oyster, toadfish). Other related areas of study, i.e. synaptic transmission (squid), are also, at times, pursued. Tools include video light microscopy, multispectral, subwavelength, and very high speed (sub-millisecond frame rate) photon counting video light microscopy, telemanipulation of living cells and tissues, and modeling of decision processes. A cornerstone of the laboratory's analytical efforts is high performance computational processing and analysis of video light microscopy images and modeling. With luminescent, fluorescent, and absorptive probes, both empirical observation and computational modeling of cellular, biochemical, and biophysical processes permit interpretation and mapping of space-time patterns of intracellular chemical reactions and calcium signaling in living cells. A variety of I'M vitro biochemical, biophysical, and immunological methods are used. In addition to fundamental biological studies, the staff designs and fabricates optical hardware, and designs software for large video image data processing, analysis, and modeling. Staff Silver, Robert. Associate Scientist Visiting Investigators Crawford, Karen. St. Mary's College Hummel. John, Argonne National Laboratory Pearson, John, Los Alamos National Laboratory Intern Deming, Nicole A., REU Intern, St. Mary's College Laboratory of Norman Wainwright The mission of the laboratory is to understand the molecular defense mechanisms exhibited by marine invertebrates in response to invasion by bacteria, fungi, and viruses. The primitive immune systems demonstrate unique and powerful strategies for survival in diverse marine environments. The key model has been the horseshoe crab LJmulus [>ol\phenn/s. Limulus hemocytes exhibit a very sensitive LPS- triggered protease cascade which results in blood coagulation. Several proteins found in the hemocyte and hemolymph display microbial binding proteins that contribute to antimicrobial defense. Commensal or symbiotic microorganisms may also augment the antimicrobial mechanisms of macroscopic marine species. Secondary metabolites are being isolated from diverse marine microbial strains in an attempt to R54 Annual Report understand their role. Microhial participation in oxidation of the toxic gas hydrogen sulfide is also being studied. Staff Wainwright, Norman, Senior Scientist Child, Alice, Research Assistant Visiting Investigator Anderson, Porter. University of Rochester Laboratory of Seymour Zigman This laboratory is investigating basic mechanisms of photooxidative stress to the ocular lens due to environmentally compatible UVA radiation. This type of oxidative stress contributes to human cataract formation. Other studies are the search for and use of chemical antioxidants to retard the damage that occurs. Cultured mammalian lens epithelial cells and whole lenses in vitro are exposed to environmentally compatible UVA radiation with or without previous antioxidant feeding. The following parameters of lens damage are examined: molecular excitation to singlet states via NADPH (the absorber); cell growth inhibition and cell death; catalase inactivation; cytoskeletal description (of actin, tubulin. integrins); and cell membrane damage (lipid oxidation, loss of gap junction integrity and intercellular chemical communications). Thus far, the most successful antioxidant to reduce these deficiencies is alpha-tocopherol ( 10 fig/ml) and tea polyphenols (especially from green tea). The preliminary phases of the research are usually carried out using marine animal eyes (i.e., smooth dogfish) as models. Our goal is to provide information that will suggest means to retard human cataract formation. Staff Seymour Zigman, Laboratory Director, Professor of Ophthalmology, Boston University Medical School Keen Rafferty, Research Associate, Boston University Medical School Nancy S. Rafferty, Research Associate, Boston University Medical School Bunnie R. Zigman, Laboratory Manager. Boston University Medical School The Marine Resources Center The Marine Resources Center (MRC) is one of the world's most advanced facilities for maintaining and culturmg aquatic organisms essential to advanced biological, biomedical, and ecological research. Service and education also play an important and complementary role in the modern, 32,000-square-foot facility. The MRC and its life support systems have already increased the ability of MBL scientists to conduct research and have inspired new concepts in scientific experiments. Vigorous research programs focusing on basic biological and biomedical aquatic models are currently being developed at the Center. The Program in Scientific Aquaculture was initiated in 1998. In addition to research, the MRC provides a variety of services to the MBL community through its Aquatic Resources Division, the Water Quality and System Engineering Division, and the Administrative Division. Research and educational opportunities are available at the facility to established investigators, postdoctoral fellows, graduate, and undergraduate students. Investigators and students will find that the MRC's unique life support and seawater engineering systems make this a favorable environment in which to conduct independent research and masters and doctoral theses using a variety of aquatic organisms and flexible tank space for customized experimentation on live animals. Prospective investigators and students should contact the Director of the MRC for further information. Staff Hanlon, Roger, Director and Senior Scientist Buresch, Kendra, Research Assistant Kuzirian, Alan, Associate Scientist Maxwell, Michael, Postdoctoral Scientist Santore, Gabrielle. Executive Assistant Shashar, Nadav, Postdoctoral Scientist Smolowitz, Roxanna, MBL Veterinarian Sussman, Raquel, Investigator Visiting Investigators Adamo, Shelley. Dalhousie University Baker, Robert, New York University Benjamins, Steven, Graduate Student. University of Groningen Boal, Jean, Adjunct Scientist Cavanaugh, Joseph. Graduate Student. Boston University Marine Program Fern, Sophie, Graduate Student, Boston University Marine Program Gilland. Edwin, Staff Scientist Hatfield, Emma, Postdoctoral Fellow Kier, William, University of North Carolina King, Alison. Graduate Student. Dalhousie University Milbury. Coren. Research Assistant Ring, Sabine, Graduate Student, University of Frankfurt Saidel. William. Rutgers University Spotte. Stephen, University of Connecticut Honors Friday Evening Lectures June 18 June 25 July 2 July 9 July 16 July 22, 23 July 30 August 6 August 13 Barbara and Bruce Furie, Harvard Medical School "Of Molluscs and Men: Vitamin K-dependent Synthesis of Gla, a Novel Amino Acid" Roderick MacKinnon. Rockefeller University "Potassium Channels" Nancy Kanwisher, Massachusetts Institute of Technology "Functional Specialization in Human Visual Cortex: Faces and Places" Judith Kimble. University of Wisconsin. Madison "Regulation of Development of C. elegans — Lessons from the Gonad" (Classman Lecture) Eric Chivian, Harvard Medical School "The Value of Plants, Animals, and Microbes to Human Health" Marianne Bronner-Fraser and Scott E. Fraser, California Institute of Technology 1. "Formation of the Neural Crest" 2. "Working with the Wiring of the Developing Brain" (Forbes Lectures) Dr. Bernd U. Budelmann. Marine Biomedical Institute. University of Texas "The Sensory World of Cephalopods" (Lang Lecture) Dr. Gerald Fischbach, National Institute of Neurological Disorders and Stroke "Neuroscience at the New Millennium" Dr. Luca Cavalli-Sforza, Stanford University School of Medicine "Crucial Times in Human Evolution" Fellowships and Scholarships In 1999. the MBL swarded research fellowships to 20 scientists from around the world. These fellows' research topics ranged from a study of how calcium enters heart and nen'e cells when a cell is stimulated to research on how the skate senses small electric potentials in surrounding seawater to locate prey. The MBL awarded scholarships to 65 students in the MBL's summer courses as well as 1 1 post course research awards. In 1999, donors provided gifts for endowed and expendable funds amounting to $103.309 in support of the research fellowships program and an additional $233,884 to provide scholarships to students im MBL courses. The individuals who received fellowships and scholarships are listed beginning on p. R56. Robert Day Allen Fellowship Fund Drs. Joseph and Jean Sanger American Society for Cell Biology Scholarships American Society for Cell Biology Frederick B. Bang Fellowship Fund Mrs. Betsy G. Bang Jean and Katsuma Dan Fellowship Fund Drs. Joseph and Jean Sanger Mrs. Eleanor Steinbach Bernard Davis Fellowship Fund Mrs. Elizabeth M. Davis Fries Fellowship Trust of Anna B. Fries Aline D. Gross Scholarship Fund Dr. and Mrs. Paul R. Gross Technic, Inc. Keffer Hartline Fellowship Fund Dr. Max Snodderly E. E. Just Research Fellowship Fund Ayco Charitable Foundation William Townsend Porter Foundation Fred Karush Endowed Library Readership Dr. and Mrs. Laszlo Lorand Dr. and Mrs. Arthur M. Silverstein S. O. Mast Founders Endowed Scholarship Fund Dr. and Mrs. John B. Buck Mrs. Louise M. Specht James A. And Faith Miller Fellowship Fund Drs. David and Virginia Miller Frank Morrell Scholarship Fund Dr. Leyla de Toledo-Morrell R55 R56 Annual Report Emily Hartshorne Mudd Scholarship Fund World Academy of Art and Science Mountain Memorial Fund Dr. and Mrs. Dean C. Allard, Jr. Dr. and Mrs. R. Walter Schlesinger Neural Systems & Behavior Scholarship Fund Dr. and Mrs. Alan Gelperin Josephine Bay Paul and C. Michael Paul Foundation Drs. Harold Zakon and Lynne McAnelly Nikon Fellowship Fund Nikon, Inc. The Ann Osterhout Edison/Theodore Miller Edison and Olga Osterhout Sears/Harold Bright Sears Endowed Scholarship Fund Ms. Nancy Miller Arnn Mr. and Mrs. Alan K. Karplus Phillip H. Presley Scholarship Fund Carl Zeiss, Inc. William Townsend Porter Scholarship Fund for Minority Students William Townsend Porter Foundation The Evelyn and Melvin Spiegel Fellowship Fund Drs. Joseph and Jean Sanger The Sprague Foundation H. B. Steinbach Fellowship Fund Mrs. Eleanor Steinhach Horace W. Stunkard Scholarship Fund Dr. Albert Stunkard and Dr. Margaret Maurin Eva Szent-Gyorgyi Scholarship Fund Dr. and Mrs. Laszlo Lorand Dr. Andrew Szent-Gyorgyi and Ms. Ursula Rowan Selman A. Waksman Endowed Scholarship in Microbial Diversity Foundation for Microbiology Walter L. Wilson Endowed Scholarship Dr. Paul N. Chervin Mrs. Rigaumont Mr. and Mrs. Leslie J. Wilson Young Scholars/Fellows Program Mr. and Mrs. David Bakalar Mrs. LeRoy Clark Mrs. George H. A. Clowes Mr. and Mrs. Daniel D. Federman Dr. and Mrs. Harold S. Ginsberg Dr. and Mrs. Robert Haselkorn Mr. and Mrs. Gary G. Hayward Dr. and Mrs. John E. Hobbie Dr. and Mrs. Edward F. MacNichol, Jr. Mr. and Mrs. William J. Pechilis Mrs. Atholie K. Rosett Dr. and Mrs. Edward A. Spiegel Mr. and Mrs. Leslie J. Wilson Drs. Jonathan and Beatrice Wittenberg Fellowships Awarded MBL Summer Research Fellows • Pavel Balaban, Ph.D.. is a Professor at the Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences in Moscow. He is interested in the mechanism of post- synaptic activation potentials in connection with synaptic plasticity, using the terrestrial mollusc. Helix, as a model organism. Dr. Balaban studies the putative command neurons, a group of serotonin-containing cells, which modulate withdrawal behavior and the activity of neurons underlying this behavior. Spikes in these neurons do not elicit behavior, yet behavioral responses evoked by noxious stimuli are changed. Dr. Balaban was funded by the Herbert W. Rand Fellowship. • Richard Cardullo, Ph.D., is an Associate Professor in the Department of Biology at the University of California, Riverside. His research project was titled: "Microscopic evaluation and functional analysis of the egg extracellular matrix." Dr. Cardullo is interested in the molecular determinants in fertilization. He uses advanced imaging technologies along with biochemical and biophysical methodologies to determine both the molecules involved in fertilization and the precise sequence of molecular events ultimately leading to the fusion of mammalian sperm and egg. Dr. Cardullo was supported by the Lucy B. Lemann Fellowship Fund, the Robert Day Allen Fellowship, the Charles R. Crane Fellowship, the John O. Crane Fellowship, and an MBL Research Fellowship. • Anthony DePass. Ph.D., is an Assistant Professor in the Biology Department at Lung Island University in Brooklyn. NY. His research focuses on how caiaurr enters heart and nerve cells when a cell is stimulated. He uses Lytechus pictus, Arbacia puncntlata (sea urchins), and Raja arinacius (skate) as biomedical models in this work. Specifically, he studies receptors that mediate Ca2+ release from intracellular stores and the second messenger pathways involved in signal transduction. Dr. DePass was a Josiah Macy, Jr. Foundation Research Fellow. • Ana S. DePina is a graduate student at Dartmouth College. Her research project this summer was titled "Actin-based movement in clam oocyte extracts." She studies vesicle transport on actin filaments in clam oocyte extracts. She wants to determine the types of inyosins that function as molecular motors for vesicle transport. Ms. DePina was sponsored by the Mi/ton L. Shifman Endowed Scholarship and the William Townsend Porter Fellowship. • Inigo Novales Flamarique, Ph.D., is a post-doctoral fellow in the Department of Biology at the University of Victoria, British Columbia. His summer's research project was "Optical recordings of UV sensitivity in the optic tectum of rainbow trout using voltage sensitive dyes." His research focuses on the chromatic organization of neural pathways in the visual systems of vertebrates. The ultraviolet sensitive cones in the retinas of some fishes disappear and are reincorporated in the photoreceptor layer at specific stages during the animal's life. As such, the ultraviolet neural pathway is a good model to study the cellular mechanisms behind cell apoptosis and regeneration. Dr. Novales Flamanque was funded by the Stephen W. Huffier Fellowship. • Elizabeth A. Jonas, MD. is in the Department of Pharmacology at the Yale University School of Medicine in New Haven. Connecticut. Her research project was titled "Activation of conductances on intracellular organelles during synaptic transmission." She is interested in measuring ionic currents on membranes of mitochondria during neurotransmission in squid. She has found that, in addition to their role Honors R57 in calcium management in cells, mitochondria! ion channels maintain electrochemical gradients that are essential to mitochondria! function as well as the regulation of the transport of peptides and metabolites between the cytosol and the inner mitochondria! matrix. Dr. Jonas was supported by the Frank R. Li/lie Fellowship Fund. * Samantha Joye, Ph.D., is an Assistant Professor in the Department of Marine Sciences at the University of Georgia in Athens. Her research project was titled "Denitritication of coastal marshes; relationship to nitrogen loading." Dr. Joye's research focuses on biogeochemical cycling in coastal environments and on understanding how humans impact coastal ecosystems. She examines how nitrogen and phosphorus cycles are altered by human activities, with a focus on developing critical new tools for evaluating how pristine environments might be affected by nitrogen loading. Dr. Joye was supported by the Lucy B. Lemann Fellowship, an MBL Associates Fellowship, and an MBL Research Fellowship. * Eileen M. Lafer, Ph.D. is Associate Professor at the Institute of Biotechnology at the University of Texas Health Science Center in San Antonio. At the MBL she studied the molecular mechanisms thai regulate neurotransmission. Her research focuses on the biochemical studies of various peptides. specifically the significance of the clathrin pathway at the squid giant synapse in an effort to understand neurotransmission in synaptic vesicles. Dr. Lafer was funded by the Ann E. Kammer Memorial Fellowship, the Frederick B. Bang Fellowship, the Evelyn and Melvin Spiegel Fellowship, and an MBL Research Fellowship. * Jennifer LaVail. Ph.D.. is a Professor of Anatomy/Ophthalmology at the University of California, San Francisco. She investigated the genetic and molecular regulation of Herpes simplex virus transport using GFP-labeled virus injected into squid axons. This movement was monitored by confocal microscopy. It is hypothesized that studying the virus transport mechanisms will shed light on transport in other classes of neurotropic viruses, and on organelle trafficking in general. Dr. LaVail was funded by the Evelyn and Melvin Spiegel Fellowship and the Frederick B. Bang Fellowship. * Jeff Magee, Ph.D., is an Assistant Professor at the Department of Neuroscience at the Louisiana State University in New Orleans. Louisiana. His research project was titled "Mechanisms of Ca2+ entry into neurons." He uses optical imaging to study varying concentrations of calcium ions in hippocampal neurons. Changes in the strength of synaptic connections are thought to form the basis of memory because they ultimately lead to changes in the firing patterns of neurons. Dr. Magee was supported by an MBL Associates Fellowship. * Guy Major. Ph.D.. is a Research Fellow at Lucent Technologies/Bell Labs in Murray Hill, NJ. His research project was titled "Voltage-sensitive dye recordings from cortical neurons." He studies how single neurons function by means of voltage-sensitive dyes and imaging. He has been successful in measuring the spread of the action potential through the axodendntic tree of the injected cell. Dr. Major was an MBL Associates Fellow. * Antonio Malgoroii. Ph.D.. is a Professor in the Neurobiology of Learning Unit in the Department of Biological and Technological Research at the Scientific Institute of San Raffaele in Milan, Italy. Dr. Malgoroii studies the cellular and molecular events that form the basis of synaptic plasticity in the hippocampus, especially as it relates to learning and memory. He investigates changes in calcium concentration in the postsynaptic neuron as a function of long-term potentiation (LTP). Dr. Malgaroli is pursuing research on the nature of pre- synaptically silent synapses that are recruited into active neurotransmission during LTP. Dr. Malgoroii was supported by the Herbert W. Rand Fellowship. * Paul McNeil, Ph.D.. is a Professor in the Department of Cellular Biology and Anatomy at the Medical College of Georgia in Augusta. Dr. McNeil studies resealing mechanisms used in the repair of large plasma membrane disruptions. He uses the sea urchin egg as a model system to define the mechanistic basis of this fundamental cell survival response. Specifically, he investigates the calcium flows that regulate the fusion of intracellular vesicles that, in turn, fuse with the plasma membrane to reseal the disruption. Dr. McNeil was sponsored by the NASA Life Science Program Fellowship and the Baxter Postdoctoral Fellowship. • David Ogden. Ph.D.. is a Principal Investigator at the National Institute for Medical Research in London. His research project was titled "Central electrosensory processing in the skate." This summer he studied how the skate senses small electric potentials in surrounding seawater to locate prey. Specifically, he investigated the area of electroreceptors in the skin of the skate that generate sensory information that is relayed to the dorsal nucleus of the brain stem. The dorsal nucleus shows organization and structural features similar to the cerebellum and it is likely that mechanisms of plasticity will prove to be similar to mammalian cerebellar learning mechanisms. Dr. Ogden was an M.G.F. Fuones Memorial Fellow and an H.B. Steinbach Fellow. • Oladele A. Ogunseitan. Ph.D., is Associate Professor in the Department of Environmental Analysis and Design at the University of California, Irvine. Dr. Ogunseitan studies bacterial populations in aquatic systems because they are highly sensitive indicators of the phyiological consequences of toxic compounds, including trace metals. Molecular analyses of these natural microbial communities provide valuable ecotoxicological information, especially when coastal habitats have been affected by human habitation resulting in dynamic shifts in chemical speciation and concentration fluxes. Dr. Ogunseitan was supported by the Jositili Mac\. Jr. Foundation Research Fund. • David Paydarfar. Ph.D.. is Associate Professor at the Department of Neurobiology at the University of Massachusetts medical School in Worcester. The title of his research project was "Can noise regulate oscillatory state'1 In ni/mero and in vitro analysis of squid axon membrane." He studies how electrical nerve activity is controlled and has found that a variety of neural oscillators can exhibit abrupt and lasting transformation of activity from an oscillatory to an arrhythmic state. Dr. Paydarfar was funded by the M.G.F. Fuones Memorial Fellowship Fund and the H. Keller Hanline Fellowship. • Edward Salmon, Ph.D., is Professor in the Department of Biology at the University of North Carolina, Chapel Hill. Dr. Salmon and members of the Cell Division Group investigated the protein assemblies that achieve accurate chromosome segregation in cell division using sand dollars and frogs as model systems. Using advanced imaging technology. Dr. Salmon and his colleagues studied mechanisms of chromosome segregation during meiosis and mitosis, pronuclear movement during fertilization, and cytokinesis at cell division. They were especially interested in developing experimental approaches to directly measure the magnitude and direction of forces associated with microtubule flux in mitosis in living cells. Dr. Salmon and the Cell Division Group were sponsored by the Nikon Fellowship. • Edgar T. Walters, Ph.D., is a Professor in the Department of Integrative Biology at the University of Texas in Houston, Texas. His research project was titled "Network representation of nociceptive memory in Aplysia." He studies the primitive neural mechanisms underlying central memory of peripheral injury, using simple molluscan preparations. He uses optical recording with voltage-sensitive dyes to compare spike activity in the neurons in the abdominal ganglion of Apl\sia californica before, during, and afer intense noxious stimulation of the siphon. Dr. Walters was funded by the James A. and Faith Miller Fellowship Fund. • Ebenezer Yamoah, Ph.D., is Assistant Professor in the Department of Anatomy and Cell Biology at the University of Cincinnati College of Medicine in Cincinnati. Ohio. Dr. Yamoah's research focuses on R58 Annual Report characterizing the role of the plasma membrane calcium pump in hair cell calcium homeostasis. This work is important because the perception of sound and the ability to balance in relation to head position depend on the proper function of hair cells in the inner ear. A better understanding of the functional determinants of hair-cell sensitivity will provide rational strategies for treating hearing and vestibular disorders. Dr. Yamoah was a Josiah Macy. Jr. Foundation Research Fellow. Grass Fellows • Matthew L. Beckman. Ph.D., University of Alabama at Birmingham. Project: "Analysis of lobster serotonin transporter expression and function in Homarus americanua." • Mathew Brock, Hopkins Marine Station, Stanford University. Project: "Block of squid axon Ik by S-nitrosidithiothreitol." • Marco Crespie, Scientific Institute S. Raffaele, Italy. Project: "Expression of LTP at CA3-CA1 hippocampal synapses: A dendritic- synaptic model to reveal contributions from recruitment of silent synapses and address spread of changes." • Frederic Doussau, Ph.D.. Laboratoire de Neurobiologie Cellulaire, France. Project: "Control of synaptic vesicle traffic by the action cytoskeleton." • Yi Han, Ph.D.. Baylor College of Medicine. Project: "Electrophysiology studies of zebrafish retinal mutants with an abnormal b-wave." • Barbara Innocenti, Ph.D., Iowa State University. Project: "Imaging of calcium-dependent glutamate release from Miiller cells." • Peter Koulen, Ph.D., Yale University School of Medicine. Project: "Differential localization of ryanodine receptor and inositol 1 ,4,5-trisphospate receptor isofornis in neurons and its relationship to the regulations of intracellular calcium. • Seth J. Ramus, Ph.D., Boston University. Project: "Learning in the Eocene ocean: The first systemic examination of learning and memory in the Nautilus (Nautilus pompilius)." ' Miduturu Srinivas, Ph.D., Albert Einstein College of Medicine. Project: "Biophysical characterization of gap junction channels in marine invertebrates." • Ayako Yamaguchi, Ph.D.. Columbia University. Project: "Neuronal coding of sexually differentiated behavior by motoneurons." • Karen Zito. Ph.D., University of California, Berkeley. Project: '7;i vitrn analysis of Drosophila neuromuscular development" and "Role of the adhesion molecule, Fascilin II, in synaptic function." • Michal Zochowski. Ph.D., Yale University School of Medicine. Project: "Investigating physiological, functional and dynamical properties of synchronous oscillatory signal in turtle olfactory system using optical techniques." MBL Science Writing Fellowships Program Fellows Ballingrud, David, St. Petersburg Times Bates, Todd, Asburv Park Press Beeman, Perry, The Des Moines Register Burns, Michael K., The Baltimore Sun Cohen, Nancy, Freelance reporter Cuthbert, Lori, Discovery Channel Online Eckelbecker, Lisa, Worcester Telegram & Ga-ette Erickson, James, Ari-ona Daily Star Grossman. Daniel, NPR's Living on Earth Hogue, Cheryl, Bureau of National Affairs. Inc. Lesser, Carolyn. Author of children's books Miller, John, Freelance medical and science reporter/producer Moran, Barbara. Freelance science writer/producer Parks. Noreen, Freelance science writer Pennybucker. Mindy, The Green Guide Potera, Carol. Freelance writer/editor Rogerio, Graciela. WABC-TV Eyewitness News Schueller. Gretel, Audubon magazine Witze. Alexandra. The Dallas Morning News Program Directors Goldman. Robert D.. Northwestern University Rensberger. Boyce. Knight Science Journalism Program Hands-On Laboratory Course Directors Chisholm. Rex, Director. Northwestern University (Biomedical) Hobbie, John E., Co-Director. Marine Biological Laboratory (Environment) Mclillo, Jerry, Co-Director, Marine Biological Laboratory (Environment) Palazzo. Robert, Associate Director, University of Kansas (Biomedical) Scholarships Awarded American Society for Cell Biology Baca. Serapio, University of California, San Diego Casillas. Lilliam. Autonomous University of the State of Pueblo Jones. Stacy. University of Virginia Macias. Chanda. Howard University Marin Bivens. Carrie. University of Massachusetts. Amherst Nzambi, Eduardo, Howard University Purves, Dianne, California State University. Sacramento Biology Club of the College of the City of New York Suadicani. Sylvia, Albert Einstein College of Medicine Honors R59 C. Lalor-Burdick Scholarship Buhimschi. Irina, University of Maryland at Baltimore Lwigale. Peter, Kansas State University Burroughs Wellcome Fund Biology of Parasitism Course Angeli, Veronique. Pasteur Institute-Lille Aviles. Hernan. Indiana State University Barragan. Antonio, Karolinska Institute Dobbin, Caroline, University of Technology, Sydney Falcone, Franco, University of Edinburgh Gavrilescu. Cristina, Cornell University Sodre. Catia, Universidade Federal do Rio de Janeiro Stem, Leah, University of California, San Francisco Wang, Zefeng, Johns Hopkins University Burroughs Wellcome Fund Frontiers in Reproduction Course Belts, Dean, University of Guelph Bos-Mich. Adriana. FUEFE-Porto Alegre-Brazil Buhimschi. Irina, University of Maryland, Baltimore Jobanputra, Vaidehi, All India Institute of Medical Sciences Lue. Yanhe, Harbor-UCLA Medical Center Marin Bivens. Carrie, University of Massachusetts, Amherst Mendeluk, Gabriela, University of Buenos Aires Natesampillai. Sekar, University of Virginia Ollero. Mario, Beth Israel Deaconess Medical Center Pritts, Elizabeth, Yale University Sprague, David, Texas A&M Burroughs Wellcome Fund Molecular Mycology Course Cowen, Leah, University of Toronto Devasamayam. Gina. Wadsworth Center Giles, Steven. University of Wisconsin. Madison Goldstein. Alan, Duke University Medical Center de Jesus-Bem'os, Marisol. University of Puerto Rico Latouche. Nicholas. Sydney University Miller. Nancy. The Johns Hopkins Medical Institute Wormley. Floyd, Louisiana State University Medical Center Gary N. Calkins Memorial Scholarship Fund Li, Dongling. University of Texas Edwin Grant Conklin Memorial Fund D'Souza, Jacinta. Tata Institute of Fundamental Research Contraceptive Research and Development Program Mendeluk, Gabriela, University of Buenos Aires William F. and Irene C. Diller Memorial Scholarship Fund Vukovich, Wolfgang, Max-Planck-Institute of Biophysical Chemistry Caswell Grave Scholarship Fund Pfeiffer. Sven, National Institute for Medical Research Daniel S. Grosch Scholarship Fund Van Lith, Yvonne, ETH Zurich Zopfi, Jakob. Max-Planck-Institute for Marine Microbiology Aline D. Gross Scholarship Fund Spengler. Tatjana. Universite Paris XI William Randolph Hearst Foundation Scholarships Jessani, Nadium, Scripps Research Institute Kimbell, Jennifer, University of Hawaii March, Tony, University of Idaho International Brain Research Organization Ashworth, Rachel. University College. London Bellefroid, Eric. Universite Libre de Bruxelles Lightfoot, Kurt. University of Witwaterstand Rinkwitz. Silke. New York University Medical Center Arthur Klorfein Scholarship and Fellowship Fund Junghlut. Benno. University of Tubingen Panopoulou. Georgia. Max-Planck-Institute fiir Molekulare Genetik Paul, Angelika, University of Otago Saiide, Leonor, National Institute for Medical Research Frank R. Lillie Fellowship and Scholarship Fund Chen. Lihong. University of North Carolina. Chapel Hill March Tony. University of Idaho van Drogen, Frank. ISREC Varshney. Anurag. National Centre for Biological Sciences Jacques Loeb Founders' Scholarship Fund van Drogen. Frank. ISREC Massachusetts Space Grant Consortium Best. Aaron A.. University of Illinois-Urbana Bouchet, Valerie B.. Boston University DiMeo. Carol A., University of Delaware Harbinski, Fred M.. Harvard University Hurtado, Luis A.. Rutgers University Mead. Louise A.. University of Massachusetts Richardson. Susan L.. Yale University Worapong. Jeerapun. Montana State University S. O. Mast Memorial Fund Jungblut, Benno. University of Tubingen Spengler, Tatjana, Universite Paris XI MBL Associates Endowed Scholarship Fund Rao, Anita. University of Maryland, College Park R60 Annual Report MBL Pioneers Scholarship Fund Glavic, Alvaro, University of Chile Lwigale. Peter, Kansas State University Pierdra. Elisa. Universidad de Cantabria, Spain Vukovich, Wolfgang, Max-Planck-Institute of Biophysical Chemistry Merck & Company, Inc. Scholarships Batchelor, Adrian, Walter and Eliza Hall Institute Bishop, Joseph, University of Alabama at Birmingham Montgomery, Jacqui, University of Melbourne Santori, Isabel, University of Buenos Aires Stem, Leah, University of California, San Francisco Toe, Laurent. World Health Organization Wang, Zefeng, Johns Hopkins University Charles Baker Metz and William Metz Scholarship Fund in Reproductive Biology Belts, Dean, University of Guelph Ollero. Mario. Beth Israel Deaconess Medical Center Sprague. David. Texas A&M Frank Morrell Endowed Memorial Scholarship Matsui. Ko, University of Tokyo Mountain Memorial Fund Scholarship Chen, Lihong. University of North Carolina, Chapel Hill Holdaway-Clarke. Teresa. University of Massachusetts, Amherst Mazzatenta. Andrea, University of Pisa Tefft, Denise, University of Southern California Varshney, Anurag, National Centre for Biological Sciences. Bangalore Ann Osterhout Edison/Theodore Miller Edison and Olga Osterhout Sears/Harold Bright Sears Endowed Scholarship Fund Avery, Jennifer. Brandeis University Pfizer Inc. Endowed Scholarship Fund Ragusa. Maria, Alberto Monroy Foundation. Palermo Planetary Biology Internship Awards Van Lith, Yvonne, ETH Zurich Phillip H. Presley Scholarship Award, Funded by Carl Zeiss, Inc. Batchelor, Adrian, Walter and Eliza Hall Institute Dobbin, Caroline, University of Technology. Sydney Franco, Peter. Harvard Medical School Krieger. Patrik, Karolinska Institute Maruska. Karen. Florida Institute of Technology Welman, Arkadiusz, Fnedneh Miescher Institute Herbert W. Rand Fellowship and Scholarship Fund Duncan. Tod. Imperial Cancer Research Laboratory Fort, Alfredo, Albert Einstein College of Medicine Franco, Peter, Harvard Medical School Holdaway-Clarke, Terena. University of Massachusetts Junghlut, Benno. University of Tubingen Mazzatenta, Andrea, University of Pisa Sawai, Satoshi, Tohoku University Tefft, Denise. University of Southern California Tidwell, Judy, Wake Forest University Ruth Sager Memorial Scholarship Haapasalo, Annakaisa. University of Kuopio Society for Developmental Biology Scholarships Clements. Wilson, University of Washington Mui, Stina, University of California. San Diego Pizer. Margaret. State University of New York at Stony Brook Robertson. Christie, University of Washington Zigler, Kirk. Duke University Society of General Physiologists' Scholarships Diana. Marco. Max-Planck-Institute of Biophysical Chemistry Duncan. Tod, Imperial Cancer Research Laboratory Greenwood, Anna, Stanford University Paul, Angelika. University of Otago Howard A. Schneiderman Endowed Scholarship Yu, Xiang, MRC Laboratory of Molecular Biology Moshe Shilo Memorial Scholarship Fund Gillor. Osnat. The Hebrew University Marjorie W. Stetten Scholarship Fund Yoon, Miri. Northwestern University Medical School Horace W. Stunkard Scholarship Fund Bos-Mikich. Adriana, FUEFE-Porto Alegre-Brazil Surdna Foundation Scholarship Abenavoli, Alessandra, Scientific Institute San Raffaele Diana. Marco, Max-Planck-Institute of Biophysical Chemistry Haapasalo. Annakaisa, University of Kuopio Matsui, Ko, University of Tokyo Rao, Anita. University of Maryland William Townsend Porter Fellowship For Minority Students Jones. Stacy. University of Virginia Macias, Chanda, Howard University Nzambi, Eduardo, Howard University William Morton Wheeler Family Founders' Scholarship Abenavoli, Alessandra. Scientific Institute San Raffaele Yu, Xiang. MRC Lab of Molecular Biology Walter L. Wilson Endowed Scholarship Fund Tidwell. Judy. Wake Forest University World Academy of Arts and Sciences Emily Mudd Scholarship el Guiziry, Dalai, Alexandria University Honors R61 Post-Course Research Awards van Drogen. Frank, ISREC (Physiology) Kimbell. Jennifer, University of Hawaii (Physiology) Mactas, Chanda, Howard University (Physiology) Mazzatenta, Andrea, University of Pisa (Physiology) Meyers. Jason, University of Virginia (Embryology) Narayan, Sujatha, Bryn Mawr College (Physiology) Niggemyer. Allison, University of Idaho (Microbiul Diversity) O'Neill. Forest, University of California. Santa Barbara (Physiology) Purves, Dianne, California State University, Sacramento (Physiology) Tidwell. Judy, Wake Forest University (Physiology) Varshney. Anuray. National Centre for Biological Sciences (Physiology) Board of Trustees and Committees Corporation Officers and Trustees Chairman of the Board of Trustees. Sheldon J. Segal, The Population Council Co-Vice Chair of the Board of Trustees, Frederick Bay. Josephine Bay Paul and C. Michael Paul Foundation Co-Vice Chair of the Board of Trustees, Mary J. Greer. New York, NY President of the Corporation, John E. Dowling, Harvard University Director and Chief Executive Officer, John E. Burris, Marine Biological Laboratory* Treasurer of the Corporation, Mary B. Conrad, Fiduciary Trust International* Clerk of the Corporation. Robert E. Mainer. The Boston Company Chair of the Science Council, Kerry S. Bloom, University of North Carolina* Class of 2003 Kelley, Darcy Brisbane. Columbia University Landeau, Laurie J.. Marinetics. Inc. Lee. Burton J. Ill, Vero Beach, FL O'Hanley, Ronald P.. Mellon Institutional Asset Mgt. Pierce, Jean, Boca Grande. FL Ryan, Vincent J., Schooner Capital LLC Honorary Trustees Cunningham, Mary Ellen. Grosse Pointe Farms, MI Ebert, James D., Baltimore, MD Golden. William T., New York. NY Grass, Ellen R.. The Grass Foundation Class of 2000 Clowes, Alexander W., University of Washington School of Medicine Landis, Story C., Case Western Reserve University Levitan, Irwin B., Brandeis University Miller. G. William. G. William Miller and Co., Inc. Press, Frank, The Washington Advisory Group Weld, Christopher M.. Sullivan and Worcester, Boston Class of 2001 Anderson. Porter W., North Miami Beach. FL Bay, Frederick. Josephine Bay Paul and C. Michael Paul Foundation, Inc. Cox, Martha W., Hobe Sound, FL Greer. Mary J.. New York. NY Steere. William C. Jr., Pfizer Inc. Weissmann, Gerald, New York University School of Medicine Class of 2002 Cone, Sydney M III, Cleary, Gottlieb, Steen and Hamilton Lakian, John R.. The Fort Hill Group, Inc. Ruderman, Joan V , lljiuiul Medical School Segal. Sheldon J., The Population Council Speck. William T., New York Presbyterian Hospital Zeien, Alfred M.. The Gillette Company / \ i '/In u> Trustees Emeriti Adelberg, Edward A., Yale University. New Haven. CT Buck. John B.. Sykesville, MD Cohen, Seymour S.. Woods Hole, MA Colwm, Arthur L., Key Biscayne, FL Colwin. Laura Hunter, Key Biscayne. FL Copeland, Donald Eugene, Woods Hole, MA Crowe!!, Sears Jr.. Indiana University. Bloomington, IN Hayashi, Teru, Woods Hole, MA Huhbard. Ruth, Cambridge. MA Kleinhol/., Lewis, Reed College, Portland, OR Krahl, Maurice. Tucson, AZ Prosser, C. Ladd, University of Illinois. Urbana, IL Russell-Hunter, W.D.. Syracuse University. Syracuse. NY Saunders. John W., Waquoit, MA Trigg. D. Thomas. Wellesley. MA Vincent, Walter S., Woods Hole. MA Directors Emeriti Ebert. James D.. Baltimore. MD Gross, Paul, Falmouth, MA Halvorson. Harlyn O., Woods Hole, MA Executive Committee of the Board of Trustees Segal, Sheldon J., Chair Bay, Frederick, Co-Vice Chair Greer. Mary J.. Co-Vice Chair Burris. John E. Bloom. Kerry S. R62 Trustees and Committees R63 Conrad. Mary B., Treasurer Mainer, Robert E. Ruderman, Joan V. Weissmann. Gerald Science Council Bloom, Kerry S., Chair (2000) Bums. John E.* Armstrong, Clay M. (2000) Armstrong, Peter (2000) Atema. Jelle (2001) Barlow, Robert B. (2001) Dawidowicz, E.A.* Haimo, Leah (2001) Hopkinson, Charles (2000) Jaffe, Launnda 12001 1 Smith, Peter J.S. (2001) Sogin. Mitchell (2000) Standing Committees of the Board of Trustees Development Speck, William. Chair Anderson, Porter W. Barlow, Robert Bay. Frederick Conrad, Mary B. Cox, Martha Cunningham, Mary Ellen Ebert, James D. Grant, Philip Lakian, John R. Lee, Burton J. Miller, G. William Pierce, Jean Steere. William C. Weld, Christopher M. Facilities and Capital Equipment Ruderman, Joan. Chair Anderson, Porter W. Bay, Frederick Cohen, Lawrence Cornell, Neal Landis, Story C. Levitan, Irwin B. Pierce. Jean Press, Frank Weld. Christopher M. Investment Conrad. Mary B., Chair Cone, Sydney M. Lakian, John R. Muiner, Robert E. Miller. G. William O'Hanley. Ronald P Ryan, Vincent J. Segal, Sheldon J. Zeien. Alfred M. Finance O'Hanley, Ronald, Chair Clowes, Alexander W. Cone, Sydney M. Conrad, Mary B. DeHart, Donald Kelley, Darcy Brisbane Lakian, John R. Landeau, Laurie J. Loewenstein, Werner Mainer, Robert E. Manz, Robert Miller, G. William Ryan, Vincent J. Zeien, Alfred M. Nominating Weissmann. Gerald, Chair Bloom, Kerry S. Clowes. Alexander W. Cox. Martha W. Greer. Mary J. Landis. Story C. Pierce, Jean Segal. Sheldon J. Speck, William T. Steere, William C. Standing Committees of the Corporation and Science Council Buildings and Grounds Cohen. Lawrence B., Chair Boyer, Barbara C. Cutler, Richard* Fleet, Barry* Hayes, Joe* McArthur, Andrew Pelerson, Bruce J. Tweedell. Kenyon S. Valiela. Ivan *£v officio R64 Annual Report Education Committee Dione, Vincent, Chair Bloom. Kerry S.* Dawidowicz. E.A.* Dunlap, Paul Fink. Rachel Hanlon, Roger Jatfe, Laurinda Mehane. Dori Chrysler* Mendelsohn, Michael Venuti, Judith Wadsworth. Patricia Zottoli. Steve MBL/WHOI Library Joint Advisory Committee Shcpro, David. Chair, MBL Ashmore, Judy.* MBL Dow. David, NMFS Fornari. Daniel. WHOI Harbison, G. Richard. WHOI Hobbie, John. MBL Hurter, Colleen,* WHOI Kurz, Mark. WHOI Alternate Norton. Cathy,* MBL Robb. James. USGS Rose, Birgit. MBL Smith, Peter J.S.. MBL Warren. Bruce, WHOI Fellowships Pederson, Thoru, Chair Dawidowicz, E.A.* Deegan. Linda Ehrlich, Barbara Kaufmann. Sandra* (Recording Secretary) Lemos, Jose Sluder. Greenfield Smith. Peter J.S. Treistman, Steven (Guest Member) Housing, Food Service and Child Care Browne. Carole, Chair Bloom, Kerry S.* King, LouAnn* Malchow. Robert P. Stuart. Ann E. Weeks. Janis C. Assisting Browne, Robert (representing MBL Child Care) Goux, Susan (Administrator) Hinklc, Pamela Clapp (representing the MBL Club) Cutler. Richard (representing Housing) Discovery: The Campaign for Science at the Marine Biological Laboratory Steering Committee Research Services and Space Laufer. Hans, Chair Armstrong. Peter B. Cornell, Neal Cutler, Richard* Dawidowicz, E.A.* Foreman. Kenneth Kerr, Louis M.* Landowne. David Mattox, Andrew* Melillo. Jerry Mizell, Merle Smith, Peter J.S. Steudler, Paul Valiela, Ivan Bay, Frederick, Campaign Chair Golden. William T., Honorary Chair Grass. Ellen R., Honorary Chan Clowes, Alexander W., Vice-Chair Cox. Martha W., Vice-Chair Miller, G. William. Vice-Chair Weissmann. Gerald. Vice-Chair Anderson, Porter W. Barlow, Robert B. Jr. Bernstein, Norman Cobb, Jewell Plummer Conrad. Mary B. Cunningham, Mary Ellen *Ex officiti Dowling, John E. Ebert. James D. Fischbach. Gerald D. Goldman, Robert D. Greer. Mary J. Jacobson. M. Howard Landeau, Laurie J. Langford. George M. Lee, Burton J. Ill Pierce, Jean Prendergast, Robert A. Shcpro, David Speck. William T. Steere. William C. Jr. Weld. Christopher M. Zeien, Alfred M. Trustees and Committees R65 Council of Visitors Norman B. Asher, Esq., Hale and Dorr. Counsellors at Law, Boston, MA Mr. Donald J. Bainton, Continental Can Co.. Boca Raton, FL Mr. David Bakalar. Chestnut Hill, MA Mr. Charles A. Baker, The Liposome Company, Inc., Princeton. NJ Dr. George P. Baker, Massachusetts General Hospital, Boston, MA Dr. Sumner A. Barenberg. Bernard Technologies, Chicago, IL Mr. Mel Burkan, The Barkan Companies, Boston, MA Mr. Robert P. Beech, Component Software International, Inc.. Mason. OH Mr. George Berkowitz, Legal Sea Foods. Allston, MA Mr. and Mrs. Nathaniel J. Bickford. New York, NY Dr. Elkan R. Blout, Harvard Medical School, Boston, MA Mr. and Mrs. Philip Bogdanovitch, Lake Clear, NY Mr. Malcolm K. Brachman, Northwest Oil Company, Dallas, TX Dr. Goodwin M. Breinin, NY University Medical Center, New York, NY Mr. John Callahan, Carpenter, Sheperd & Warden, New London, NH Mrs. Elizabeth Campanella, West Falmouth, MA Thomas S. Crane, Esq., Mint/ Levin Cohen Ferris Glovsky & Popeo, PC, Boston, MA Dr. Stephen D. Crocker, Cyber Cash Inc.. Reston, Virginia Mrs. Lynn W. Piasecki Cunningham, Film and Videomaker, Piasecki Productions, Brookline, MA Dr. Anthony J. Cutaia, Anheuser-Busch. Inc., St. Louis, MO Mrs. Sara Greer Dent, Chevy Chase, MD Mr. D. H. Douglas-Hamilton, Hamilton Thorne Research, Beverly, MA Mr. Benjamin F. du Pont. Du Pont Company. Deepwater, NJ Dr. Sylvia A. Earle, Founder, Deep Ocean Engineering. Oakland, CA Mr. and Mrs. Hoyt Ecker, Vero Beach. FL Mr. Anthony B. Evnin, Venrock Associates, New York, NY Stuart Feiner, Esq.. Inco Limited, Toronto, ON, Canada Mrs. Hadley Mack French, Edsel & Eleanor Ford House, Grosse Pointe Farms. MI Mr. and Mrs. Huib Geerlings. Boston, MA Mr. William J. Gilbane. Jr.. Gilbane Building Company, Providence, RI Dr. Michael J. Goldblatt. Intelligent Biocides, Tewksbury, MA Mr. Maynard Goldman. President, Maynard Goldman & Associates, Boston, MA Ms. Charlotte I. Hall, Edgartown, MA Drs. Linda Hirshman. Brandeis University. Waltham, MA, and David Forkosh, FMH Foundation Mr. Thomas J. Hynes, Jr., Meredith & Grew, Inc., Boston, MA Mr. M. Howard Jacobson. Bankers Trust, Westborough, MA Mrs. Elizabeth Ford Kontulis. New Canaan, CT Mr. and Mrs. Robert Lambrecht. Boca Grande. FL Dr. Catherine C. Lastavica. Tufts University School of Medicine. Boston. MA Mr. Joel A. Leavitt, Boston, MA Mr. Stephen W. Leibholz. TechLabs, Inc., Huntingdon. PA Mrs. Margarei Lilly, West Falmouth, MA Mr. Richard Lipkin. ResponseLogic. Inc., New York, NY Mr. George W. Logan, Valley Financial Corp., Roanoke, VA Mr. Michael T. Martin. SportsMark, Inc.. New York. NY Mrs. Christy Swift Maxwell. Grosse Pointe Farms, MI Dr. Georges de Menil. DM Foundation, New York, NY Mr. Ambrose Monell, G. Unger Vetlesen Foundation, Palm Beach. FL Dr. Mark Novilch, Washington, DC Ms. Julie Packard. Monterey Bay Aquarium. Monterey, CA Mr. David R. Palmer, David Ross Palmer & Associates, Waquoit, MA Dr. Roderic B. Park, Richmond. CA Mr. Santo P. Pasqualucci, Falmouth Co-Operative Bank. Falmouth, MA Mr. Robert Pierce, Jr.. Pierce Aluminum Co.. Canton, MA Mr. Richard Reston, Vineyard Ga/ette. Edgartown, MA Mr. Marius A. Robinson, Fundamental Investors Ltd., Key Biscayne, FL John W. Rowe, M.D., Mt. Sinai School of Medicine & Mt. Sinai Medical Center, New York, NY Mr. Edward Rowland, Tucker, Anthony, Inc., Boston, MA Mr. Gregory A. Sandomirsky, Mintz Levin Cohen Ferris Glovsky & Popeo. PC. Boston. MA Mrs. Mary Schmidek, Marion. MA Dr. Cecily C. Selby, New York, NY Mr. Robert S. Shifman, St. Simon's Island. Georgia Mr. and Mrs. Gregory Skau. Grosse Pointe Farms. Ml Mr. Malcolm B. Smith, General American Investors Co.. New York. NY Mr. John C. Stegeman, Campus Rentals, Ann Arbor. MI Mr. Joseph T. Stewart. Jr.. Skillman, NJ Mr. John W. Stroh. III. The Stroh Brewery Company, Detroit, MI Mr. Gerard L. Swope, Washington, DC Mr. John F. Swope. Concord. NH Mr. and Mrs. Stephen E. Taylor. Milton, MA Mrs. Donna Vanden Bosch-Flynn. Spring Lake. NJ Mrs. Carolyn W. Verbeck, Vineyard Haven, MA Mr. Benjamin S. Warren III, Grosse Pointe Farms, MI Nancy B. Weinstein, R.N.. The Hospice, Inc., Glen Ridge. NJ Stephen S. Weinstein. Esq., Morristown, NJ Mr. Frederick J. Weyerhaeuser. Beverly, MA Mr. Tony L. White. The Perkin Elmer Corporation, Norwalk, CT Dr. Torslen N. Wiesel, The Rockefeller University, New York. NY Administrative Support Staff1 Biological Bulletin Greenberg, Michael J.. Editor-in-Chief Hinkle, Pamela Clapp. Managing Editor Burns. Patricia Gibson. Victoria R. Schachinger. Carol H. Director's Office Burris. John E.. Director and Chief Executive Officer Donovan. Marcia H. Equal Employment Opportunity MacNeil, Jane L. Veterinarian Sen'ices Champagne. Jaimie2 Reynolds, Martha Smolowitz, Roxanna Stukey. Jetley Ecosystems Center Administrative Staff Berthel. Dorothy J. Donovan, Suzanne J. Nunez. Guillermo Seifert, Mary Ann External Affairs Carotenuto, Frank C.. Director Butcher. Valerie Faxon. Wendy P. Johnson. A. Kristine Martin. Theresa H. Patch-Wing, Dolores Quigley. Barbara A. Shaw. Kathleen L. Associates Pn H; liim Bohr, Kendall I! Zeida, Robert ' Including persons who joined or left the staff during 2 Summer or temporary. IW9. Communications Office Hinkle. Pamela Clapp, Director Cox. Sarah2 Flynn, Bridget Furfey. Susan2 Joslin, Susan Liles. Beth R. Noonan, Ryan2 Schanhacher, Jennifer2 Financial Sen'ices Office Lane, Homer W. Jr.. Chief Financial Officer Bowman, Richard, Controller Afonso, Janis Barry, Maureen Crosby, Kenneth Eidelman, Dana Lancaster. Cindy Livingstone, Suzanne McLaughlin. Rebecca Jill Ran/inger. Laura Stellrecht. Lynette Stock Room Schorer. Timothy M.. Supervisor Brmson. Robert2 Burnette. Donald O'Connor-Lough, Susan Purchasing Hall. Lionel E. Jr., Supervisor Gannon, Katherine2 Shamon. Lynne R. Housing and Conferences King. LouAnn D., Director Grasso, Deborah Hanlon. Arlene K.2 Johnson-Horman, Frances N. Masse, Todd C. Perito. Diana Ridley, Alberta2 R66 Administrative Support Staff R67 Human Resources Goux, Susan P., Director Houser. Carmen Josephine Bay Paul Center for Comparative Molecular Biology and Evolution Administrative Staff Harris. Marian Lini. Pauline Cc/>v Center Mounttord. Rebecca J., Supervisor Abisla, Richard L.2 Adams. Jessica2 Churchill, Elizabeth2 Clark. Tamaru L. Cosgrove, Nancy Kefeauver, Lee Mancini. Mary E. Waterbury. Andrew2 Journal of Membrane Biology Loewenstein. Werner R.. Editor Fay, Catherine H. Howard Isenberg. Linda L. Lvnch. Kathleen F. Marine Resources Center Hanlon. Roger T, Director Santore. Gabnelle Aquatic Resources Department Enos, Jr., Edward G., Superintendent Bourque, Ryan M.2 Chappell, P. Dreux2 DeGiorgis, Joseph A.2 Grossman, William M. Gudas. Christopher N.2 Kilpatrick. Brian2 Klimm. Henry W. Ill Reynolds. Justin2 Sexton. Andrew W. Smith. Gary2 Sullivan. Daniel A. Tassinari. Eugene MRC Life Support System Mebane, William N., Systems Operator Carroll, James Clark. Heidi2 Hanley. Janice S. Kuzirian, Alan Till. Geoffrey A. MBUWHOI Library Norton. Catherine N.. Director Ashmore. Judith A. Buckingham. Andrew2 Costa. Marguerite E. Crocker, Daniel2 Deveer. Joseph M. Farrar. Stephen R.L. Fredericks. Julie2 Medeiros, Melissa Monahan. A. Jean Moniz. Kimberly L. Moore. Laurel E. Nelson. Heidi Person, Matthew Riley. Jacqueline Shinkle. Chelsea2 Information Systems Division In/ina, Barbara, Network Manager Berrios, Kelly2 Cohen, Alex2 Milliard. Channing2 Kokmeyer. Remmert2 Malchow, Robert2 Mountford, Rebecca J. Moynihan, James V. Purdy, Heather2 Remsen. David P. Renna. Denis J. Space, David B. Wheeler. Patrick NASA Center for Advanced Studies in the Space Life Sciences Blazis. Diana, Administrator Amit, Udeni P. Golden. Catherine Oldham. Pamela Research Administration & Educational Programs Dawidowicz, Eliezar A.. Director Hamel, Carol C. Holzworth, Kelly Kaul'mann, Sandra J. Kleinhenz, Andrea Lynn. Rebecca Mebane, Dorianne C. White, Laurie Central Microscopy Facility and General Use Rooms Kerr. Louis M., Supervisor Luther, Herbert Matthews, Douglas2 Peterson. Martha B. Safety Sen'ices Mattox, Andrew H.. Environmental. Health, and Safety Manager Lingler. Nicholas2 Normand. Danielle2 Satellite/Periwinkle Children 's Programs Robinson, Paulina H.2 Borst, Douglas2 Browne. Jennifer L.2 Curran. Kelly2 David, Amy2 R68 Annual Report Fitzelle. Annie" Gallant, Cynthia2 Guiftrida. Beth2 Halter. Sarah2 Hike. Kelly2 Mekelatos, Sharon2 Noonan. Brendan" Robbins, Sarah2 Shanley, Jennifer2 Sen'ice, Projects and Facilities Cutler. Richard D.. Director Enos, Joyce B. Guarente, Jeffrey Apparatus Baptiste, Michael G. Barnes. Franklin D. Haskins, William A. Building .SVrnVc.v & Grounds Hayes. Joseph H.. Superintendent Anderson, Lewis B. Atwood. Paul R. Baker, Harrison S. Barnes, Susan M. Beernik, Daniel Berthel, Frederick Billings. Julia2 Boucher. Richard L. Brereton, Richard S.2 Callahan. John J. Cameron, Lawrence M.2 Chen, Zhi Xm Clayton. Daniel Collins, Paul J. Cowan. Matthew B.2 Cutler. Matthew D.2 Cutillo, David Dimond, Jay2 Doherty. Garrett2 Dorris, John .1. Eldridge. Myles2 Elias, Michael Fernandez, Peter R.2 Foster, Martin2 Gibbons, Roberto G. Hannigan. Catherine Illgen. Robert F. Joyner. Matthew2 Ledwell, L. Patrick2 Lynch. Henry L. MacDonald, Cynthia C. Malchow, Kate2 McCarthy, Michael McDonnell. Gregory McNamara. Moreen M, McQuillan. Jeffrey2 Parker, Karen2 Plant, Stephen W. Pratt, Barry Stites, Clint2 Thiele, Tod2 Ware, Lynn M. Plnnt Openilitms untl Maintenance Fleet, Barry M., Manager Cadose, James W.. Maintenance Supervisor Barnes, John S. Blunt. Hugh F. Bourgoin. Lee E. Callahan, John Carroll, James R. Davis, Douglas Fish. David L. Jr. Fuglister. Charles K. Goehl. George Gonsalves, Walter W. Jr. Hathaway. Peter J. Henderson, Jon R. Kelley. Kevin L'Abbe, Robert Langill, Richard Lochhead, William M. McAdams, Herbert M. Ill McHugh, Michael O. Mills, Stephen A. Olive, Charles W. Jr. Rattacasa, Frank2 Rozum. John Schoepf. Claude Settlemire, Donald Shepherd. Denise M. Toner, Michael Wetzel. Ernest D.2 Members of the Corporation Life Members Acheson, George H., 25 Quissett Avenue. Woods Hole, MA 02543 Adelberg, Edward A., 204 Prospect Street, New Haven. CT 065 1 1 - 2107 Afzelius, Bjorn, University of Stockholm, Wenner-Gven Institute. Department of infrastructure Research, Stockholm, Sweden Amatniek. Ernest, (address unknown) Arnold, John M., 329 Sippewissett Road. Falmouth, MA 02540 Bang, Betsy G., 76 F. R. Lillie Road. Woods Hole. MA 02543 Bartlett, James H., University of Alabama, Department of Physics, Box 870324, Tuscaloosa, AL 35487-0324 Berne, Robert M., University of Virginia School of Medicine, Department of Physiology, Box 1116, MR4, Charlottesville, VA 22903 Bernheimer, Alan W., New York University Medical Center. Department of Microbiology, 550 First Avenue. New York, NY 10016 Bertholf, Lloyd M., Westminster Village. #21 14, 2025 East Lincoln Street. Bloomington, IL 61701-5995 Bosch, Herman F., 163 Elm Road, Falmouth, MA 02540-2430 Buck, John B., Fairhaven C-020. 7200 Third Avenue, Sykesville, MD 21784 Burbanck, Madeline P., P.O Box 15134, Atlanta, GA 30333 Burbanck, William D., P.O. Box 15134. Atlanta. GA 30333 Clark, Arnold M., 53 Wilson Road, Woods Hole, MA 02543 Clark, James M., 258 Wells Road, Palm Beach, FL 33480-3625 Cohen, Seymour S., 10 Carrot Hill Road, Woods Hole, MA 02543- 1206 Colwin, Arthur L., 320 Woodcrest Road, Key Biscayne, FL 33149- 1322 Colwin, Laura Hunter, 320 Woodcrest Road, Key Biscayne, FL 33149-1322 Cooperstein, Sherwin J., University of Connecticut, School of Medicine, Department of Anatomy, Farmington, CT 06030-3405 Copeland, D. Eugene, Marine Biological Laboratory, Woods Hole, MA 02543 Corliss, John O., P.O. Box 2729, Bala Cynwyd, PA 19004-2116 Costello, Helen M., Carolina Meadows, Villa 137, Chapel Hill, NC 27514-8512 Crouse, Helen, Rte. 3. Box 213, Hayesville, NC 28904 DeHaan, Robert L., Emory University School of Medicine, Department of Anatomy and Cell Biology, 1648 Pierce Drive, Room 108, Atlanta. GA 30322 Dudley, Patricia L., 3200 Alki Avenue SW. #401, Seattle. WA 98116 Edwards, Charles, 3429 Winding Oaks Drive, Longboat Key, FL 34228 Elliott, Gerald F., The Open University Research Unit. Foxcombe Hall, Berkeley Road, Boars Hill. Oxford OX1 5HR, United Kingdom Failla, Patricia M., 2149 Loblolly Lane. Johns Island, SC 29455 Ferguson, James K. W., 56 Clarkehaven Street, Thomhill, Ontario L4J 2B4, Canada (deceased 1999) Gabriel, Mordecai L., Brooklyn College, Department of Biology, 2900 Bedford Avenue, Brooklyn, NY 11210 Glusman, Murray, New York State Psychiatric Institute, 722 W. 168th St.. Unit #70, New York. NY 10032 Graham, Herbert, 36 Wilson Road, Woods Hole, MA 02543 Hamburger, Viktor, Washington University, Department of Biology, 740 Trinity Avenue, St. Louis, MO 63130 Hamilton, Howard L., University of Virginia, Department of Biology, 238 Gilmer Hall. Charlottesville, VA 22901 Harding, Clifford V. Jr., 54 Two Ponds Road. Falmouth. MA 02540 Haschemeyer, Audrey E. V., 2 1 Glendon Road, Woods Hole, MA 02543-1406 Hauschka, Theodore S., 333 Fogler Road. Bremen. ME (deceased 1999) Hayashi, Teru. 15 Gardiner Road. Woods Hole, MA 02543-1 1 13 Hisaw, Frederick L., (address unknown) Hoskin, Francis C. G., c/o Dr. John E. Walker, U.S. Army Natick RD&E Center, SAT NC-YSM, Kansas Street. Natick. MA 01760- 5020 Hubbard, Ruth, Harvard University, Biological Laboratories, Cambridge, MA 02138 Humes. Arthur G., Marine Biological Laboratory, Boston University Marine Program, Woods Hole, MA 02543 (deceased 1999) Hunter, W. Bruce, 305 Old Sharon Road, Peterborough, NH 03458- 1736 Hurwitz, Charles, Stratton VA Medical Center, Research Service, Albany, NY 12208 Katz, George, Merck. Sharp and Dohme. Fundamental and Experimental Research Laboratory. PO Box 2000. Rahway, NJ 07065 Kingsbury, John M., Cornell University, Department of Plant Biology, Plant Science Building. Ithaca, NY 14853 Kleinholz, Lewis, Reed College. Department of Biology. 3203 SE Woodstock Boulevard. Portland. OR 97202 Kusano, Kiyoshi, National Institutes of Health, Building 36, Room 4D- 20. Bethesda. MD 20892 Laderman, Ezra, Yale University, New Haven. CT 06520 R69 R70 Annual Report LaMarche, Paul H., Eastern Maine Medical Center, 489 State Street, Bangor, ME 04401 Lauffer, Max A., Penn State University Medical Center. Department of Biophysics and Physiology, Hershey, PA 17033 Lochhead, John H., 49 Woodlawn Road, London SW6 6PS. United Kingdom Loewus, Frank A., Washington State University, Institute of Biological Chemistry. Pullman. WA 99164 Loftfield, Robert B., University of New Mexico, School of Medicine. 915 Stanford Drive. Albuquerque, NM 87131 Lorand, Laszlo, Northwestern University Medical School, CMS Biology, Searle 4-555, 303 East Chicago Avenue, Chicago. IL 60611- 3008 Malkiel, Saul, 174 Queen Street, #9 A. Falmouth, MA 02540 Marsh, Julian B., 9 Eliot Street, Chestnut Hill, MA 02467-1407 Martin, Lowell V., 10 Buzzards Bay Avenue, Woods Hole. MA 02543 Mathews, Rita W., East Hill Road. P.O. Box 237, Southfield, MA 01259-0237 Moore, John A., University of California, Department of Biology. Riverside, CA 92521 Moore, John W., Duke University Medical Center, Department of Neurobiology, Box 3209, Durham, NC 2771(1 Moscona, Aron A., University of Chicago, Department Molecular Genetics and Cell Biology, Chicago, IL 60637 Musacchia, X. J., P.O. Box 5054, Bella Vista, AR 72714-0054 Nasatir, Maimon, P.O. Box 379, Ojai, CA 93024 Passano, Leonard M., University of Wisconsin, Department of Zoology, Birge Hall, Madison. WI 53706 Price, Carl A., 20 Maker Lane, Falmouth, MA 02540 Prosser, C. Ladd, University of Illinois, Department of Physiology, 524 Burrill Hall. Urbana, IL 61801 Prytz, Margaret McDonald, (Address unknown) Ratner, Sarah, Public Health Research Institute, Department of Biochemistry. 455 First Avenue, New York, NY 10016 (deceased 1999) Renn, Charles E., (Address unknown) Reynolds, George T., Princeton University, Department of Physics, Jadwin Hall. Princeton, NJ 08544 Rice, Robert V., 30 Burnham Drive, Falmouth. MA 02540 Rockstein, Morris, 600 Biltmore Way, Apt. 805, Coral Gables, FL 33134 Ronkin, Raphael R., 3212 McKinley Street. NW. Washington. DC 20015-1635 Roslansky, John D., 57 Buzzards Bay Avenue, Woods Hole, MA 02543 Roslansky, Priscilla F., Associates of Cape Cod. Inc.. P.O. Box 224. Woods Hole, MA 02543-0224 Sanders, Howard L., Woods Hole Oceanographic Institution. Woods Hole. MA 02543 Sato, Null . N igova University, 3-24-101, Oakinishi Machi, Toba Mie 517-0023. Japan Schlesinger, R. Walter, 7 Langley Road, Falmouth, MA 02540-1809 Scott, Allan C., Colby College, Waterville, ME 04901 Silverstein, Arthur M., Johns Hopkins University. Institute of the History of Medicine. 1900 E. Monument Street. Baltimore, MD 21205 Sjodin, Raymond A., 3900 N. Charles Street, Apt. #1301. Baltimore, MD 21218-1719 Smith, Paul F., P.O. Box 264, Woods Hole, MA 02543-0264 Speer, John W., 293 West Main Road. Portsmouth. RI 02871 Sperelakis, Nicholas, University of Cincinnati, Department of Physiology/Biophysics. 231 Bethesda Avenue. Cincinnati, OH 45267- 0576 Spiegel. Evelyn, Dartmouth College. Department of Biological Sciences, 204 Oilman, Hanover, NH 03755 Spiegel, Melvin, Dartmouth College, Department of Biological Sciences, 204 Oilman. Hanover, NH 03755 Stephens, Grover C., University of California. School of Biological Sciences. Department of Ecology and Evolution/Biology, Irvine, CA 92717 Strehler, Bernard L., 42903 Moulton Parkway. #248. Laguna Hills, CA 92653-6425 Sussman. Maurice, 72 Carey Lane, Falmouth, MA 02540 Sussman, Raquel B., Marine Biological Laboratory. Woods Hole, MA 02543 Szent-Gyorgyi, Gwen P., 45 Nobska Road, Woods Hole, MA 02543 Thorndike, W. Nicholas, Wellington Management Company, 200 State Street, Boston, MA 02109 Trager, William, The Rockefeller University, 1230 York Avenue. New York, NY 10021-6399 Trinkaus, J. Philip, 870 Moose Hill Road, Guilford, CT 06437 Villee, Claude A. Jr., Harvard Medical School, Carrel L. Countway Library, 10 Shattuck Street, Boston, MA 021 15 Vincent, Walter S., 16 F.R. Lillie Road, Woods Hole, MA 02543 Waterman, Talbot H., Yale University. Box 208103, 912 KBT Biology Department, New Haven. CT 06520-8103 Wigley, Roland L., 35 Wilson Road, Woods Hole, MA 02543 Members Abt, Donald A., Aquavet, University of Pennsylvania, School of Veterinary Medicine, 230 Mam Street, Falmouth, MA 02540 Adams. James A., 3481 Paces Ferry Road, Tallahassee, FL 32308 Adelman, William J., 160 Locust Street, Falmouth, MA 02540 Alkon, Daniel L., National Institutes of Health, Laboratory of Adaptive Systems, 36 Convent Drive, MSC 4124, 36/4A21, Bethesda, MD 20892-4124 Allen, Garland E., Washington University, Department of Biology. Box 1137. One Brookings Drive, St. Louis. MO 63130-4899 Allen, Nina S., North Carolina State University. Department of Botany. Box 7612, Raleigh, NC 27695 Alliegro, Mark C., Louisiana State University Medical Center, Department of Cell Biology and Anatomy, 1901 Perdido Street. New Orleans, LA 70112 Anderson, Everett, Harvard Medical School. Department of Cell Biology. 240 Longwood Avenue, Boston, MA 021 15-6092 Anderson, John M., 110 Roat Street, Ithaca. NY 14850 Anderson, Porter W., 100 Bayview Drive. #2224. North Miami Beach. FL 33160 Armett-Kibel, Christine, University of Massachusetts, Dean of Science Faculty, Boston, MA 02125 Armstrong, Clay M., LIniversity of Pennsylvania School of Medicine. B701 Richards Building, Department of Physiology, 3700 Hamilton Walk, Philadelphia. PA 19104-6085 Armstrong, Ellen Prosser, 57 Millfield Street. Woods Hole, MA 02543 Arnold. William A., Oak Ridge National Laboratory, Biology Division, 102 Balsalm Road, Oak Ridge, TN 37830 Ashton, Robert W., Bay Foundation, 1 7 West 94th Street, New York, NY 10025 Members of the Corporation R71 Atema, Jelle, Boston University Marine Program, Marine Biological Laboratory. Woods Hole. MA 02543 Baccetti, Baccio, University of Sienna. Institute of Zoology, 53100 Siena, Italy Baker. Robert G., New York University Medical Center, Department Physiology and Biophysics, 550 First Avenue. New York, NY 10016 Baldwin, Thomas O., University of Arizona, Department of Biochemistry, P.O. Box 210088. Tucson, AZ 85721-0088 Baltimore, David, California Institute of Technology. 1200 East California Boulevard, Pasadena. CA 91 125 Barlow, Robert B., SUNY Health Science Center at Syracuse, 750 East Adams Street. Center for Vision Research, 3258 Weiskotten Hall, Syracuse, NY 13210 Barry, Daniel T., National Aeronautics and Space Administration, Lyn B. Johnson Space Center, 2101 NASA Road 1, Houston, TX 77058 Barry, Susan R., Mount Holyoke College. Department of Biological Sciences. South Hadley, MA 01075 Bass, Andrew H., Cornell University, Department of Neurobiology and Behavior, Seely Mudd Hall. Ithaca. NY 14853 Battelle, Barbara-Anne, University of Florida, Whitney Laboratory. 9505 Ocean Shore Boulevard, Augustine, FL 32086 Bay, Frederick, Bay Foundation. 17 W. 94th Street. First Floor, New York. NY 10025-7116 Baylor, Martha B., P.O. Box 93, Woods Hole, MA 02543 Bearer, Elaine L., Brown University, Division of Biology and Medicine. Department of Pathology. Box G, Providence, RI 02912 Beatty, John M., University of Minnesota. Department of Ecology and Behavioral Biology, 1987 Conner, Street Paul, MN 55108 Beauge, Luis Alberto, Instituto de Investigacion Medica, Department of Biophysics. Casilla de Correo 389. Cordoba 5000. Argentina Begenisich, Ted, University of Rochester, Medical Center, Box 642, 601 Elmwood Avenue, Rochester. NY 14642 Begg, David A., University of Alberta. Faculty of Medicine, Department of Cell Biology and Anatomy, Edmonton, Alberta T6G 2H7. Canada Bell, Eugene, 305 Commonwealth Avenue, Boston, MA 02115 Benjamin, Thomas L., Harvard Medical School, Pathology, D2-230, 200 Longwood Avenue. Boston. MA 021 15 Bennett, Michael V. L., Albert Einstein College of Medicine, Department of Neuroscience. 1300 Morris Park Avenue, Bronx. NY 10461 Bennett, Miriam F., Colby College. Department of Biology. Waterville. ME 04901 Bennett, R. Suzanne, Albert Einstein College of Medicine, Department of Neuroscience. 1300 Morris Park Avenue. Bronx, NY 10461 Berg, Carl J. Jr., P.O. Box 681, Kilauea. Kauai, HI 96754-0681 Berlin. Suzanne T., 5 Highland Street. Gloucester, MA 01930 Bernstein. Norman, Columbia Realty Venture. 5301 Wisconsin Avenue, NW, #600, Washington, DC 20015-2015 Bezanilla, Francisco, Health Science Center. Department of Physiology, 405 Hilgard Avenue. Los Angeles, CA 90024 Biggers, John D., Harvard Medical School, Department of Physiology, Boston, MA 02115 Bishop, Stephen H., Iowa State University, Department of Zoology, Ames, I A 50010 Blaustein, Mordecai P., University of Maryland, School of Medicine. Department of Physiology, Baltimore, MD 21201 Blennemann, Dieter, 1117 East Putnam Avenue. Apt. #174. Riverside, CT 06878-1333 Bloom, George S., The University of Texas Southwestern Medical Center, Department of Cell Biology and Neuroscience, 5323 Harry Hines Boulevard, Dallas, TX 75235-9039 Bloom, Kerry S., University of North Carolina. Department of Biology. 623 Fordham Hall CB#3280. Chapel Hill. NC 27599-3280 Bodznick, David A., Wesleyan University, Department of Biology. Lawn Avenue, Middletown, CT 06497-0170 Boettiger, Edward G., 17 Eastwood Road, Storrs, CT 06268-2401 Boolootian, Richard A., Science Software Systems, Inc., 3576 Woodcliff Road, Sherman Oaks, CA 91403 Borgese, Thomas A., Lehman College. CUNY. Department of Biology. Bedford Park Boulevard, West, Bronx, NY 10468 Borst, David W. Jr., Illinois State University, Department of Biological Sciences, Normal, IL 61790-4120 Bowles, Francis P., Marine Biological Laboratory, Ecosystems Center. Woods Hole. MA 02543 Boyer, Barbara C., Union College. Biology Department. Schenectady. NY 12308 Brandhorst, Bruce P., Simon Fraser University. Institute of Molecular Biology/Biochemistry. Bamaby, B.C. V5A 1S6, Canada Brinley, F. J. Jr., N1NCDS/NIH, Neurological Disorders Program. Room 812 Federal Building, Bethesda, MD 20892 Bronner-Fraser, Marianne, California Institute of Technology. Beckman Institute Division of Biology. 139-74, Pasadena, CA 91125 Brown, Stephen C., SUNY. Department of Biological Sciences, Albany, NY 12222 Brown, William L., 80 Black Oak Road, Weston, MA 02193 Browne, Carole L., Wake Forest University. Department of Biology, Box 7325 Reynolds Station, Winston-Salem, NC 27109 Browne, Robert A., Wake Forest University. Department of Biology, Box 7325, Winston-Salem. NC 27109 Bucklin, Anne C., University of New Hampshire. Ocean Process Analysis Laboratory, 142 Morse Hall, Durham, NH 03824 Bullis. Robert A., Oceanic Institute of Applied Aquaculture, 41-202 Kalanianaole Highway, Waimanalo, HI 96795 Burger, Max M., Friedrich Miescher-Institute, P.O. Box 2543. CH- 4002 Basel, Switzerland Burgess, David R., Boston College, Bourneuf House. 84 College Road. Chestnut Hill, MA 02467-3838 Burgos, Mario H., IHEM Medical School, UNC Conicet, Casilla de Correo 56. 5500 Mendoza, Argentina Burky, Albert, University of Dayton, Department of Biology, Dayton, OH 45469 Burris, John E., Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Burstyn, Harold Lewis, United States Air Force, Air Force Materiel Command, Rome Research Site RL/JA, 26 Electronic Parkway, Rome, NY 13441-4514 Bursztajn, Sherry, LSU Medical Center, 1501 Kings Highway, Building BRIF 6-13, Shreveport, LA 71 130 Calabrese, Ronald L., Emory University. Department of Biology. 1510 Clifton Road. Atlanta, GA 30322 Callaway, Joseph C., New York Medical College, Department of Physiology. Basic Sciences Building, Valhalla, NY 10595 Cameron, R. Andrew, California Institute of Technology, Division of Biology 156-29. Pasadena. CA 91 125 Campbell, Richard H., Bang-Campbell Associates, Eel Pond Place, Box 402, Woods Hole, MA 02543 Candelas, Graciela C., University of Puerto Rico, Department of Biology. P.O. Box 23360, UPR Station, San Juan, PR 00931-3360 Cariello, Lucio, Stazione Zoologica "A. Dohrn," Villa Comunale. 80121 Naples. Italy Case, James F., University of California, Marine Science Institute. Santa Barbara, CA 93106 Cassidy, Father Joseph D., Providence College, Priory of St. Thomas Aquinas, Providence, RI 02918-0001 Cavanaugh, Colleen M., Harvard University, Biological Laboratories. 16 Divinity Avenue. Cambridge, MA 02138 R72 Annual Report Chaet, Alfred B., University of West Florida, Department of Cell and Molecular Biology, 1 1000 University Parkway, Pensacola. FL 32514 Chambers, Edward L., University of Miami School of Medicine, Department of Physiology and Biophysics P.O. Box 016430, Miami, FL 33101 Chang, Donald C., Hong Kong University, Science and Technology, Department of Biology, Clear Water Bay, Kowloon, Hong Kong Chappell, Richard L., Hunter College. CUNY. Department of Biological Sciences, Box 210. 695 Park Avenue, New York, NY 10021 Child, Frank M., 28 Lawrence Farm Road, Woods Hole. MA 02543- 1416 Chisholm, Rex Leslie, Northwestern University, Medical School. Department of Cell Biology, Chicago, IL 6061 1 Citkowitz, Elena, Hospital of St. Raphael, Lipid Disorders Clinic, 1450 Chapel Street, New Haven. CT 06511 Clark, Eloise E., Bowling Green State University, Biological Sciences Department, Bowling Green, OH 43403 Clark, Hays, 150 Gomez Road, Hobe Sound, FL 33455 Clark. Wallis H. Jr., 12705 NW 1 12th Avenue, Alachua, FL 32615 Claude, Philippa, University of Wisconsin, Department of Zoology, Zoology Research Building 125, 1 1 17 W Johnson Street, Madison, WI 53706 Clay, John R., National Institutes of Health, N1NDS, Building 36, Room 2-CO2, Bethesda, MD 20892 Clowes, Alexander W., University of Washington, School of Medicine, Department of Surgery, Box 356410, Seattle. WA 98195-6410 Cobb, Jewel Plummer, California State University, 5151 University Drive, Health Center 205, Los Angeles, CA 90032-8500 Cohen, Carolyn, Brandeis University, Rosenstiel Basic Medical. Sciences Research Center, Waltham, MA 02254 Cohen, Lawrence B., Yale University School of Medicine, Department of Physiology, 333 Cedar Street, New Haven, CT 06520 Cohen, Maynard M., Rush Medical College, Department of Neurological Sciences, 600 South Paulina, Chicago, IL 60612 Cohen, William D., Hunter College, Department Biological Sciences, New York, NY 10021 Coleman, Annette W., Brown University, Division of Biology and Medicine, Providence, Rl 02912 Colinvaux, Paul, Marine Biological Laboratory, Woods Hole, MA 02543 Collier, Jack R., 3431 Highway, #107, P.O. Box 139, Effie, LA 71331 Collier, Marjorie McCann, 3431 Highway 107. P.O. Box 139, Effie. LA 71331 Cook, Joseph A., Edna McConnell Clark Foundation. 250 Park Avenue. New York. NY 10177-0026 Cornell, Neal W., Marine Biological Laboratory, Woods Hole, MA 02543 (deceased, 2000) Cornwall, Melvin C. Jr., Boston University School of Medicine. Department of Physiology L714, Boston, MA 02118 Corson, D. Wesley Jr., Storm Eye Institute, Room 537, 171 Ashley Avenue. Charleston. SC 29425 Corwin, Jeffrey T., University of Virginia, School of Medicine, Department Otolaryngology and Neuroscience, Box 396, Charlottesville, VA 22908 Couch, Ernest F., Texas Christian University, Department of Biology, TCU Box 298930, Fort Worth. TX 76129 Cox, Rachel Llanelly, Woods Hole Oceanographic Institute, Biology Department, Woods Hole, MA 02543 Crane, Sylvia E., 438 Wendover Drive. Princeton, NJ 08540 Cremer-Bartels, Gertrud, Horstmarer Landweg 142, 48149 Muenster. Germany Crow, Terry J., University of Texas Medical School, Department of Neurobiology and Anatomy, Houston, TX 77225 Crowell, Sears, Indiana University. Department of Biology. Bloomington. IN 47405 Crowther, Robert J., Shriners Hospitals for Children, 51 Blossom Street, Boston, MA 02114 Cummings, Michael P., Marine Biological Laboratory. Bay Paul Center, Woods Hole, MA 02543 Cunningham, Mary-Ellen, 62 Cleverly Road. Grosse Pointe Farms. MI 48236-3313 Cutler, Richard D., Marine Biological Laboratory. Woods Hole. MA 02543 Davidson, Eric H., California Institute of Technology, Division of Biology 156-29, 391 South Holliston, Pasadena. CA 91125 Davison, Daniel B., Bristol-Myers Squibb PRI. Bioinformatics Department, 5 Research Parkway, Wallingford, CT 06492 Daw, Nigel W., 5 Old Pawson Road, Branford, CT 06405 Dawidowicz, Eliezar A., Marine Biological Laboratory, Office of Research Administration and Education, Woods Hole, MA 02543 De Weer, Paul J., University of Pennsylvania, B400 Richards Building. Department of Physiology. 3700 Hamilton Walk, Philadelphia, PA 19104-6085 Deegan, Linda A., Marine Biological Laboratory. The Ecosystems Center. Woods Hole, MA 02543 DeGroof, Robert C., 145 Water Crest Drive, Doylestown, PA 18901- 3267 Denckla, Martha Bridge, Johns Hopkins University, School of Medicine, Kennedy-Kneger Institute, 707 North Broadway. Baltimore. MD 21205 DePhillips, Henry A., Trinity College, Department of Chemistry. 300 Summit Street, Hartford. CT 06106 DeSimone, Douglas W., University of Virginia, Department of Cell Biology, Box 439, Health Sciences Center, Charlottesville, VA 22908 Dettbarn, Wolf-Dietrich, 4422 Wayland Drive, Nashville. TN 37215 Dionne, Vincent E., Boston University Marine Program, Marine Biological Laboratory. Woods Hole. MA 02543 Dowling, John E., Harvard University. Biological Laboratories. 16 Divinity Street, Cambridge, MA 02138 Drapeau, Pierre, Montreal General Hospital, Department of Neurology. 1650 Cedar Avenue. Montreal. Quebec H3G 1A4, Canada DuBois. Arthur Brooks, John B. Pierce Foundation Laboratory, 290 Congress Avenue. New Haven. CT 06519 Duncan, Thomas K., Nichols College, Environmental Sciences Department. Dudley, MA 01571 Dunham. Philip B., Syracuse University, Department of Biology, 130 College Place. Syracuse. NY 13244-1220 Dunlap. Paul V., University of Maryland Biotechnology Institute, Center of Marine Biotechnology, Columbus Center. Suite 236. 701 East Pratt Street, Baltimore, MD 2 1 202 Ebert, James D., The Johns Hopkins University, Department of Biology, Homewood. 3400 North Charles Street, Baltimore, MD 21218-2685 Eckberg, William R., Howard University. Department of Biology, P.O. Box 887, Administration Building, Washington, DC 20059 Edds, Kenneth T., R & D Systems, Inc., Hematology Division, 614 McKinley Place. NE. Minneapolis, MN 55413 Eder, Howard A., Albert Einstein College of Medicine, 1300 Morris Park Avenue. Bronx. NY 10461 Edstrom, Joan, 53 Two Ponds Road. Falmouth, MA 02540 Egyud, Laszlo G., Cell Research Corporation, P.O. Box 67209. Chestnut Hill. MA 02167-0209 Khrlich, Barbara E., Yale University Medical School, Department of Pharmacology. New Haven, CT 06473 Members of the Corporation R73 Eisen, Arthur Z., Washington University, Division of Dermatology, St. Louis, MO n3l 10 Eisen, Herman N., Massachusetts Institute of Technology, Center for Cancer Research, El 7- 1 28. 77 Massachusetts Avenue, Cambridge. MA 02 1 39-4307 Elder, Hugh Young. University of Glasgow. Institute of Physiology, Glasgow G 1 2 8QQ, Scotland Englund, Paul T., Johns Hopkins Medical School. Department of Biological Chemistry, 725 North Wolfe Street. Baltimore. MD 21205 Epel, David, Stanford University. Hopkins Marine Station. Ocean View Boulevard, Pacific Grove, CA 93950 Epstein, Herman T., 18 Lawrence Farm Road. Woods Hole. MA 02543 Epstein, Ray L., 701 Winthrop Street. #311. Taunton. MA 02780-2187 Farb, David H., Boston University School of Medicine, Department of Pharmacology L603, 80 East Concord Street, Boston, MA 021 18 Farmanfarmaian, A. Verdi, Rutgers University. Department of Biological Sciences. Nelson Biology Laboratory FOB 1059. Piscataway. NJ 08855 Feldman, Susan C., University of Medicine and Dentistry. New Jersey Medical School. 100 Bergen Street. Newark, NJ 07103 Festoff, Barry William. VA Medical Center, Neurology Service (151). 4801 Linwood Boulevard. Kansas City. MO 64128 Fink, Rachel D., Mount Holyoke College. Department of Biological Sciences. Clapp Laboratories. South Hadley, MA 01075 Finkelstein, Alan, Albert Einstein College of Medicine, 1300 Morris Park Avenue. Bronx, NY 10461 Fischbach, Gerald D., National Institute of Health, Neurological Disorders and Strokes, 3 1 Center Drive, MSC 2540, Bldg 3 1 , Rm 8A03. Bethesda, MD 20892-2540 Fishman, Harvey M., University of Texas Medical Branch, Department of Physiology and Biophysics, 301 University Boulevard, Galveston. TX 77555-0641 Flanagan, Dennis, 12 Gay Street, New York. NY 10014 Fluck, Richard Allen, Franklin and Marshall College. Department of Biology. Box 3003, Lancaster, PA 17604-3003 Foreman, Kenneth H., Marine Biological Laboratory. Woods Hole. MA 02543 Fox, Thomas Oren, Harvard Medical School, Division of Medical Sciences, MEC 435. 260 Longwood Avenue. Boston. MA 021 15 Franzini-Armstrong, Clara, University of Pennsylvania, School of Medicine. 330 South 46th Street, Philadelphia. PA 19143 Fraser, Scott, California Institute of Technology, Beckman Institute 139-74, 1201 East California Boulevard, Pasadena. CA 91 125 Frazier, Donald T., University of Kentucky Medical Center. Department of Physiology and Biophysics. MS501 Chandler Medical Center, Lexington, KY 40536 French, Robert J., University of Calgary, Health Sciences Centre, Alberta, T2N 4NI, CANADA Fulton, Chandler M., Brandeis University. Department of Biology. MS 008. Waltham. MA 02454-91 II) Furie, Barbara C.. Beth Israel Deaconess Medical Center. BIDMC Cancer Center, Kirstein 1, 330 Brookline Avenue, Boston. MA 02215 Furie, Bruce. Beth Israel Deaconess Medical Center. BIDMC Cancer Center, Kirstein 1. 330 Brookline Avenue. Boston. MA 02215 Furshpan, Edwin J., Harvard Medical School. Department of Neurobiology. 220 Longwood Avenue, Boston. MA 021 15 Futrelle, Robert P., Northeastern University. College of Computer Science, 360 Huntington Avenue. Boston. MA 021 15 Gabr, Howaida, Sue/, Canal University, Department of Marine Science, Faculty of Science, Ismailia, Egypt Gadsby, David C., The Rockefeller University, Laboratory of Cardiac- Physiology. 1230 York Avenue. New York. NY 10021-6399 Gainer, Harold, National Institutes of Health. NINDS, BNP, DIR, Neurochemistry. Building 36, Room 4D20. Bethesda, MD 20892- 4130 Galatzer-Levy, Robert M., 534 Judson Avenue, Evanston, IL 60202 Gall, Joseph G., Carnegie Institution, I 15 West University Parkway. Baltimore. MD 21210 Garber, Sarah S.. Allegheny University of the Health Sciences. Department of Physiology, 2900 Queen Lane, Philadelphia, PA 19129 Gascoyne, Peter, University of Texas. M. D. Anderson Cancer Center. Experimental Pathology. Box 89, Houston, TX 77030 Gelperin, Alan, Bell Labs Lucent. Department Biology Comp.. Rm 1C464. 600 Mountain Avenue. Murray Hill, NJ 07974 German, James L. Ill, The New York Blood Center, Laboratory of Human Genetics. 310 East 67th Street. New York. NY 10021 Gibbs, Martin, Brandeis University. Institute for Photobiology of Cells and Organelles. Waltham, MA 02254 ( .ililin. Anne E., Marine Biological Laboratory, The Ecosystems Center, Woods Hole, MA 02543 Gibson, A. Jane, Cornell University, Department of Biochemistry. Biotech Building. Ithaca. NY 14850 Gifford, Prosser, Library of Congress, Madison Building LM605, Washington DC 20540 Gilbert, Daniel L., National Institutes of Health. Biophysics Sec.. BNP, Building 36. Room 5A-27. Bethesda, MD 20892 Giudice, Giovanni, Universita di Palermo. Dipartimento di Biologia, Cellulare e Dello Sviluppo, 1-90123 Palermo. Italy Giuditta, Antonio, University of Naples. Department of General Physiology. Via Mezzocannone 8. Naples. 80134, Italy Glynn, Paul, P.O. Box 6083. Brunswick, ME 04011-6083 Golden, William T., Chairman Emeritus, American Museum of Natural History, 500 Fifth Avenue, 50th Floor, New York, NY 101 10 Goldman. Robert D., Northwestern University Medical School, Department of Cell and Molecular Biology, 303 E. Chicago Avenue. Chicago. IL 60611-3008 Goldsmith, Paul K., National Institutes of Health. Building 10. Room 9C-101. Bethesda, MD 20892 Goldsmith, Timothy H., Yale University. Department of Biology, New Haven. CT06510 Goldstein, Jr., Moise H., The Johns Hopkins University. ECE Department. Barton Hall. Baltimore, MD 2121X Gould, Robert Michael, NYS Institute of Basic Research, 1050 Forest Hill Road. Staten Island, NY 10314-6399 Govind, C. K., Scarborough College, Life Sciences Division. 1 265 Military Trail. West Hill. Ontario MIC IA4, Canada Grace, Dick, Doreen Grace Fund, The Brain Center, Promontory Point. New Seabury. MA 02649 Graf, Werner M., College of France, 1 1 Place Marcelin Berthelot. 7523 1 Paris Cedex 05, France Grant, Philip, National Institutes of Health, NINDS\BN\DIR- Neurochemistry. Building 36, Room 4D20, Bethesda. MD 20892- 4130 Grass, Ellen R., The Grass Foundation. 77 Reservoir Road. Quincy. MA 02170-3610 Grassle, Judith P.. Rutgers University. Institute of Marine and Coastal Studies. Box 231. New Brunswick. NJ 08903 Graubard, Katherine G., University of Washington, Department of Zoology, NJ-15, Box 351800, Seattle, WA 98195-1800 Greenberg, Everett Peter, University of Iowa, College of Medicine, Department of Microbiology. Iowa City, IA 52242 Greenberg, Michael J., University of Florida. The Whitney Laboratory. 9505 Ocean Shore Boulevard, St. Augustine, FL 32086-8623 R74 Annual Report Greer, Mary J., 176 West 87th Street. #12A, New York, NY 10024- 2902 Griffin, Donald R., Harvard University. Concord Field Station. Old Causeway Road. Bedford, MA 01730 Gross, Paul R., 123 Perkins Street. Jamaica Plain, MA 02130 Grossman, Albert, New York University Medical Center, 550 First Avenue, New York, NY 10016 Grossman, Lawrence, The Johns Hopkins University, Hygiene Building, Room W8306, Baltimore, MD 21205 Gruner, John A., Cephalon. Inc., 145 Brandy wine Parkway, West Chester, PA 19380-4245 Gunning, A. Robert, P. O. Box 165. Falmouth. MA 02541 Gwilliam, G. Francis, Reed College, Department of Biology, Portland, OR 97202 Haimo, Leah T., University of California, Department of Biology, Riverside. CA 92521 Hajduk, Stephen L., University of Alabama. School of Medicine/Dentistry. Department of Biochemistry/Molecular Genetics, University Station. Birmingham. AL 35294 Hall, Linda M., Shriners Hospital for Children, 2425 Stockton Boulevard. Sacramento, CA 95X17 Hall, Zach W., University of California, Department of Physiology, San Francisco, C A 941 14 Halvorson, Harlyn O., University of Massachusetts, Policy Center for Marine Biosciences and Technology. 100 Morrissey Boulevard, Boston, MA 02 1 25-3393 Haneji, Tatsuji, The University of Tokushima, Department of Histology and Oral Histology, School of Dentistry. 18-15. 3 Kuramoto-cho. Tokushima 770-8504, Japan Hanlon, Roger T., Marine Biological Laboratory, Woods Hole, MA 02543 Harosi, Ferenc, New College of the USF. Division of Natural Sciences, 5700 North Tamiami Trail. Sarasota, FL 34243-2197 Harrigan, June F., 7415 Makaa Place, Honolulu. HI 96825 Harrington, Glenn W., Weber State University. Department of Microbiology, Ogden, UT 84408 Harrington, John P., University of South Alabama, Department of Chemistry, Mobile, AL 36688 Harrison, Stephen C., Harvard University. Department of Molecular and Cell Biology, 7 Divinity Avenue, Cambridge, MA 02138 Haselkorn, Robert, Llniversity of Chicago, Department of Molecular Genetics and Cell Biology, Chicago, IL 60637 Hastings, J. Woodland, Harvard University. The Biological Laboratories. 16 Divinity Avenue. Cambridge. MA 02138-2020 Haydon-Baillie, Wensley G., Porton Institute. 2 Lowndes Place, London SW1X 8Dd. United Kingdom Hayes, Raymond L. Jr., Howard University, College of Medicine, 520 W Street. NW. Washington, DC 20059 Heck, Diane E., Rutgers University, Department of Pharmacology/Toxicology, 681 Frelinghuysen Road, Piscataway, NJ 08855 Henry, Jonathan Joseph, University of Illinois, Department of Cell and Structural Biology. 601 South Goodwin Avenue #B107, Urbana. IL 61801-3709 Hepler, Peter K , iMiy of Massachusetts. Department of Biology. Morrill III, Amiu-i M\ 01003 Herndon, Walter K., n isiiy of Tennessee, Department of Botany, Knoxville. TN >.7'" Hersliko, Avram, Technion IM:J<-| Institute of Technology, Unit of Biochemistry. The Bruce Rappaport Faculty of Medicine. Haifa 31096. Israel Herskovits, Theodore T., Fordham University. Department of Chemistry. John Mulcahy Hall, Room 638. Bronx. NY 10458 Hiatt, Howard H., Bngham and Women's Hospital, Department of Medicine, 75 Francis Street. Boston. MA 021 15 Highstein, Stephen M., Washington University. Department of Otolaryngology. Box 8115, 4566 Scott Avenue, St. Louis. MO 63110 Hildebrand, John G., University of Arizona. ARL Division of Neurobiology, P.O. Box 210077, Tucson, AZ 85721-0077 Hill, Richard W., Michigan State University. Department of Zoology, East Lansing. MI 48824 Hill, Susan D., Michigan State University. Department of Zoology. East Lansing. MI 48824 Hillis, Llewellya W., Marine Biological Laboratory. Woods Hole. MA 02543 Hinchcliffe, Edward H., University of Massachusetts Medical School, Department of Cell Biology. 377 Plantation Street. Worcester, MA 01605 Hinkle, Gregory J., Bioinformatics Group, Cereon Genomics, One Kendall Square, Building 200. Cambridge, MA 02139 Hinsch, Gertrude W., University of South Florida, Department of Biology, Tampa. FL 33620 Hinsch, Jan, Leica. Inc.. 1 10 Commerce Drive, Allendale, NJ 07401 Hobbie, John E., Marine Biological Laboratory, The Ecosystems Center. Woods Hole, MA 02543 Hodge, Alan J., 3843 Mount Blackburn Avenue. San Diego, CA 921 1 1 Hoffman, Joseph F., Yale University School of Medicine. Cellular and Molecular Physiology, 333 Cedar Street, New Haven, CT 06520-8026 Hollyneld, Joe G., The Cleveland Clinic, Ophthalmic Research, 9500 Euclid Avenue. Cleveland, OH 44195 Holz, George G. IV, New York Llniversity Medical Center. Medical Sciences Building. Room 442, 550 First Avenue, New York, NY 10016 Hopkinson, Charles S. Jr., Marine Biological Laboratory, Woods Hole. MA 02543 Houk. James C., Northwestern University Medical School, 303 East Chicago Avenue, Ward 5-315. Chicago. IL 60611-3008 Hoy, Ronald R., Cornell University. Section of Neurobiology and Behavior. 215 Mudd Hall. Ithaca. NY 14853 Huang, Alice S., California Institute of Technology. Mail Code 1-9, Pasadena, CA 91125 Hufnagel-Zackroff, Linda A., University of Rhode Island, Department of Microbiology, Kingston, RI 02881 Hummon, William D., Ohio University. Department of Biological Sciences, Athens, OH 45701 Humphreys, Susie H., Food and Drug Administration. HFS-308. 200 C Street, SW. Washington. DC 20204-0001 Humphreys, Tom, University of Hawaii. Kewalo Marine Laboratory, 41 Ahui Street. Honolulu. HI 96813 Hunt, Richard T., ICRF. Clare Hall Laboratories. South Mimms Potter's Bar. Herts EN6-3LD, England Hunter, Robert D., Oakland University, Department of Biological Sciences, Rochester, MI 48309-4401 Huxley. Hugh E., Brandeis University. Rosenstiel Center. Biology Department. Waltham, MA 02154 Ilan, Joseph, Case Western Reserve University. School of Medicine, Department of Anatomy, Cleveland. OH 44 1 (Id Ingoglia, Nicholas A., New Jersey Medical School. Department of Pharmacology/Physiology, 185 South Orange Avenue. Newark. NJ 07103 Inoue. Saduyki, McGill Llniversity. Department of Anatomy. 3640 University Street. Montreal. PQ H3A 2B2, Canada Inoue, Shinya, Marine Biological Laboratory. Woods Hole, MA 02543 Isselbacher, Kurt J., Massachusetts General Hospital Cancer Center, Charlestown. MA 02 1 29 Issidorides, Marietta Radovic, University of Athens. Department of Psychiatry. Monis Petraki 8. Athens. 140. Greece Members of the Corporation R75 Izzard, Colin S., SUNY-Albany, Department of Biological Sciences, 1400 Washington Avenue, Albany. NY 12222 Jacobs. Neil, Hale and Dorr, 60 State Street. Boston, MA 02109 Jaffe, Laurinda A., University of Connecticut Health Center, Department of Physiology. Farmington Avenue. Farmington. CT 06032 Jaffe. Lionel, Marine Biological Laboratory. Woods Hole, MA 02543 Jeffery, William R., University of Maryland. Department of Biology, College Park, MD 20742 Johnston, Daniel, Baylor College of Medicine. Division of Neuroscience. Baylor Plaza, Houston. TX 77030 Josephson. Robert K., University of California. School of Biological Science, Department of Psychobiology. Irvine. CA 92697 Kaczmarek, Leonard K., Yale University School of Medicine, Department of Pharmacology. 333 Cedar Street. New Haven. CT 06520 Kaley, Gabor, New York Medical College. Department of Physiology. Basic Sciences Building. Valhalla, NY 10595 Kaltenbach. Jane, Mount Holyoke College. Department Biological Sciences. South Hadley, MA 01075 Kaminer, Benjamin, Boston University Medical School. Physiology Department, 80 East Concord Street, Boston, MA 02 1 I S Kaneshiro, Edna S., University of Cincinnati. Biological Sciences Department. JL 006. Cincinnati, OH 45221-0006 Kaplan, Ehud, Mount Sinai School of Medicine. 1 Gustave Levy Place. Box 1 1S3. New York, NY 10029 Karakashian, Stephen J., Apartment 16-F. 165 West 91st Street. New York. NY 10024 Karlin, Arthur, Columbia University, Center for Molecular Recognition, 630 West 168th Street, Room 11-401. New York. NY 10032 Karnovsky, Morris John, Harvard Medical School, Department of Pathology. 200 Longwood Avenue. Boston. MA 02 1 1 5 Keller, Hartmut Ernst, Carl Zeiss, Inc.. One Zeiss Drive, Thornwood. NY 10594 Kelley, Darcy B., Columbia University, Department of Biological Sciences, 91 1 Fairchild, Mailcode 2432, New York. NY 10027 Kelly, Robert E., 5 Little Harbor Road, Woods Hole. MA 02543 Kemp. Norman E., University of Michigan, Department of Biology. Ann Arbor. MI 48109 Kendall. John P., Faneuil Hall Associates. 176 Federal Street. 2nd Floor, Boston. MA 021 10 Kerr, Louis M., Marine Biological Laboratory, Woods Hole. MA 02543 Keynan, Alexander, Israel Academy of Science and Humanity, P.O. Box 4040, Jerusalem. Israel Khan, Shahid M. M., Albert Einstein College of Medicine. Department of Physiology and Biophysics. 1 300 Morris Park Avenue. Room U273. Bronx, NY 10461 Khodakhah. Kamran, University of Colorado School of Medicine, Department of Physiology and Biophysics. 4200 East 9th Avenue, C-240. Denver, CO 80262 Kiehart, Daniel P., Duke University Medical Center. Department of Cell Biology. Box 3709, 308 Nanaline Duke Building, Durham. NC 27710 Kleinfeld, David, University of California. Department of Physics. 0319 9500 Oilman Drive. La Jolla, CA 92093 Klessen, Rainer, (address unknown) Klotz, Irving M., Northwestern University. Department of Chemistry, Evanston. 1L 60201 Knudson, Robert A., Marine Biological Laboratory. Woods Hole. MA 02543 Koide, Samuel S., The Rockefeller University, The Population Council. 1230 York Avenue. New York. NY 10021 Kornberg, Hans, Boston University. The University Professors. 745 Commomveath Avenue, Boston. MA 02215 Kosower, Edward M., Tel-Aviv University. Department of Chemistry. Ramat-Aviv. Tel Aviv. 69978, Israel Krahl. Maurice E., 2783 West Casas Circle. Tucson. AZ 85741 Krane, Stephen M., Massachusetts General Hospital, 55 Fruit Street, Bulf-165. Boston, MA 021 14 Krauss, Robert, P.O. Box 291, Denton, MD 21629 Kravitz, Edward A., Harvard Medical School, Department of Neurobiology. 220 Longwood Avenue. Boston. MA 021 15 Kriebel. Mahlon E., SUNY Health Science Center. Department of Physiology. Syracuse. NY 13210 Kristan, William B. Jr., University of California, Department of Biology 0357, 9500 Oilman Drive. La Jolla. CA 92093-0357 Kropin.ski, Andrew M., Queen's University. Department of Microbiology/Immunology. Kingston, Ontario K7L 3N6. Canada Kuffler, Damien P., Institute of Neurobiology. 201 Boulevard del Valle, San Juan 00901. PR Kuhns, William J., Hospital for Sick Children. Biochemistry Research, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada Kunkel, Joseph G., University of Massachusetts, Department of Biology. Amherst, MA 01003 Kuzirian, Alan M., Marine Biological Laboratory, Woods Hole, MA 02543-1015 Laderman, Ainilee D., Yale University, School of Forestry and Environmental Studies, 370 Prospect Street, New Haven, CT 0651 1 I .mil. ;m, Laurie J., Listowel, Inc., 2 Park Avenue, Suite 1525, New York. NY 10016 Landis, Dennis M. D., University Hospital of Cleveland. Department Neurology, 1 1 100 Euclid Avenue. Cleveland. OH 44106 Landis, Story C.. National Institutes of Health. Building 36. Room 5A05. 36 Convent Drive. Bethesda, MD 20892-4150 Landowne, David, University of Miami Medical School, Department of Physiology, PO Box 016430, Miami, FL 33101 Langford, George M., Dartmouth College, Department of Biological Sciences. 6044 Oilman Laboratory. Hanover. NH 03755 Laskin, Jeffrey, University of Medical and Dentistry of New Jersey, Robert Wood Johnson Medical School. 675 Hoes Lane. Piscataway, NJ 08854 Lasser-Ross. Nechama, New York Medical College. Department of Physiology. Valhalla, NY 10595 Laster, Leonard, University of Massachusetts Medical School, 55 Lake Avenue, North, Worcester. MA 01655 Laties, Alan, Scheie Eye Institute, Myrin Circle, 51 North 39th Street, Philadelphia. PA 19104 Laufer, Hans, University of Connecticut. Department of Molecular and Cell Biology. U- 1 25. 75 North Eagleville Road Storrs. CT 06269- 3125 Lazarow, Paul B., Mount Sinai School of Medicine. Department of Cell Biology and Anatomy. 1190 Fifth Avenue. Box 1007, New York. NY 10029-6574 Lazarus, Maurice, Federated Department Stores, Sears Crescent, City Hall Plaza. Boston. MA 02108 Leadbetter, Edward R., University of Connecticut. Department of Molecular and Cell Biology. U-131, Beach Hall, Room 249, 354 Mansfield Road, Storrs. CT 06269-2131 Lederberg, Joshua, The Rockefeller University, Suite 400 (Founders Hall). 1230 York Avenue. New York. NY 10021 Lee, John J., City College of CUNY, Department of Biology. Convent Avenue and 138th Street. New York. NY 10031 Lehv, Donald B., 35 Willow Field Drive. North Falmouth. MA 02556 R76 Annual Report Leighton, Joseph. Aeron Biotechnology, Inc.. 1933 Davis Street #310, San Leandro, CA 44577 (deceased 1999) Leighlon, Stephen B., National Institutes of Health. Building 13. 3W13. Bethesda, MD 20892 Lemos, Jose Ramon. University of Massachusetts Medical Center. Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury. MA 01545-2737 Lerner, Aaron B., Yale University School of Medicine, Department of Dermatology. P.O. Box 3333. New Haven. CT 06510 Levin, Jack, Veterans Administration. Medical Center, 1 1 1 H2. 4150 Clement Street. San Francisco, C A 94121 Levine, Michael, University of California, Department MCB, 401 Barker Hall. Berkeley. CA 94720 Levine, Richard B., University of Arizona, Division of Neurobiology, Room 61 1. Gould Simpson Building. PO Box 210077. Tucson. AZ 85721-0077 Levinthal. Francoise, Columbia University, Department of Biological Sciences. Broadway and 1 16th Street, New York, NY 10026 Levitan, Herbert, National Science Foundation, 4201 Wilson Boulevard. Room 835, Arlington, VA 22230 Levitan, Irwin B., University of Pennsylvania. School of Medicine. 218 Stemmler Hall, 3450 Hamilton Walk. Philadelphia, PA 19104-6074 Linck, Richard VV., University of Minnesota School of Medicine, Cell Biology and Neuroanatomy Department. 4-135 Jackson Hall. 321 Church Street. Minneapolis. MN 55455 Lipicky, Raymond J., Food and Drug Administration. CDER/ODE1/ HFD-1 10, 5600 Fishers Lane. Rockville. MD 20857 Lisman, John E., Brandeis University, Molecular and Cell Biology. 415 South Street. Waltham. MA 02454-91 10 Liuzzi, Anthony, 180 Beacon Street. #80. Boston. MA 021 16 Llinas, Rodolfo R., New York University Medical Center, Department of Physiology/Biophysics, 550 First Avenue. Room 442. New York. NY 10016 Lohel, Phillip S., Boston University Marine Program, Marine Biological Laboratory, Woods Hole. MA 02543 Loew, Franklin M., Becker College. 61 Sever Street, Worcester, MA 01615-0071 Loewenstein, Birgit Rose, Marine Biological Laboratory. Woods Hole, MA 02543 Loewenstein, Werner R., Marine Biological Laboratory, Woods Hole, MA 02543 London, Irving M., Harvard-MIT. Division. E-25-551, Cambridge. MA 02 1 39 Longo, Frank J., University of Iowa, Department of Anatomy, Iowa City. IA 52442 Luckenhill, Louise M., Ohio University. Department of Biological Sciences. Irvine Hall. Athens. OH 45701 Macagno, Eduardo R., Columbia University. 109 Low Memorial Library, Mail Code 4306. New York. NY 10027 MacNichol. Edward F. Jr., Boston University School of Medicine. Department of Physiology, 80 East Concord Street, Boston, MA 02 1 IS Maglott-Dultield, Donna R., American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852-1776 Maienschein, Jane Ann, Arizona State University, Department of Philosophy, PO B. - S72004. Tempe, AZ 85287-2004 Mainer, Robert ¥.., ":\tL Boston Company, Inc.. One Boston Place. OBP-I5-D. Boston. MA 02108 Malhon, Craig C., SUNY, University Medical Center. Pharmacology- HSC. Stony Brook. NY I l?'>4-Sf>5l Malchow, Robert P., UimciMly ol Illinois, Department of Ophthalmology, 1855 West Taylor Street N/C 648, Chicago. IL (,0(i I 2 Manalis, Richard S., Indiana-Purdue University, Department of Biological Science, 2101 Coliseum Boulevard East, Fort Wayne, IN 46805 Manz, Robert D., 304 Adams Street. Milton. MA 02186 Margulis, Lynn, University of Massachusetts. Department of Geosciences, Morrill Science Center. Box 35820. Amherst, MA 01003-5820 Marinucci, Andrew C., 102 Nancy Drive, Mercerville, NJ 08619 Martinez, Joe L. Jr., The University of Texas, Division of Life Sciences, 6900 North Loop 1604 West, San Antonio, TX 78249-0662 Martinez-Palomo, Adolfo, CINVESTAV-IPN, Sec. de Patologia Experimental, 07000 Mexico, D.F.A.P. 140740, Mexico Mastroianni, Luigi Jr., Hospital of University of Pennsylvania, 106 Dulles, 3400 Spruce Street. Philadelphia, PA 19104-4283 Mauzerall, David, Rockefeller University, 1230 York Avenue, New York. NY 10021 McAnelly. M. Lynne, University of Texas, Section of Neurobiology, School of Life Sciences. Austin, TX 78712 McCann, Frances V., Dartmouth Medical School, Department of Physiology, Lebanon. NH 03756 McLaughlin, Jane A., Marine Biological Laboratory. Woods Hole. MA 022543 McMahon, Robert F., University of Texas. Arlington, Department of Biology, Box 19498. Arlington. TX 76019 Meedel, Thomas, Rhode Island College, Biology Department. 600 Mount Pleasant Avenue, Providence. RI 02908 Meinertzhagen, Ian A., Dalhousie University. Department of Psychology, Halifax, NS B3H 4J1, Canada Meiss, Dennis E., Immunodiagnostic Laboratories, 488 McCormick Street, San Leandro, CA 94577 Melillo, Jerry M., Marine Biological Laboratory, Ecosystems Center, Woods Hole. MA 02543 Mellon, DeForest Jr., University of Virginia, Department of Biology, Gilmer Hall. Charlottesville. VA 22903 Mellon. Richard P., P.O. Box 187, Laughlintown, PA 15655-0187 Mendelsohn, Michael E., New England Medical Center, Molecular Cardiology Laboratory, NEMC Box 80. 750 Washington Street, Boston. MA 02 1 1 I Mensinger, Allen F., Washington University School of Medicine. Department of Otolarynology. 4566 Scott Avenue. St. Louis, MO 63110 Merriman, Melanie Pratt, 7511 Beach View Drive. North Bay Village. FL 33141 Meselson, Matthew, Harvard University. Fairchild Biochemistry Building. 7 Divinity Avenue. Cambridge. MA 02138 Metuzals, Janis, University of Ottawa Faculty of Medicine, Department of Pathology and Laboratory Medical, 45 1 Smyth Road, Ottawa, Ontario K1H 8M5, Canada Miledi, Ricardo, University of California, Irvine. Department of Psychobiology. 2205 Biology Science II. Irvine, CA 92697-4550 Milkman, Roger D., University of Iowa. Department of Biological Sciences. Biology Building, Room 318, Iowa City, IA 52242-1324 Miller. Thomas J., Analogic. 8 Centennial Drive, Peabody. MA 01960 Mills. Robert, 10315 44th Avenue, W 12 H Street, Brandenton. FL 34210 Misevic. Gradimir, University Hospital of Basel, Department of Research, Mebelstr. 20. CH-4031 Basel. Switzerland Mitchell. Ralph, Harvard University, Division of Applied Sciences, 29 Oxford Street, Cambridge, MA 02138 Miyakawa, Hiroyoshi, Tokyo College of Pharmacy. Laboratory of Cellular Neurobiology. 1432-1 Horinouchi. Hachiouji, Tokyo 192-03, Japan Miyamoto, David M., Drew University, Department of Biology, Madison, NJ 07940 Members of the Corporation R77 Mi/ell. Merle, Tulane University, Department of Cell and Molecular, Biology, New Orleans. LA 701 18 Moreira. Jorge E., National Institutes of Health. NICHD. Department of Cell and Molecular Biol., Bethesda. MD 20852 Morin, James G., Cornell University, Dept. of Ecology & Evolutionary Biology, G14 Stimson Hall, Ithaca. NY 14853-2801 Morrcll. Leyla de Toledo, Rush-Presbyterian-St. Lukes Medical Center. 1653 West Congress Parkway. Chicago. IL 60612 Morse, M. Patricia, National Science Foundation, Room 885, Esie, Arlington. VA 22230 Morse, Stephen S., DARPA/DSO, 3701 North Fairfax Dnve, Arlington, VA 22203-1714 Mote, Michael I., Temple University, Department of Biology, Philadelphia. PA 19122 Muller, Kenneth J., University of Miami School of Medicine, Department of Physiology and Biophysics, 1600 NW 10th Avenue. R-430, Miami, FL 33136 Murray, Andrew W., University of California. Department of Physiology. Box 0444. 513 Parnassus Avenue. San Francisco. CA 94143-0444 Nabrit. Samuel M., 686 Beckwith Street, SW, Atlanta, GA 30314 Nadelhoffer. Knute J., Marine Biological Laboratory. 7 MBL Street, Woods Hole. MA 02543 Nagel, Ronald L., Albert Einstein College of Medicine. 1300 Morris Park Avenue, Bronx. NY 10461 Naka. Ken-ichi, 2-9-2 Tatumi Higashi. Okazaki. 444, Japan Nakajima, Yasuko, University of Illinois, College of Medicine, Anatomy and Cell Biology Department, M/C 512, Chicago, IL 60612 Narahashi, Toshio, Northwestern University Medical School, Department of Pharmacology, 303 East Chicago Avenue, Chicago, IL 60611 Nasi, Enrico, Boston University School of Medical, Department of Physiology, R-406, 80 East Concord Street. Boston. MA 02 1 1 8 Neill, Christopher, Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Nelson, Leonard, Medical College of Ohio, Department of Physiology, CS 10008. Toledo. OH 43699 (deceased 1999) Nelson, Margaret C., Cornell University, Section of Neurobiology and Behavior, Ithaca, NY 14850 Nicholls, John G., SISSA. Via Beirut 2, 1-34014 Trieste. Italy Nickerson. Peter A., SUNY at Buffalo. Department of Pathology. Buffalo. NY 14214 Nicosia, Santo V., University of South Florida, College of Medicine, Box 1 1, Department of Pathology, Tampa, FL 33612 Noe, Bryan D., Emory University School of Medicine, Department of Anatomy and Cell Biology, Atlanta, GA 30322 Norton, Catherine N., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Nusbaum, Michael P., University of Pennsylvania School of Medicine, Department of Neuroscience, 215 Stemmler Hall, Philadelphia, PA 19104-6074 O'Herron, Jonathan, Lazard Freres and Company, 30 Rockefeller Plaza, 59th Floor, New York, NY 10020-1900 Obaid, Ana Lia, University of Pennsylvania School of Medicine, Neuroscience Department, 234 Stemmler Hall, Philadelphia, PA 19104-6074 Ohki, Shinpei, SUNY at Buffalo, Department of Biophysical Sciences, 224 Cary Hall. Buffalo. NY 14214 Oldenbourg, Rudolf, Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Olds, James L., George Mason University. Krasnow Institute for Advanced Studies, Mail Stop 2A1, Fairfax, VA 22030-4444 Olins, Ada L.. 45 Eastern Promenade. #7-D, Portland. ME 04101 Olins. Donald E.. 45 Eastern Promenade. #7-D. Portland. ME 04101 Oschman, James L., Nature's Own Research Association, P.O. Box 5101. Dover. NH 03820 Palazzo, Robert E., University of Kansas. Department of Physiology and Cell Biology. Lawrence, KS 66045 Palmer, John D., University of Massachusetts, Department of Zoology, 221 Merrill Science Center, Amherst. MA 01003 Pant, Harish C., National Institutes of Health. NINCDS, Laboratory of Neurochemistry, Building 36. Room 4D20. Bethesda, MD 20892 Pappas, George D., University of Illinois, College of Medicine, Department of Anatomy. Chicago, IL 60612 Pardee, Arthur B., Dana-Farber Cancer Institute, D810, 44 Binney Street, Boston, MA 021 15 Pardy, Rosevelt L., University of Nebraska, School of Life Sciences, Lincoln, NE 68588 Parmentier, James L., AstraZeneca, 725 Chesterbrook Boulevard, Wayne, PA 19087-5677 Pederson, Thoru, University of Massachusetts Medical Center. Worcester Foundation Campus, 222 Maple Avenue, Shrewsbury, MA 01545 Perkins, Courtland D., 400 Hilltop Terrace. Alexandria. VA 22301 Person, Philip, 137-87 75th Road. Flushing. NY 11367 Peterson, Bruce J., Marine Biological Laboratory. 7 MBL Street. Woods Hole, MA 02543 Pethig. Ronald, University College of North Wales, School of Electronic Engineering, Bangor, Gwynedd, LL 57 IUT, United Kingdom Pfohl, Ronald J., Miami University, Department of Zoology. Oxford, OH 45056 Pierce, Sidney K. Jr., University of South Florida, 4202 East Fowler Avenue. Tampa, FL 33620 Pleasure, David E., Children's Hospital. Neurology Research. 5th Floor, Ambramson Building, Philadelphia. PA 19104 Poindexter. Jeanne S.. Barnard College, Columbia University, 3009 Broadway, New York, NY 10027-6598 Pollard, Harvey B., U.S.U.H.S., 4301 Jones Bridge Road. Bethesda. MD 20814 Pollard, Thomas D., Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037 Porter, Beverly H., 5542 Windysun Court, Columbia, MD 21045 Porter, Mary E., University of Minnesota, Department of Cell Biology and Neuroanatomy. 4-135 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455 Potter, David D., Harvard Medical School, Department of Neurobiology, 25 Shattuck Street, Boston. MA 021 15 Potts, William T., University of Lancaster. Department of Biology, Lancaster, England Powers, Maureen K., University of California, Department of Molecular & Cellular Biology. Life Sciences Addition, Berkeley, CA 94720 Prendergast. Robert A., 38 Pondlet Place, Falmouth. MA 02540 Prior, David J., Northern Arizona University. Arts and Sciences Dean's Office. Box 5621. Flagstaff, AZ 8601 1 Prusch. Robert D., Gonzaga University, Department of Life Sciences. Spokane, WA 99258 Purves. Dale. Duke University Medical Center. Department of Neurobiology, Box 3209. 101 -I Bryan Research Building. Durham, NC 27710 Quigley, James P., The Scnpps Research Institute, Department of Vascular Biology, 10550 N. Torrey Pines Road VB-1, La Jolla, CA 92037 R78 Annual Report Rabb, Irving W., 1010 Memorial Drive, Cambridge. MA 02138 Rabin, Harvey, 1 102 Ralston Road. Rockville. MD 20852 Rabinowitz, Michael B., Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Rafferty, Nancy S., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Rakowski, Robert F., Finch University of Health Sciences, The Chicago Medical School, Department of Physiology and Biophysics, 3333 Greenbay Road. N. Chicago, 1L 60064 Ramon, Fidel, Universidad Nacional Autonoma de Mexico, Division EStreet Posgrado E Invest., Facultad de Medicina, 04510, D.F . Mexico Rastetter, Edward B., Marine Biological Laboratory, The Ecosystems Center. Woods Hole. MA 02543 Rebhun, Lionel I., University of Virginia. Department of Biology, Gilmer Hall 45, Charlottesville, VA 22901 Reddan, John R., Oakland University. Department of Biological Sciences. Rochester. MI 48309-4401 Reese, Thomas S., National Institutes of Health, NINDS, Building 36, Room 2A29, Bethesda, MD 20892 Reinisch, Carol L., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Rickles, Frederick R.. 2633 Danforth Lane. Decatur, GA 30033 Rieder, Conly L., Wadsworth Center. Division of Molecular Medicine. P.O. Box 509, Albany, NY 12201-0509 Riley, Monica, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Ripps, Harris, University of Illinois at Chicago, Department of Ophthalmology/Visual Sciences, 1855 West Taylor Street. Chicago, IL60612 Rjtchie, J. Murdoch, Yale University School of Medicine, Department of Pharmacology, 333 Cedar Street, New Haven, CT 06510 Rome, Lawrence C., University of Pennsylvania, Department of Biology, Philadelphia, PA 19104 Rosenbluth, Jack, New York University School of Medical, Department of Physiology and Biophysics, RR 714. 400 East 34ih Street. New York, NY 10016 Rosenbluth, Raja, Simon Fraser University, Institute of Molecular Biology and Biochemistry, Burnaby, BC V5A 1S6. Canada Rosenfield, Allan, Columbia University School of Public Health, 600 West 168th Street, New York, NY 10032-3702 Rosenkranz, Herbert S., 130 Desoto Street, Pittsburgh, PA 15213-2535 Ross, William N., New York Medical College, Department of Physiology, Valhalla, NY 10595 Roth, Jay S., 26 Huettner Road, P. O. Box 692, Woods Hole, MA 02543-0692 Rottenfusser, Rudi, Marine Biological Laboratory, 7 MBL Street, Woods Hole. MA 02543 Rowland, Lewis P., Neurological Institute. 710 West 168th Street, New York. NY 10032 Ruderman, Joan V., Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston. MA 021 15 Rummel, John D., NASA Headquarters, Office of Space Science, Washington, D.C. 20546 Rushforth, Norman B., Case Western Reserve University. Department of Biolo;jy, Ci.-veland, OH 44106 Russell-Hunter, V. illiam D., 711 Howard Street, Easton, MD 21601- 3934 Saffo, Mary Beth, AM ia State University West, Life Science Department, MC 2352. I'.O. Box 37100, Phoenix, AZ 85069-7100 Salama, Guy, University of Pittsburgh, Department of Physiology, Pittsburgh. PA 15261 Salmon, Edward D., University of North Carolina, Department of Biology. CB 3280. Chapel Hill, NC 27514 Salyers, Abigail, University of Illinois. Department of Microbiology, 407 South Goodwin Avenue, Urbana. IL 61801 Salzherg, Brian M., University of Pennsylvania School of Medicine, Department of Neuroscience, 215 Stemmler Hall. Philadelphia, PA 19104-6074 Sanger, Jean M., University of Pennsylvania School of Medicine. Department of Anatomy, 36th and Hamilton Walk, Philadelphia. PA 19104 Sanger, Joseph W., University of Pennsylvania Medical Center, Department of Cell and Developmental Biology. 36th and Hamilton Walk. Philadelphia, PA 19104-6058 Saunders, John W. Jr.. P.O. Box 3381, Waquoit. MA 02536 Schachman. Howard K., University of California. Molecular and Cell Biology Department, 229 Stanley Hall, #3206, Berkeley, CA 94720- 3206 Schatten, Gerald P., Oregon Health Sciences University, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue. Beaverton, OR 97006 Schatten, Heide, University of Wisconsin, Department of Zoology, Madison, WI 53706 Schmeer, Arlene C., Mercenene Cancer Research Institute, 790 Prospect Street, New Haven, CT 0651 1 Schuel. Herbert, SUNY at Buffalo. Department of Anatomy/Cell Biology, Buffalo, NY 14214 Schwartz, James H., New York State Psychiatric Institute, Research Annex, 722 West 168th Street, 7th floor. New York. NY 10032 Schwartz, Lawrence, University of Massachusetts. Department of Biology. Morrill Science Center. Amherst, MA 01003 Schweitzer, A. Nicola, Brigham and Women's Hospital. Immunology Division, Department of Pathology, 221 Longwood Avenue. LMRC 521. Boston. MA 02115 Segal. Sheldon J., The Population Council. One Dag Hammarskjold Plaza. New York, NY 10036 Senft, Stephen Lamont, Yale University, Neuroengineering/Neuroscience Center, P.O. Box 208205, New Haven. CT 06520-8205 Shanklin, Douglas R., University of Tennessee. Department of Pathology, Room 576. 800 Madison Avenue, Memphis. TN 381 17 Shashar, Nadav, The Interuniversity Institute of Eilat. P.O. Box 469, Eilat 88103. Israel Shashoua, Victor E., Harvard Medical School. Ralph Lowell Labs. McLean Hospital. I 15 Mill Street, Belmont. MA 02178 Shaver, Gaius R., Marine Biological Laboratory. The Ecosystems Center, Woods Hole, MA 02543 Shaver, John R., Michigan State University, Department of Zoology, East Lansing, MI 48824 Sheetz, Michael P., Duke University Medical Center, Department of Cell Biology, Bx 3709, 388 Nanaline Duke Building, Durham. NC 27710 Shepro, David, Boston University, CAS Biology, 5 Cummington Street, Boston, MA 02215 Shimomura, Osamu, Marine Biological Laboratory. 7 MBL Street, Woods Hole, MA 02543 Shipley, Alan M., P.O. Box 943. Forestdale. MA 02644 Silver, Robert B., Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543 Siwicki, Kathleen K., Swarthmore College, Biology Department, 500 College Avenue. Swarthmore, PA 19081-1397 Skinner. Dorothy M., 24 Gray Lane, Falmouth, MA 02540 Sloboda, Roger D., Dartmouth College. Department of Biological Science, 6044 Oilman, Hanover, NH 03755-1893 Sluder. Greenfield, University of Massachusetts Medical School, Room 324, 377 Plantation Street, Worcester, MA 01605 Members of the Corporation R79 Smith. Peter J.S.. Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Smith, Stephen J., Stanford University School of Medicine. Department of Molecular and Cellular Physiology. Beckman Center. Stanford. CA 94305 Smolowitz, Roxanna S., Marine Biological Laboratory, 7 MBL Street. Woods Hole. MA 02543 Sogin. Mitchell L., Marine Biological Laboratory. 7 MBL Street, Woods Hole. MA 02543 Sorenson, Martha M.. Cidade Universitaria-UFRJ. Department Bioquimica Medica-ICB, 21941-590 Rio de Janerio. Brazil Speck, \\illiam T.. Massachusetts General Hospital. 55 Fruit Street. Boston, MA 021 14 Spector, Abraham, Columbia University. Department of Ophthalmology. 630 West 168th Street, New York, NY 10032 Speksnijder, Johanna E., University of Groningen. Department of Genetics, Kerklaan 30, 975 1 NN Haren, The Netherlands Spray. David C., Albert Einstein College of Medicine. Department of Neuroscience, 1300 Moms Park Avenue, Bronx, NY 10461 Spring. Kenneth R., National Institutes of Health. 10 Center Drive. MSC 1598. Building 1(1. Room 6N260. Bethesda, MD 20892-1603 Steele, John H., Woods Hole Oceanographic Institution. Woods Hole. MA 02543 Steinacker, Antoinette, University of Puerto Rico, Instituet of Neurobiology. 201 Boulevard Del Valle. San Juan, PR 00901 Steinberg, Malcolm. Princeton University, Department of Molecular Biology, M-18 Moffett Laboratory, Princeton. NJ 08544-1014 Stemmer, Andreas C., Institut fur Robotik. ETH-Sentrum. 8092 Zurich. Switzerland Stenflo, Johan, University of Lund. Department of Clinical Chemistry, Malmo General Hospital. S-205 02 Malmo. Sweden Stetten, Jane Lazarow. 4701 Willurd Avenue. #1413, Chevy Chase, MD 20815-4627 Steudler. Paul A., Marine Biological Laboratory. The Ecosystems Center. Woods Hole. MA 02543 Stokes, Darrell R., Emory University. Department of Biology, 1510 Clifton Road NE. Atlanta, GA 30322-1 100 Stommel, Elijah W., Darmouth Hitchcock Medical Center, Neurology Department, Lebanon. NH 03756 Stracher, Alfred, SUNY Health Science Center. Department of Biochemistry, 450 Clarkson Avenue, Brooklyn, NY 1 1203 Strumwasser, Felix, P.O. Box 2278, East Falmouth, MA 02536-2278 Stuart, Ann E.. 1818 North Lakeshore Drive. Chapel Hill, NC 27514 Sugimori, Mutsuyuki, New York University Medical Center. Department of Physiology and Neuroscience, Room 442. 550 First Avenue, New York. NY 10016 Summers, William C., Western Washington University, Huxley College of Environmental Studies. Bellingham. WA 982259 1 8 1 Suprenant, Kathy A., University of Kansas. Department of Physiology and Cell Biology, 4010 Haworth Hall, Lawrence, KS 66045 Swenson, Katherine I., Duke University Medical Center, Department of Molecular Cancer Biology, Box 3686, Durham. NC 27710 Sydlik, Mary Anne, Hope College, Peale Science Center. 35 East 1 2th St./PO Box 9000, Holland, MI 49422 Szent-Gyorgyi, Andrew G., Brandeis University. Molecular and Cell Biology, 415 South Street, Waltham, MA 02454-91 10 Tamm, Sidney L., Boston University. CAS Biology, 5 Cummington Street, Boston, MA 022 1 5 Tanzer, Marvin L., University of Connecticut School of Dental Medicine, Department of Biostructure and Function, Farmington. CT 06030-3705 Tasaki, Ichiji, National Institutes of Health, NIMH. Laboratory of Neurobiology, Building 36, Room 2B-16, Bethesda, MD 20892 Taylor, D. Lansing, Carnegie Mellon University. Center for Flurorescence Research. 4400 Fifth Avenue. Pittsburgh, PA 15213 Taylor, Edwin W., University of Chicago. Department of Molecular Genetics. 920 E. 58th Street. Chicago. IL 60637 Teal, John M., Woods Hole Oceanographic Institution. Department of Biology. Woods Hole. MA 02543 Telfer, William H., University of Pennsylvania, Department of Biology, Philadelphia. PA I1) 1 04 Telzer, Bruce, Pomona College. Department of Biology. Thille Building. 175 West bin Street. Claremont. CA 91711 Terasaki. Mark, University of Connecticut Health Center. Department of Physiology. 263 Farmington Avenue, Farmington. CT 06032 Townsel, James G., Meharry Medical College. Department of Physiology. 1005 DB Todd Boulevard. Nashville. TN 37208 Travis, David M., 19 High Street. Woods Hole, MA 02543-1221 Treistman, Steven N., University of Massachusetts Medical Center, Department of Pharmacology, 55 Lake Avenue North, Worcester, MA 01655 Trigg. D. Thomas, One Federal Street, 9th Floor. Boston. MA 022 1 1 Troll, \Valter, NYU Medical Center, Department of Environmental Medicine. 550 First Avenue. New York, NY 10016 Troxler, Robert F., Boston University School of Medicine. Department of Biochemistry. 80 East Concord Street. Boston. MA 021 18 Tucker, Edward B., Baruch College. CUNY. Department of Natural Sciences, 17 Lexington Avenue, New York, NY 10010 Turner, Ruth D., Harvard University. Museum of Comparative Zoology. Mollusk Department, Cambridge. MA 02138 Tweedell, Kenyon S., University of Notre Dame. Department of Biological Sciences. Notre Dame. IN 46556-0369 Tykocinski, Mark L., Case Western Reserve University, Institute of Pathology, 2085 Adelbert Road. Cleveland, OH 44106 Tytell, Michael, Wake Forest University. Bowman Gray School of Medicine. Department of Anatomy and Neurobiology. Winston- Salem. NC 27157 Ueno, I In osln. Kyoto University, AGR Chemistry, Faculty of Agriculture, Sakyo, Kyoto 606-8502, Japan Valiela, Ivan, Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 Vallee, Richard, University of Massachusetts Medical Center. Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury. MA 01545 Valois, John J., 420 Woods Hole Road, Woods Hole. MA 02543 Van Dover, Cindy Lee, The College of William and Mary, Biology Department, 328 Millington Hall, Williamsburg, VA 23187 Van Holde, Kensal E., Oregon State University, Biochemistry and Biophysics Department, Corvallis, OR 97331-7503 Vogl, Thomas P., Environmental Research Institute of Michigan, 1101 Wilson Boulevard. Arlington. VA 22209 Waimvright, Norman R., Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543 Waksman, Byron H., New York University Medical Center, Department of Pathology, 550 First Avenue. New York. NY 10016 Wall, Betty, 9 George Street. Woods Hole, MA 02543 Wang, Hsien-Yu, State University of New York, University Medical Center. Physiology and Biophysics-HSC. Stony Brook. NY 1 1794- 8633 Wangh, Lawrence J., Brandeis University. Department of Biology, 415 South Street. Waltham. MA 02254 Warner, Robert C., 1609 Temple Hills Drive. Laguna Beach. CA 9265 1 R80 Annual Report Warren, Leonard, Wistar Institute. 36th and Spruce Streets, Philadelphia. PA 19104 Waterbury, John B., Woods Hole Oceanographic Institution, Department of Biology. Woods Hole, MA 02543 Waxman, Stephen G., Yale Medical School. Neurology Department, 333 Cedar Street. P.O. Box 208018. New Haven. CT 06510 Webb, H. Marguerite, 184 Chestnut Street. Foxhoro. MA 02035-1548 (deceased 1999) Weber, Annemarie, University of Pennsylvania School of Medicine. Department of Biochemistry and Biophysics. Philadelphia, PA 19066 Weeks, Janis C., University of Oregon, Institute of Neuroscience. Eugene. OR 97403-1254 Weidner, Earl, Louisiana State University, Department of Biological Sciences, 508 Life Sciences Building, Baton Rouge, LA 70803-1715 Weiss, Alice Sara, 105 University Boulevard West. Silver Spring. MD 20901 Weiss, Dieter G., University of Rostock. Institute of Zoology. D- 18051 Rostock. Germany Weiss, Leon P., University of Pennsylvania School of Veterinary Medicine, Department of Animal Biology. Philadelphia, PA 19104 Weiss, Marisa C., Paoli Memorial Hospital, Department of Radiation Oncology. 255 W. Lancaster Avenue. Paoli. PA 19301 Weissmann, Gerald, New York University Medical Center, Department of Medicine/Division Rheumatology, 550 First Avenue, New York, NY 10016 Westerfield, Monte, University of Oregon. Institute of Neuroscience, Eugene, OR 97403 Whittaker, J. Richard, University of New Brunswick. Department of Biology, BS 4511, Frederiction, NB E3B 6E1. Canada Wiesel, Torsten N., Rockefeller University, 1230 York Avenue. New York. NY 10021 Wilkens, Lon A., University of Missouri, Department of Biology, 8001 Natural Bridge Road, St. Louis, MO 63121-4499 Wilson, Darcy B., Torrey Pines Institute, 3550 General Atomics Court, Building 2, Room 138, San Diego, C A 92121 Wilson, T. Hastings, Harvard Medical School, Department of Physiology, 25 Shattuck Street, Boston. MA 021 15 Witkovsky, Paul. NYLI Medical Center, Department of Ophthalmology, 550 First Avenue, New York. NY 10(116 MBL Associates Wittenberg, Beatrice, Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, NY 10461 Wittenberg. Jonathan B., Albert Einstein College of Medicine, Department of Physiology and Biophysics. Bronx, NY 10461 Wolken, Jerome J., Carnegie Mellon University. Department of Biological Sciences, 440 Fifth Avenue. Pittsburgh, PA 15213 (deceased 1999) Wonderlin, William ¥., West Virginia University, Pharmacology and Toxicology Department, Morgantown, WV 26506 Worden, Mary Kate, University of Virginia, Department of Neuroscience. McKim Hall Box 230, Charlottesville, VA 22908 Worgul, Basil V., Columbia University, Department of Ophthalmology, 630 West 168 Street, New York. NY 10032 Wu, Chau Hsiung, Northwestern University Medical School, Department of Pharmacology (S215), 303 East Chicago Avenue, Chicago. IL 60611-3008 Wyttenbach, Charles R., University of Kansas, Biological Sciences Department. 2045 Haworth Hall. Lawrence. KS 66045-2106 Yen, Jay Z., Northwestern University Medical School, Department of Pharmacology, Chicago. IL 60611 Zacks, Sumner I., 65 Saconesset Road, Falmouth, MA 02540-1851 (deceased 2000) Zakon, Harold H., University of Texas, Section of Neurobiology, School of Life Science, Austin. TX 78712 Zigman, Seymour, Marine Park Condominiums. 174 Queen Street. Unit 10-F. Falmouth. MA 02540 Zigmond, Michael J., University of Pittsburgh, S-526 Biomedical Science Tower, 3500 Terrace Street, Pittsburgh. PA 15213 Zimmerherg, Joshua J., National Institutes of Health, LCMB. NICHD, Building 10. Room 10D14, 10 Center Drive, Bethesda. MD 20892 Zottoli, Steven J., Williams College. Department of Biology, Williamstown, MA 01267 Zucker, Robert S., University of California. Neurohiology Division. Molecular and Cellular Biology Department, Berkeley, CA 94720 Executive Board Ruth Ann Laster. President Jack Pearce. Vice President Kitty Brown. Treasurer Molly Cornell, Secretary Elizabeth Farnham, Membership Chair Tammy Smith Amon Duncan Aspinwall Barbara Atwood Kitty Brown Julie Child Seymour Cohen Michael Fenlon Sallie Giffen Alice Knowles Rebecca Lash Cornelia Hanna McMurlne Joan Pearl man Virginia R. Reynolds Volker Ulbrich John Valois Associates Liaison/Gift Sliop Coordinator Kendall B. Bohr Supporting Associate Mrs. George H. A. Clowes Dr. and Mrs. James D. Ebert Mr. and Mrs. David Fausch Mrs. Janet F. Gillette Drs. Luigi and Elaine Mastroianni Ms. Linda Sallop and Mr. Michael Fenlon Mrs. Anne W. Sawyer Dr. John Tochko and Mrs. Christina Myles-Tochko Mr. and Mrs. John J. Valois Mr. and Mrs. Leslie J. Wilson Sustaining Associate Dr. and Mrs. James J. Ferguson. Jr. Mr. and Mrs. Lon Hocker Members of the Corporation R81 Mr. Robert A. Jaye Dr. and Mrs. Edward F. MacNichol. Jr. Fainilv Membership Dr. Frederick W. Ackroyd Dr. and Mrs. Edward A. Adelberg Dr. and Mrs. Dean C. Allard, Jr. Mr. and Mrs. Douglas F. Allison Drs. Peggy and Fred Alsup Drs. James and Helene Anderson Dr. and Mrs. Samuel C. Armstrong Mr. and Mrs. Duncan P. Aspinwall Mr. and Mrs. Donald R. Aukamp Mr. and Mrs. John M. Baitsell Mr. and Mrs. David Bakalar Mr. and Mrs. William L. Banks Dr. and Mrs. Robert B. Barlow, Jr. Mr. and Mrs. John E. Barnes Dr. and Mrs. Robert M. Berne Drs. Harriet and Alan Bernheimer Mr. and Mrs. Robert O. Bigelow Dr. and Mrs. Edward G. Boettiger Mr. and Mrs. Kendall B. Bohr Dr. and Mrs. Thomas A. Borgese Mr. and Mrs. Richard M. Bowen Dr. and Mrs. Francis P. Bowles Dr. and Mrs. John B. Buck Dr. and Mrs. John E. Burns Mr. and Mrs. William O. Burwell Mr. and Mrs. G. Nathan Calkins, Jr. Mr. and Mrs. D. Bret Carlson Prof, and Mrs. James F. Case Dr. and Mrs. Richard L. Chappell Dr. and Mrs. Frank M. Child Mr. and Mrs. Thomas Claflin Dr. and Mrs. Arnold M. Clark Mrs. LeRoy Clark Mr. and Mrs. James Cleary Dr. and Mrs. Laurence P. Cloud Mr. and Mrs. Harrington T. Collins Dr. and Mrs. Neal W. Cornell (Dr. Cornell deceased, 2000) Mr. and Mrs. Norman C. Cross Dr. and Mrs. John M. Cummings Mr. and Mrs. Bruce G. Daniels Mr. and Mrs. Joel P. Davis Mr. and Mrs. Richard C. Dierker Mr. and Mrs. F. Gerald Douglass Dr. and Mrs. Arthur Brooks DuBois Dr. and Mrs. Michael J. Fishbein Mr. and Mrs. Harold Frank Mr. and Mrs. Howard G. Freeman Dr. and Mrs. Robert A. Frosch Dr. and Mrs. John J. Funkhouser Dr. and Mrs. Mordecai L. Gabriel Dr. and Mrs. Sydney Gellis Dr. and Mrs. James L. German. Ill Dr. and Mrs. Harold S. Ginsberg Dr. and Mrs. Murray Glusman Drs. Alfred and Joan Goldberg Mrs. Mary L. Goldman Mr. and Mrs. Charles Goodwin, III Dr. and Mrs. Philip Grant Mr. and Mrs. Anthony D. Green Dr. and Mrs. Thomas C. Gregg Dr. Newton H. Gresser Dr. and Mrs. Antoine F. O. Hadamard Mr. and Mrs. Peter A. Hall Dr. and Mrs. Harlyn O. Halvorson Drs. Alexander and Carol Hannenberg Dr. and Mrs. Richard Bennet Harvey Dr. and Mrs. J. Woodland Hastings Mr. and Mrs. Gary G. Hayward Dr. and Mrs. Howard H. Hiatt Mr. and Mrs. David Hibbitt Dr. and Mrs. John E. Hobbie Drs. Francis C. G. Hoskin and Elizabeth M. Farnham Dr. and Mrs. Robert J. Huettner Dr. and Mrs. Shinya Inoue Dr. and Mrs. Kurt J. Isselbacher Dr. and Mrs. Gary Jacobson Mrs. Mary D. Janney Mr. and Mrs. DeWitt C. Jones. Ill Dr. and Mrs. Benjamin Kanuner Mr. and Mrs. Paul W. Knaplund Mr. and Mrs. A. Sidney Knowles. Jr. Sir and Lady Hans Kornberg Dr. and Mrs. S. Andrew Kulin Mr. and Mrs. Trevor Lambert Dr. and Mrs. George M. Langford Dr. and Mrs. Leonard Laster Dr. and Mrs. Hans Laufer Mr. William Lawrence and Mrs. Barbara Buchanan Dr. and Mrs. Berton J. Leach Mr. and Mrs. Stephen R. Levy Mr. and Mrs. Robert Livingstone. Jr. Dr. and Mrs. Laszlo Lorand Mr. and Dr. Bernard Manuel Mr. and Mrs. Joseph C. Martyna Mr. and Mrs. Frank J. Mather. Ill Mr. and Mrs. John E. Matthews Dr. and Mrs. Robert T. McCluskey Mr. Paul McGonigle Dr. and Mrs. Jerry M. Melillo Mr. and Mrs. Wesley J. Merritt Mr. and Mrs. Richard Meyers Mr. and Mrs. Charles A. Mitchell Dr. and Mrs. Merle Mi/ell Dr. and Mrs. Charles H. Montgomery Mr. and Mrs. Stephen A. Moore Mr. and Mrs. Charles F. Murphy Dr. and Mrs. John E. Naugle Dr. Pamela Nelson and Mr. Christopher Olmsted Mr. and Mrs. Frank L. Nickerson Dr. and Mrs. Clifford T. O'Connell Mr. and Mrs. James J. O'Connor Mr. and Mrs. David R. Palmer Mr. and Mrs. Robert Parkinson Mr. and Mrs. Richard M. Paulson. Jr. Dr. and Mrs. John B. Pearce Mr. and Mrs. William J. Pechilis Mr. and Mrs. John B. Pen Dr. and Mrs. Courtland D. Perkins Dr. and Mrs. Philip Person Mr. and Mrs. Frederick S. Peters Mr. and Mrs. George H. Plough Dr. and Mrs. Aubrey Pothier, Jr. Dr. and Mrs. Carl A. Price Mr. and Mrs. Allan Putnam Dr. and Mrs. Lionel I. Rebhun Dr. and Mrs. George T. Reynolds Dr. and Mrs. Harris Ripps Ms. Jean Roberts Drs. Priscilla and John Roslansky Mr. and Mrs. John D. Ross Dr. and Mrs. John W. Saunders, Jr. Dr. and Mrs. R. Walter Schlesinger Mr. and Mrs. Harold H. Sears Mr. John Seder and Ms. Frances Plough Dr. and Mrs. Sheldon J. Segal Dr. and Mrs. David Shepro Mr. and Mrs. Bertram R. Silver Mr. and Mrs. Jonathan O. Simonds Drs. Frederick and Marguerite Smith Dr. and Mrs. William K. Stephenson Mr. and Mrs. E. Kent Swift, Jr. Mr. and Mrs. Gerard L. Swope, III Mr. Norman N. Tolkan Dr. and Mrs. Walter Troll Mr. and Mrs. Volker Ulbrich Ms. Susan Veeder Drs. Claude and Dorothy Villee Dr. and Mrs. Gerald Weissmann Dr. and Mrs. Paul S. Wheeler Dr. Martin Keister White Mr. and Mrs. Geoffrey G. Whitney, Jr. Mr. and Mrs. Lynn H. Wilke Dr. and Mrs. T. Hastings Wilson Mr. and Mrs. Dick Yeo Dr. and Mrs. Sumner I. Zacks (Dr. Zacks deceased. 2000) Dr. Linda and Mr. Erik Zettler Individual Membership Mrs. Jean E. Ahearn Mr. Henry Albers Dr. Nina S. Allen Mrs. Tammy Amon Mr. Dean N. Arden Mrs. Ellen Prosser Armstrong Mrs. Kimball C. Atwood. Ill Dr. Serena Baccetti Mr. Everett E. Bagley Dr. Millicent Bell Mr. C. John Berg Dr. Thomas P. Bleck Ms. Avis Blomberg Mr. Theodore A. Bonn Mr. James V. Bracchitta Mrs. Jennie P. Brown Mrs. M. Kathryn S. Brown Dr. Robert H. Broyles Ms. Henriete Bull Dr. Alan H. Burghauser Mrs. Barbara Gates Burwell Mr. Bruce E. Buxton Mr. Patrick J. Calie R82 Annual Report Mrs. Anne M. Campbell Dr. Graciela C. Candelas Mr. Frank C. Camtenuto Dr. Roben H. Carrier Mrs. Patricia A. Case Ms. Mia D. Champion Dr. Sallie Chisholm Mrs. Octavia C. Clement Mr. Allen W. Clowes Mrs. Margaret H. Cohurn Dr. Seymour S. Cohen Ms. Anne S. Concannon Prof. D. Eugene Copeland Dr. Vincent Cowling Mrs. Marilyn E. Crandall Ms. Dorothy Crossley Ms. Helen M. Crossley Mrs. Villa B. Crowell Mr. Norman Dana Dr. Morton Davidson Mrs. Elizabeth M. Davis Ms. Carol Reimann DeYoung Mrs. Shirley Dierolf Mr. David L. Donovan Ms. Su/.anne Droban Mr. Roy A. Duffus Mrs. Charles Eastman Dr. Frank Egloff Mr. Raymond Eliott Ms. Judy Ernst Mrs. Ruth Alice Fitz Mr. Robert Fitzpatrick Ms. Sylvia M. Flanagan Mr. John W. Folino, Jr. Dr. Krystyna Frenkel Mr. Paul J. Freyheit Mrs. Ruth E. Fye Mr. Joseph C. Gallagher Miss Eleanor Garfield Mrs. Ruth H. Garland Mr. John Garnett Ms. Sallie A. Giffen Mr. Charles Gifford Mrs. James R. Glazebrook Mr. Michael P. Goldring Mrs. Phyllis Goldstein Mrs. DeWitt S. Goodman Ms. Muriel Gould Mrs. Rose Grant Mrs. Jeanne B. Griffith Mrs. Valerie A. Hall Dr. Peter J. Hamre Ms. Mary Eli/.aheth Hamstrom Ms. Elizabeth E. Hathaway Dr. Robert R. Haiilun h Mrs. Jane M. Heakl Mrs. Nathan Hir.schfeld Mr. Roger W. Hubhell Miss Elizabeth B. Jackson Mr. Raymond L. Jewett Mrs. Barbara W. Jones Mrs. Megan H. Jones Mrs. Joan T. Kanwisher Mrs. Sally Karush Mrs. Marcella Katz Ms. Patricia E. Keoughan Dr. Peter N. Kivy Dr. Bruno P. Kremer Mr. Bernard H. Labitt Mrs. Janet W. Larcom Dr. Marian E. LeFevre Dr. Mortimer Levitz Mr. Edwin M. Libbin Mr. Lennart Lindberg Mrs. Barbara C. Little Mrs. Sarah J. Loessel Mrs. Ermine W. Lovell Mr. Richard C. Levering Mrs. Victoria H. Lowell Mrs. Margaret M. MacLeish Ms. Anne Camille Maher Mrs. Annemarie E. Mahler Mr. Patrick J. Mahoney Dr. Saul Malkiel Ms. Diane Maranchie Mr. Daniel R. Martin Dr. Miriam Jacob Mauzerall Mrs. Mary Hartwell Mavor Mrs. Nella W. McElroy (deceased 1999) Dr. Susan Gerbi Mcllwam Ms. Mary W. McKoan Ms. Jane A. McLaughlin Ms. Louise McManus Ms. Cornelia Hanna McMurtrie Mrs. Ellen L. Meigs Mr. Ted Melillo Martin Mendelson. M.D.. Ph.D. Ms. Carmen Merryman Mrs. Grace S. Metz Mrs. Mary G. Miles Mrs. Florence E. Mixer Mr. John T. Moakley Mr. Lawrence A. Monte Mrs. Mary E. Montgomery Ms. Cynthia Moor Mr. Alan F. Morrison Dr. M. Patricia Morse Mrs. Eleanor M. Nace Mr. William G. Neall Mrs. Anne Nelson Mr. Edmund F. Nolan Ms. Catherine N. Norton Mr. John J. O'Connor (deceased 1999) Dr. Renee Bennett O'Sullivan Miss Carolyn L. Parmenter Mrs. Dolores Patch-Wing Ms. Joan Pearlman Mr. Raymond W. Peterson Ms. Victoria A. Powell Ms. Elizabeth T. Price Ms. Dianne Purves Mrs. Julia S. Rankin Mr. Fred J. Ravens. Jr. Ms. Anecia Kathy Regis Ms. Mary W. Rianhard Dr. Renato A. Ricca (deceased 1<> members, the Chairperson of the Board, the Treasurer and the Chairperson of the Audit Committee, together with such Trustees as may be required for not less than two-thirds of the Investment Committee to consist of Trustees. Except as otherwise provided by these Bylaws or determined by the Trustees, any such committee may make rules for the conduct of its business, but, unless otherwise provided by the Trustees 01 in such rules, its business shall be conducted as nearly as possible in the same manner as is provided by these Bylaws for the Trustees. F. Actions Without a Meeting. Any action required or permitted to be taken at any meeting of the Executive Committee or any other committee elected by the Trustees may be taken without a meeting if all members of such committees consent to the action in writing and such written consents are filed with the records of meetings. Members of the Executive Committee or any other committee elected by the Trustees may also participate in any meeting by means of a telephone conference call, or otherwise take action in such a manner as may. from time to time, be permitted by law. G. Manual of Procedures. The Board of Trustees, on the recommendation of the Executive Committee, shall establish guidelines and modifications thereof to be recorded in a Manual of Procedures. Guidelines shall establish procedures for: ( 1 ) Nomination and election of members of the Corporation, Board of Trustees and Executive Committee; (2) Election of Officers; (3) Formation and Function of Standing Committees. ARTICLE VI— OFFICERS A Enumeration- The officers of the Corporation shall consist of a President, a Treasurer and a Clerk, and such other officers having the powers of President, Treasurer and Clerk as the Board may determine, and a Director of the Laboratory The Corporation may have such other officers and assistant officers as the Board may determine, including (without limitation) a Chairperson of the Board, Vice Chairper- son and one or more Vice Presidents, Assistant Treasurers or Assistant Clerks. Any two or more offices may be held by the same person. The Chairperson and Vice Chan-person of the Board shall be elected by and from the Trustees, but other officers of the Corporation need not be Trustees or Members. If required by the Trustees, any officer shall give the Corporation a bond for the faithful performance of his or her duties in such amount and with such surety or sureties as shall be satisfactory to the Trustees. B. Tenure. Except as otherwise provided by law, by the Articles of Organization or by these Bylaws, the President, Treasurer, and all other officers shall hold office until the first meeting of the Board following the annual meeting of Members and thereafter, until his or her successor is chosen and qualified. C. Resignation. Any officer may resign by delivering his or her written resignation to the Corporation at its principal office or to the President or Clerk and such resignation shall be effective upon receipt unless it is specified to be effective at some other time or upon the happening of some other event. D. Removal. The Board may remove any officer with or without cause by a vote of a majority of the entire number of Trustees then in office, at a meeting of the Board called for that purpose and for which notice of the purpose thereof has been given, provided that an officer may be removed for cause only after having an opportunity to be heard by the Board at a meeting of the Board at which a quorum is personally present and voting. E. Vacancy. A vacancy in any office may be filled for the unexpired balance of the term by vote of a majority of the Trustees present at any meeting of Trustees at which a quorum is present or by written consent of all of the Trustees, if less than a quorum of Trustees shall remain in office. F. Chairperson. The Chairperson shall have such powers and duties as may be determined by the Board and, unless otherwise determined by the Board, shall serve in that capacity for a term coterminous with his or her term as Trustee. G. Vice Chairperson. The Vice Chairperson shall perform the duties and exercise the powers of the Chairperson in the absence or disability of the Chairperson, and shall perform such other duties and possess such other powers as may be determined by the Board. Unless otherwise determined by the Board, the Vice Chairperson shall serve for a one-year term. H. Director. The Director shall be the chief operating officer and. unless otherwise voted by the Trustees, the chief executive officer of the Corporation. The Director shall, subject to the direction of the Trustees, have general supervision of the Laboratory and control ot the business of the Corporation. At the annual meeting, the Director shall submit a report of the operations of the Corporation for such year and a statement of its affairs, and shall, from time to time, report to the Board all matters within his or her knowledge which the interests of the Corporation may require to be brought to its notice. I. Deputy- Dirt-dor. The Deputy Director, if any, or if there shall be more than one. the Deputy Directors in the order determined by the Trustees, shall, in the absence or disability of the Director, perform the duties and exercise the powers of the Director and shall perform such other duties and shall have such other powers as the Trustees may. from time to time, prescribe. J. President. The President shall have the powers and duties as may be vested in him or her by the Board. K, Treasurer and Assistant Treasurer. The Treasurer shall, subject to the direction of the Trustees, have general charge of the financial affairs of the Corporation, Bylaws of the Corporation R87 including its long-range financial planning, and shall cause to be kept accurate books of account. The Treasurer shall prepare a yearly report on the financial status of the Corporation to be delivered al the annual meeting. The Treasurer shall also prepare or oversee all filings required by the Commonwealth of Massachusetts, the Internal Revenue Service, or other Federal and State Agencies. The account of the Treasurer shall be audited annually by a certified public accountant The Assistant Treasurer, if any, or if there shall be more than one, the Assistant Treasurers in the order determined by the Trustees, shall, in the absence or disability of the Treasurer, perform the duties and exercise the powers of the Treasurer, shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. L. Clerk and Assistant Clerk. The Clerk shall be a resident of the Commonwealth of Massachusetts, unless the Corporation has designated a resident agent in the manner provided by law. The minutes or records of all meetings of the Trustees and Members shall be kept by the Clerk who shall record, upon the record books of the Corporation, minutes of the proceedings at such meetings. He or she shall have custody of the record books of the Corporation and shall have such other powers and shall perform such other duties as the Trustees may, from time to time, prescribe. The Assistant Clerk, if any, or if there shall be more than one, the Assistant Clerks in the order determined by the Trustees, shall, in the absence or disability of the Clerk, perform the duties and exercise the powers of the Clerk and shall perform such other duties and shall have such other powers as the Trustees may, from time to time, prescribe. In the absence of the Clerk and an Assistant Clerk from any meeting, a temporary Clerk shall be appointed al the meeting. M. Other Powers and Duties. Each officer shall have in addition to the duties and powers specifically set forth in these Bylaws, such duties and powers as are custom- arily incident to his or her office, and such duties and powers as the Trustees may, from time to time, designate. ARTICLE VII— AMENDMENTS These Bylaws may be amended by the affirmative vote of the Members at any meeting, provided that notice of the substance of the proposed amendment is stated in the notice of such meeting. As authorized by the Articles of Organization, the Trustees, by a majority of their number then in office, may also make, amend or repeal these Bylaws, in whole or in part, except with respect to (a) the provisions of these Bylaws governing (i) the removal of Trustees and (ii) the amendment of these Bylaws and (b) any provisions of these Bylaws which by law, the Articles of Organization or these Bylaws, requires action by the Members. No later than the time of giving notice of meeting of Members next following the making, amending or repealing by the Trustees of any Bylaw, notice thereof stating the substance of such change shall be given to all Members entitled to vote on amending the Bylaws. Any Bylaw adopted by the Trustees may be amended or repealed by the Members entitled to vote on amending the Bylaws. ARTICLE VIII— INDEMNITY Except as otherwise provided below, the Corporation shall, to the extent legally permissible, indemnify each person who is, or shall have been, a Trustee, director or officer of the Corporation or who is serving, or shall have served at the request of the Corporation as a Trustee, director or officer of another organization in which the Corporation directly or indirectly has any interest as a shareholder, creditor or otherwise, against all liabilities and expenses (including judgments, fines, penalties, and reasonable attorneys' fees and all amounts paid, other than to the Corporation or such other organization, in compromise or settlement) imposed upon or incurred by any such person in connection with, or arising out of, the defense or disposition of any action, suit or other proceeding, whether civil or criminal, in which he or she may be a defendant or with which he or she may be threatened or otherwise involved, directly or indirectly, by reason of his or her being or having been such a Trustee, director or officer. The Corporation shall provide no indemnification with respect to any matter as to which any such Trustee, director or officer shall be finally adjudicated in such action, suit or proceeding not to have acted in good faith in the reasonable belief that his or her action was in the best interests of the Corporation. The Corporation shall provide no indemnification with respect to any matter settled or comprised unless such matter shall have been approved as in the best interests of the Corporation, after notice that indemnification is involved, by (i) a disinterested majority of the Board of the Executive Committee, or (ii) a majority of the Members. Indemnification may include payment by the Corporation of expenses in defending a civil or criminal action or proceeding in advance of the final disposition of such action or proceeding upon receipt of an undertaking by the person indemnified to repay such payment if it is ultimately determined that such person is not entitled to indemnification under the provisions of this Article VIII. or under any applicable law As used in the Article VIII, the terms "Trustee," "director," and "officer" include their respective heirs, executors, administrators and legal representatives, and an "interested" Trustee, director or officer is one against whom in such capacity the proceeding in question or another proceeding on the same or similar grounds is then pending. To assure indemnification under this Article VIII of all persons who are determined by the Corporation or otherwise to be or to have been "fiduciaries" of any employee benefits plan of the Corporation which may exist, from time to time, this Article VIII shall be interpreted as follows: (i) "another organization" shall be deemed to include such an employee benefit plan, including without limitation, any plan of the Corpo- ration which is governed by the Act of Congress entitled "Employee Retirement Income Security Act of 1974," as amended, from time to time, ("ERISA"); (ii) "Trustee" shall be deemed to include any person requested by the Corporation to serve as such for an employee benefit plan where the performance by such person of his or her duties to the Corporation also imposes duties on. or otherwise involves services by, such person to the plan or participants or beneficiaries of the plan; (iii) "fines" shall be deemed to include any excise tax plan pursuant to ERISA; and (iv) actions taken or omitted by a person with respect to an employee benefit plan in the performance of such person's duties tor a purpose reasonably believed by such person to be in the interest of the participants and beneficiaries of the plan shall be deemed to be for a purpose which is in the best interests of the Corporation. The right of indemnification provided in this Article VIII shall not be exclusive of or affect any other rights to which any Trustee, director or officer may be entitled under any agreement, statute, vote of Members or otherwise. The Corporation's obligation to provide indemnification under this Article VIII shall be offset to the extent of any other source of indemnification of any otherwise applicable insurance coverage under a policy maintained by the Corporation or any other person. Nothing contained in the Article shall affect any rights to which employees and corporate personnel other than Trustees, directors or officers may be entitled by contract, by vote of the Board or of the Executive Committee or otherwise. ARTICLE IX— DISSOLUTION The consent of every Trustee shall be necessary to effect a dissolution of the Marine Biological Laboratory. In case of dissolution, the property shall be disposed of in such a manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Trustees then in office in accordance with the laws of the Com- monwealth of Massachusetts. ARTICLE X— MISCELLANEOUS PROVISIONS A. Fiscal Year. Except as otherwise determined by the Trustees, the fiscal year of the Corporation shall end on December 31st of each year. B. Seal. Unless otherwise determined by the Trustees, the Corporation may have a seal in such form as the Trustees may determine, from time to time. C. Execution of Instruments. All checks, deeds, leases, transfers, contracts, bonds, notes and other obligations authorized to be executed by an officer of the Corporation in its behalf shall be signed by the Director or the Treasurer except as the Trustees may generally or in particular cases otherwise determine. A certificate by the Clerk or an Assistant Clerk, or a temporary Clerk, as to any action taken by the Members. Board of Trustees or any officer or representative of the Corporation shall as to all persons who rely thereon in good faith be conclusive evidence of such action. D. Corporate Records. The original, or attested copies, of the Articles of Organi- zation, Bylaws and records of all meetings of the Members shall be kept in Massa- chusetts at the principal office of the Corporation, or at an office of the Corporation's Clerk or resident agent. Said copies and records need not all be kept in the same office. They shall be available at all reasonable times for inspection by any Member for any proper purpose, but not to secure a list of Members for a purpose other than in the interest of the applicant, as a Member, relative to the affairs of the Corporation. E. Articles of Organization. All references in these Bylaws to the Articles of Organization shall be deemed to refer to the Articles of Organization of the Corpo- ration, as amended and in effect, from time to time. F. Transactions with Interested Parties. In the absence of fraud, no contract or other transaction between this Corporation and any other corporation or any firm, association, partnership or person shall be affected or invalidated by the fact that any Trustee or officer of this Corporation is pecuniarily or otherwise interested in or is a director, member or officer of such other corporation or of such firm, association or partnership or in a party to or is pecuniarily or otherwise interested in such contract or other transaction or is in any way connected with any person or person, firm, association, partnership, or corporation pecuniarily or otherwise interested therein; provided that the fact that he or she individ- ually or as a director, member or officer of such corporation, firm, association or R88 Annual Report partnership in such a party or is so interested shall be disclosed to or shall have been authorizing any such contract or transaction with like force and effect as if he/she were not known by the Board of Trustees or a majority of such Members thereof as shall be present so interested, or were not a director, member or officer of such other corporation, firm, at a meeting of the Board of Trustees at which action upon any such contract or association or partnership, provided that any vote with respect to such contract or transaction shall be taken; any Trustee may be counted in determining the existence of a transaction must be adopted by a majority of the Trustees then in office who have no quorum and may vote at any meeting of the Board of Trustees for the purpose of interest in such contract or transaction. 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Hooke: 43 years later, the compound microscope reveals living things are composed of cells. Leeuwenhoek: invents the first practical microscope and discovers bacteria. Armed with the knowledge and inspiration of these and other Olympians, secure in the history of our own accomplishments and aware that the mind's eye should always focus on challenging the enigmatic as well as the undeniable, Olympus has created the BX2 series of clinical and research microscopes, noteworthy for their enhancements in optics, imaging and ergonomics. And each so individual, it deserves the nomenclature My Microscope. From possibility to actuality, it is wonderful to behold. A posse ad esse est mirabile visu. TheBX4l The new standard *"»* ^ in laboratory r microscopy vis-a-vis " ; '. ' performance, ver- satility, operation. Uncanny digitized imaging: enhanced with the DP11 digital camera, delivering high-resolution image recording beyond the nonn. Unusually i optics: obse magnificatic 1.25x to 10— without changing the condenser. Choice of condenser includes Abbe, swing . out, pnase-c' and darkfiel Unerringly precise- motion stage: selectable for right/ left hand. Disrnvpr speed, ease, comfort, am accuracy. Uncommonly easy-to-operate controls: extended fine-focus knob; •warded-positioned illumination intensity dial; tactile X-Y control. Welcome to the n Series The new standard in ergonomic microscopy. A dramatic metamor- phosis: innova- <£?$ tive, intelligent, insightful design improvements specifically addressing the needs of those spending hours looking through a microscope. The lowest low-position stage: three inches lower than standard microscopes, offering the promise of minimum effort and hand movement when changing specimens. The tilting, telescoping observation tube: a marvel of adjustability, it can be changed to accommodate any '" ""mm higher d 70mm closer to the operator than a convention- al tilting tube. 1 And the eye- re J forward by 45mm . from 0-25 degrees. The new stan- dard in research microscopy. The words "ne^ and "standard" really don't do it justice. How about "ingenious" or "original," "paragon" or "prototypical." Prepare yourself for a higher power. The 6-position filter cube turret: Not 4, not 5, but 6, so single and multi- band imaging of new fluorochromes and fluorescent proteins is faster, simpler and exclusively tailored. The rectangular field stop: ours alone. By cleverly matching the camera's field of view, only the area requiring fluorescence excita- tion is exposed. Which means that the surrounding areas are protected from photo bleaching. The fluorescence excitation bal- ancers: another Olympus exclusive. A continuously variable excitation bandwidth. Visualization is enhanced, differentiation of multi-labels is immeasurably improved, capturing of images is at an unheard-of level. Triple Labeled Specimen (1 to r): nt FITC with prevalent DAPI and Cy£ • with balanced fluorescence emission wider mirror aspnencai lens The aspherical collector lens: sui generis, in a class by itself. Specimens appear brighter and more evenly illuminated; and achromatic performance now incorporates wavelengths from UV to IR. The universal ^ condenser: with 8 positions, it can accept optical inserts for DIG, phase, brightfield and dark- field illumination. And the advanced Nomarski DIC system has been expanded and optimized to encompass prisms for enhanced contrast and image resolution. OLYMPUS FOCUS ON LIFE Visit us at www.olympusamericacom or call 1-800-455-8236 © Olympus America Inc. THE BIOLOGICAL BULLETIN OCTOBER 2000 Editor Associate Editors Section Editor Online Editors Editorial Board Editorial Office MICHAEL J. GREENBERG Louis E. BURNETT R. 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Santa Barbara University of California, Los Angeles Managing Editor Staff Editor Editorial Associate Subscription & Advertising Secretary Published by MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS »ne Biological Laboratory ' .'3phicinsi,:tuiio,i Library NOV Wooc': 2000 Cover Fission yeast (Saccharomyces pombe) — in contrast to budding yeast — is a rod-shaped cell that divides by cleaving medially. The nucleus is located at the geometric center of the cell, where it is attached to multiple bundles of dynamic microtubules that push on it. As shown on the cover, both the nuclear membrane and the tubulin bundles can be visual- ized by fluorescence microscopy in a strain of fis- sion yeast that expresses a pair of proteins fused to green fluorescent protein (GFP): nucleoporin-GFP and GFP-tubulin. P.T. Tran, V. Doye, F. Chang, and S. Inoue have used this fluorescent strain of fission yeast to test the hypothesis that the microtubular bundles deter- mine the central position of the nucleus which, in turn, determines the position of the cleavage plane and the septum that forms between the daughter cells. The details were reported at the General Sci- entific Meetings of the Marine Biological Labora- tory in August, 2000 (see Tran el al., p. 205 in this issue). The images on the cover were produced at room temperature by time-lapse fluorescence micros- copy. The panel on the left comprises successive images — taken at intervals of 2 hours — of untreated cells undergoing cell division. During interphase, the microtubules span the length of the cell. As the cells grow, the nucleus comes to lie at the center of the cell, where subsequent cell division and septa- tion occurs, creating two daughter cells of approx- imately equal length. Note that, during mitosis, the interphase microtubules disappear from the cell cy- toplasm, whilst the mitotic spindle appears promi- nently inside the cell nucleus. The right panel on the cover shows images of cells treated with MBC, a drug that depolymerizes mi- crotubules. Without microtubules, the nuclei are offset, the cell cycle is delayed, and no spindles form. Subsequent division planes and septa are formed at the location of the offset nuclei, creating "cut" nuclei in daughter cells of unequal length. These treated cells do not survive. Cover design b\ Beth Liles CONTENTS VOLUME 199. No. 2: OCTOBER 2000 RESEARCH NOTE Hourdez, Stephana, Jason Lamontagne, Pat Peterson, Roy E. Weber, and Charles R. Fisher Hemoglobin from a deep-sea hvdrothermal-vent copepod 95 PHYSIOLOGY Marsh, Adam G., Patrick K.K. Leong, and Donal T. Manahan Gene expression and enzvme activities of the sodium pump during sea urchin development: implications for indices of phvsiological state 100 Hill, Richard W., John W.H. Dacey, and Ahser Edward Dimethylsulfoniopropionate in giant clams (Tridac- nidae) . 10S ECOLOGY AND EVOLUTION Baker, Shirley M., Jeffrey S. Levinton, and J. Evan Ward Particle transport in the zebra mussel, Dreissena poly- morpha (Pallas) 116 Lajeimesse, T.C., and R.K. Trench Biogeography of nvo species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthoplfura ekgantissima (Brandt) 126 NEUROBIOLOGY AND BEHAVIOR Rosenthal, Joshua J.C., and Francisco Bezanilla Seasonal variation in conduction velocity of action potentials in squid giant axon 135 DEVELOPMENT AND REPRODUCTION Misamore, M.J., andJ.W. Lynn Role of the cytoskeleton in sperm entry during fertilization in the freshwater bivalve Dreissena polymorpha 144 SHORT REPORTS FROM THE 2000 GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY /•AM REPORT The Editors Introduction to the featured report. On mapping odor qualitv ................................ 161 Wachowiak, Matt, Michal Zochowski, Lawrence B. Cohen, and Chun X. Falk The spatial representation of odors by olfactory re- ceptor neuron input to the olfactory bulb is concen- tration invariant. . 162 NEUROBIOLOGY Landowne, David Heavy water (D2O) alters the sodium channel gating current in squid giant axons 164 White, Thomas H., Harris Ripps, Miduturu Srinivas, and Roberto Bruzzone Voltage gating properties of channels formed by a skate retinal connexin 165 Molina, Anthony J.A., Peter J.S. Smith, and Robert Paul Malchow Hydrogen ion fluxes from isolated retinal horizontal cells: modulation bv glutamate 168 Wang. Jing W. Odor-induced oscillatory activity in Drtnophila CNS ... 170 Hitt, James M., Frederick A. Dodge, Ehud Kaplan, and Robert B. Barlow Orcadian rhythms in the receptive fields of the Limu- lus lateral eye 171 Fay, Richard R.. and Peggy L. Edds-Walton Frequency response of auditor)1 brainstem units in toadfish (Opsanus tau) 173 Yamaguchi, Ayako, Leonard K. Kaczmarek, and Darcy B. Kelley Intrinsic membrane properties of laryngeal mo- toneurons that control sexually differentiated vocal behavior in African clawed frogs, Xenopus laevis .... 1 75 Atherton, Jillian L., Matthew A. Krntky, James M. Hitt, Frederick A. Dodge, and Robert B. Barlow Optic nerve responses of Limulux in its natural habi- tat at night 1 76 Krutky, Matthew A., Jillian L. Atherton, Spence Smith, Frederick A. Dodge, and Robert B. Barlow Do the properties of underwater lighting influence the visually guided behavior of Limulus? 178 Hale, Melina E. Startle responses of fish without Mauthner neurons: escape behavior of the lumpfish (Cyclopterus liimpus) 180 Epstein, David A., Herman T. Epstein, Frank M. Child, and Alan M. Kuzirian Memory consolidation in Hermissenda crassicornis ... 182 Abenavoli, A., L. Forti, and A. Malgaroli Mechanisms of spontaneous miniature activity at CA3-CA1 synapses: evidence for a divergence from a random Poisson process 184 PHYSIOLOGY AND BIOCHEMISTRY Novales Flamarique, Inigo, Kristiina Ovaska, and Theodore M. Davis UV-B induced damage to the skin and ocular system of amphibians 187 Harrington, John M., and Peter B. Armstrong Initial characterization of a potential anti-fouling sys- tem in the American horseshoe crab, Limulus polyphemus 189 Asokan, Rengasamy, Margaret T. Armstrong, and Peter B. Armstrong Association of a.,-macroglobulin with the coagulin clot in the American horseshoe crab, Limulii* polyphemus: a potential role in stabilization from pro- teolysis 190 Kuhns, William J., Max M. Burger, Mohan Sarker, Xavier Fernandez-Busquets, and Tracy Simpson Enzymatic biosynthesis of N-linked glycan by the ma- rine sponge Microciona prolifera 192 Armstrong, Peter B., and Rengasamy Asokan A Ca+2-independent cytolytic system from the blood of the marine snail Bus^cmi canaliculum 194 Heck, Diane E., Lydia Louis, Michael A. Gallo, and Jeffrey D. Laskin Modulation of the development of plutei by nitric oxide in the sea urchin Arbacia punctuhna 195 Jung, Sung-Kwon, Katherine Hammar, and Peter J.S. Smith Development of self-referencing oxygen microsensor and its application to single pancreatic HIT cells: effects of aclenylate cyclase activator forskolin on ox- vgen consumption 197 Hanselmann, Rhea, Roxanna Smolowitz, and Daniel G. Gibson Identification of proliferating cells in hard clams. . . 199 Brothers, Christine, Ernest Marks III, and Roxanna Smolowitz Conditions affecting the growth and zoospoi illation of the protistan parasite O_PX in culture 200 CELL BIOLOGY Sandberg, Leslie, Phillip Stafford, and George M. Langford Effects of myosin-II antibody on actin-dependent ves- icle transport in extracts of clam oocytes 202 Stafford, Phillip, Jeremiah Brown, and George M. Langford Interaction of actin- and microtubule-based motors in squid axoplasm probed with antibodies to myosin V and kinesin 203 Tran, P.T., V. Doye, F. Chang, and S. Inoue Microtubule-dependent nuclear positioning and nu- clear-dependent septum positioning in the fission yeast, Sacrhammws pombe 205 Crawford, Karen The role of microtuhules during blastodisc forma- tion of the squid Loligo pealei 207 Weidner, Earl Cytoplasmic proteins on the surface of discharged microsporidian sporoplasms 208 MacKenzie, Roger, David Newman, Max M. Burger, Rene Roy, and William J. Kuhns Adhesion of a viral envelope protein to a non-self- binding domain of the aggregation factor in the marine sponge Micwriona prolifera 209 Goda, Makoto, Mario H. Burgos, and Shinya Inoue Fertilization-induced changes in the fine structure of stratified Arbacia eggs. I. Observations on live cells with the centrifuge polarizing microscope 212 Burgos, Mario H., Makoto Goda, and Shinya Inoue Fertilization-induced changes in the fine structure of stratified Arbacta eggs. II. Observations with electron microscopy 213 Gould, Robert M., Concetta M. Freund, John Engler, and Hilary G. Morrison Optimization of hornogenization conditions used to isolate mRNAs in processes of myelinating oligoden- drocytes 215 ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY Kirkby, Ryan, Luc Claessens, Charles Hopkinson, Jr., Edward Rastetter, and Joseph Vallino Modeling the effects of land-use change on nitrogen biogeochemistry in the Ipswich watershed, Massachu- setts 218 Perring, Anne, Michael Williams, Charles Hopkinson, Jr., Edward Rastetter, and Joseph Vallino Solute dynamics in storm flow of the Ipswich River Basin: effects of land use 219 Westgate, Elizabeth J., Kevin D. Kroeger, Wendy J. Pabich, and Ivan Valiela Fate of anthropogenic nitrogen in a nearshore Cape Cod aquifer 221 Denault, Michelle, Erica Stieve, and Ivan Valiela Effects of nitrogen load and irradiance on photosyn- thetic pigment concentrations in Cladophora vaga- bundaand Gracilaria tikvahiae'm estuaries of Waquoit Bav 223 Greenbaum, Adena, and Anne Giblin Differences in properties of salt marsh sediment be- tween haved and reference sites 225 Chikarmane, Hemant M., Alan M. Ku/irian. Robbin Kozlowski, Mark Kuzirian, and Tony Lee Population genetic structure of the goosefish, Lo- phnif nmmranus 227 ORAL PRESENTATIONS Published bv title only. . 229 THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is published MX times a year by the Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. Subscriptions and similar matter should be addressed to Subscription Manager. THE BIOLOGICAL BULLETIN. Marine Biological Laboratory. 7 MBL Street. Woods Hole. Massachusetts 02543. 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All such footnotes should appear on the title page. Footnotes are not normally permitted in the body of the text. 5. Literature cited. In the text, literature should be cited by the Harvard system, with papers by more than two authors cited as Jones et = -0.9). B. spiciilifer he- moglobin thus does not show any homotropic or hetero- tropic interactions and behaves like a myoglobin. Temperature has a strong effect on B. spiculifer hemoglo- bin, as shown by the apparent AH value of -69 kJ • mole"' (Fig. 3B). This value is higher than that of other arthropod hemoglobins (Table 1 ) and is consistent with the absence of a Bohr effect (oxygenation-linked proton dissociation that is endothermic and decreases the overall exothermic heat of oxygenation) in Benthoxynus hemoglobin (11). To determine the in vim hemoglobin concentration, a small group of copepods (about 400 animals) was weighed and homogenized in a ground-glass tissue homogenizer, and the hemoglobin content of the homogenate was determined using the cyan-met-hemoglobin method and a millimolar absorption coefficient of 11 cm1 at 540 nm (12). The estimated in vivo concentration of 0.95 mM heme explains the conspicuous red color of the animals. Large hemoglobin pools can play an important role in oxygen storage in some situations. However, assuming a respiratory rate similar to that of littoral harpacticoid copepods (13) and an abrupt switch from aerobiosis to anaerobiosis, we estimate that the quantity of hemoglobin present would support aerobic res- piration for less than 2 min at 15°C and about 30 s at 25°C. Thus the hemoglobin pool is insufficient to allow the cope- pod to make more than short forays into anaerobic micro- habitats without relying on anaerobic respiration. Another role of high affinity hemoglobins has been theorized to be detoxification of free radicals from oxygen or nitrogen monoxide (14). Although free radicals do form in sulfidic systems, and some vent animals have detoxification mech- anisms (15), we consider it more likely that the hemoglobin of B. spiculifer functions primarily in oxygen acquisition from the environment. The other hemoglobin-containing copepods identified by Fox (2) were collected from muddy and reduced environments with low levels of oxygen and high levels of sulfide. The adaptive significance of hemo- globin for acquisition of oxygen in these environments is S. HOLIRDEZ ET AL. 100 A Benthoxynus spiculifer 3.3 3.4 3.5 1/1000T -0.5 o 0? -1.0 -1.5 B 30°C * 10°C • 0.5 en E 0.1 0.05 2 - 2 o IO 1 _ i^fs *' ' — •\ c O 0 • i i i i i i i , i , i i i i . i 0 6.5 7.0 7.5 8.0 PH o 10 0.5 1.0 PO2 (mmHg) Figure 3. (A) Oxygen equilibrium curves of Benthoxynus spiculifer hemoglobin at 10°. 20°, and 30°C, measured as previously described (5), and (inset) arrhenius plot showing calculated values of the apparent oxygenation enthalpy values (AH). (B) Variation of P5n and n,n values with pH and temperature. apparent, and the very high affinity of the hemoglobin in B. spiculifer probably also reflects the very low oxygen ten- sions this species experiences in its hydrothermal vent mi- crohabitat. In this context it is relevant that hemoglobins or hemocyanins with high oxygen affinity characterize many hydrothermal vent animals (10). Acknowledgments We thank the captains, pilots, and crews of the R/V Atlantis and DSRV Alvin for their assistance at sea; Verena Tunnicliffe and Maia Tsurumi for identifying B. spiculifer. Anny Bang for assistance with the oxygen equilibrium experiments; and Veronique Robigou and the REVEL Project for providing science teachers with research oppor- tunities. This project was supported by NSF OCE 9633105 and the REVEL Program (NSF OCE-98 14073 to J.R. Delaney, with additional support from the Pennsylvania State University and the American Museum of Natural History). Literature Cited 1. Sarradin, P. M., J. C. Caprais, P. Briand, F. Gaill, B. Shillito, and D. Desbruyeres. 1998. Chemical and thermal description of the environment of the Genesis hydrothermal vent community (13 degrees N. EPR). Cah. Biol. Mar. 39(2): 159-167. 2. Fox, H. M. 1957. Haemoglobin in the Crustacea. Nature 179: 148. 3. Humes. A. G. 1994. How many copepods? Hydrobiologia 293: 1-7. 4 Sarrazin, J., V. Robigou, S. K. Juniper, and J. R. Delaney. 1997. Biological and geological dynamics over four years on a high temper- ature sulfide structure at the Juan de Fuca Ridge hydrothermal obser- vatory. Mar. Ecol. Prog. Ser. 153: 5-24. 5. Hourdez, S., F. H. Lallier, V. Martinjezequel, R. E. Weber, and A. Toulmond. 1999. Characterization and functional properties of (he extracellular coelomic hemoglobins from the deep-sea, hydrother- mal vent scaleworm Branchipolynoe symmytilida. Proteins Struct. Fund. Genet. 34(4): 435-442. 6. Weber, R. E. 1981. Cationic control of O, affinity in lugworm erythrocruorin. Nature 292: 386-387. 7. Sick, H., and K. Gersonde. 1969. Method of continuous registra- tion of O2 binding curves of hemoproteins by means of a diffusion chamber. Anal. Biochem. 32: 362-376. 8. Toulmond, A. 1992. Properties and functions of extracellular heme pigments. Pp. 231-256 in Blood anil Tissues Oxygen Carriers. C.P. Mangum, ed. Springer. Berlin. 9. Amos, A. R., and A. Shejter. 1970. Isolation and properties of the hemoglobin of the clam shrimp Cyzicus cf. hierosolymitanus (S. Fischer). Cimip. Biochem. Physio/. 33: 481-490. 10. Childness, J. J., and C. R. Fisher. 1992. The biology of hydrother- mal vent animals: physiology, biochemistry and autotrophic symbio- ses. Oceaniigr. Mar. Biol. Anna. Rev. 30: 337-341. 1 1 . Perutz, M. 1990. Mechanisms of cooperativity and allostenc regu- COPEPOD HEMOGLOBIN 99 lation in proteins. Cambridge University Press, Cambridge, UK. Pp. 1-101. 12. Van Assendelft, O. W. 1970. Spectrophotometry of haemoglobin derivatives. Royal Vangorcum. Assen, The Netherlands. 152 pp. 13. Coull, B. C., and W. B. Vernberg. 1970. Harpacticoid copepod respiration: Enhydrosoma propinquum and Longipedia helgolandica. Mar. Biol. 5: 341-344. 14. Minning, D. M., A. J. Gow, J. Bonaventura, R. Braun, M. Dewhirst, D. E. Goldberg, and J. S. Stamler. 1999. Ascaris hae- moglobin is a nitric oxide-activated 'deoxygenase'. Nature 401: 497- 502. 15. Tapley, D. W., J. M. Shick, and G. R. Buettner. 1992. Free radical production in hydrothermal vent invertebrates during sulfide oxidation. Am. Zool. 32(5): 67A. 16. Terwilliger, N. B. 1992. Molecular structure of the extracellular heme proteins. Pp. 87-1 16 in Oxygen Carriers in Blood and Tissues, Vol. 13, Advances in Comparative and Environmental Physiology, C.P. Mangum. ed. Springer, Berlin. 17. Amiconi, G.. E. Antonini. M. Brunori, H. Formaneck, and R. Huber. 1972. Functional properties of native and reconstituted hemoglobins from Chironomus thummi thummi. Eur. J. Biochem. 31: 52-58. 18. Weber, R. E., G. Braunitzer, and T. Kleinschmidt. 1985. Func- tional multiplicity and structural correlations in the hemoglobin system of larvae of Chironomus thummi thummi (Insecta, Dipteral: Hb com- ponents CTT I, CTT II , CTT III, CTT IV, CTT VI, CTT VIIB, CTT IX and CTT X. Comp. Biochem. Physiol. B 80: 747-753. 19. D'Hondt, J., L. Moens, J. Help, A. D'Hondt, and M. Kondo. 1978. Oxygen-binding characteristics of three extracellular haemoglobins of Anemia sa/ina. Biochem. J. 171: 705-710. 20. Dangott, L. J., and R. C. Terwilliger. 1992. Structural studies of a branchiopod crustacean (Lepidurus bilobatus) extracellular hemoglo- bin. Evidence for oxygen-binding domains. Biochim. Biophys. Ada 579: 452-461. 21. Sugano, H., and T. Hoshi. 1970. Purification and properties of blood hemoglobin from the fresh-water cladocera, Moina macropopa and Daphnia magna. Biochim. Biophys. Acta 229: 349-358. 22. Dangott, L. J., and R. C. Terwilliger. 1981. Arthropod extracellu- lar hemoglobins: structural and functional properties. Comp. Biochem. Physiol.B 70: 549-557. Reference: Biol. Bull. 199: 100-107. (October 2000) Gene Expression and Enzyme Activities of the Sodium Pump During Sea Urchin Development: Implications for Indices of Physiological State ADAM G. MARSH*. PATRICK K.K. LEONGt, AND DONAL T. MANAHANi Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0371 Abstract. The sodium pump consumes a large portion of the metabolic energy (407r ) in sea urchin larvae. Under- standing the developmental regulation of ion pumps is im- portant for assessing the physiological state of embryos and larvae. We sequenced a partial cDNA clone (1769 bp) from the sea urchin Strongylocentrotiis pitrpuratits and found it to contain the C-terminal portion of an open reading frame coding for 195 amino acids that exhibited high sequence similarity (897r) to invertebrate a-subunits of the Na+,K+- ATPase sodium pump. Northern blots using the 3' untrans- lated region of this cDNA specifically recognized a 4.6-kbp transcript under high stringency. During embryonic devel- opment, a rapid increase in levels of this mRNA transcript during gastrulation (25 h postfertilization) was paralleled by a concomitant increase in the total enzymatic activity of Na+,K+-ATPase. Expression of this subunit during gastru- lation increased to a maximum at 36 h, followed by a rapid decline to trace levels by 60 h. The rate of removal of the transcript from the total RNA pool after 36 h closely fol- lowed a first-order exponential decay model (r= 0.988), equivalent to a degradation rate of 7.87r h" . By 83 h. transcription of the a-subunit gene was low. yet sodium pump activity remained high. Molecular assays for the expression of this gene would underestimate sodium pump activities for assessing physiological state because of the temporal separation between maximal gene expression in a Received 29 February 2000; accepted 31 July 2000. *Present address: College of Marine Studies. University of Delaware. Lewes, DE 19958. tPresent address: Department of Physiology and Biophysics. University of Southern California School ot Medicine. Los Angeles. CA 90033. tTo whom correspondence should be addressed. E-mail: manahan @ usc.edu gastrula and maximal enzyme activities in the later larval stage. This finding illustrates the difficulty of using molec- ular probes for assessing the physiological state of inverte- brate larvae. Introduction Maintaining Na+ and Kf ion gradients is one of the most energetically demanding processes of an organism's main- tenance physiology. In general, animal cells routinely ex- pend 20%-30% of their total metabolic energy on the ac- tivity of a single protein complex, the sodium pump (Na + ,K + -ATPase; Siems et at., 1982. 1992), and for adult marine invertebrates, the sodium pump can potentially ac- count for 30%-70% of tissue metabolism (Baker and Con- nelly, 1966: Lucu and Pavicic. 1995). The ion gradients established by the sodium pump are critical for maintaining a cell's osmotic balance and resting membrane potential, as well as providing the electrochemical gradient necessary for the uptake of other ions, sugars, amino acids, and neuro- transmitters via Na+ coupled co-transporters (Blanco and Mercer, 1998). The requirements for ion regulation change rapidly dur- ing embryonic development. The increase in cell number during early embryogenesis and the consequent increase in cellular-membrane surface area necessitates the production of more sodium pumps to regulate intracellular ion flux. The in vivo physiological activity of Na+,K+-ATPase has been characterized during early development in the sea urchins Stwngylocentrotus purpiiratus and Lytechimis pic- tns (Leong and Manahan, 1997). Using stlRb+ as a radio- active tracer for K+ ion transport, Leong and Manahan (1997) described the ontogenetic changes in activity of 100 SODIUM PUMPS IN SEA URCHIN EMBRYOS 101 Na+,K+-ATPase in living embryos. They ton nil a large increase in activity — from nondetectable levels prior to fer- tilization to a high level accounting for 40% of total meta- bolic energy consumption at the pluteus larval stage (72 h postfertilization). By the same radiotracer techniques, the metabolic energy demand of Na+,K+-ATPase activity in the Antarctic sea urchin Sterechinus neumayeri was found to be as high as 80% of total metabolism at the pluteus larval stage at -1.5°C (Leong and Manahan, 1999). In the sea urchin Hemicentrotus pulcherrimus, the total protein activity and gene expression of Na+,K+-ATPase increases rapidly during gastrulation (Mitsunaga-Nakatsubo et til., 1992a. b). Overall, the physiological importance of Na+,K+-ATPase activity during embryogenesis in sea ur- chins has significant implications for metabolic energy con- sumption during development. This universal importance of Na^.K^-ATPase in animals suggests that measurements of this enzyme could be a useful indicator of physiological state. For larval stages in which direct enzyme assays are limited by the small amount of protein in an individual, measurements of gene expres- sion might provide the sensitivity necessary to assay small amounts of tissue. Functional Na+.K+-ATPase pumps are a heterodimer (a, )3 subunits; Jorgensen and Skou. 1969). with the a-subunit possessing the ATP binding site and catalytic activity (Kyte, 1971 ). In this study, we describe the timing between transcription of the a-subunit and the ap- pearance of functional sodium pumps during the develop- ment of Strongylocentrotus purpuratus. We also describe the ontogenetic changes in expression of the a-subunit to determine the developmental timing between increases in enzyme activity and the potential for using these measures as an index of physiological state in embryos and larvae. Materials and Methods Embryo cultures Adult Strongylocentrotus purpuranis were induced to release gametes (injections of 0.5 M KC1), and fertilized eggs were divided into six 20-liter culture containers at a concentration of about 20 individuals per milliliter of fil- tered seawater (0.2 jum). Culture temperatures were main- tained at 15°C during development. Embryos were main- tained in suspension by paddles connected to slow stirring motors (—30 rpm). For the gene expression analysis, time- course samples were collected throughout development at the following times from an egg to a 4-arm pluteus larva: 0, 6, 8, 10. 12, 14. 16. 18, 20, 25, 31. 36, 42, 48, 60, 72, and 83 h postfertilization (n = 17). For each sample, about 100,000 embryos were removed by sieving (80-jtun mesh) and pelleted by centrifugation ( 1000 X g) into 50-ml screw- cap tubes. Embryos were immediately dissolved in an acid- guanidinium buffer (4 M guanidinium isothiocyanate, 25 mM Na-citrate, 0.2% Sarkosyl and 215 mM /3-mercapto- ethanol: pH 5.2; Chomzinsky and Sacchi, 1987) and frozen at -80°C. cDNA clone: sequencing and analvsis An expressed sequence tag (EST) library from activated coelomocytes of adult S. purpuratus was prepared by Smith et al. ( 1996), and a sequence fragment of one cDNA clone (#020) was found to have a high nucleotide similarity to the bovine a-subunit of Na+,K+-ATPase. We sequenced this clone (provided by C. L. Smith and E. H. Davidson) by random transposon insertion in a modified pBluescript (Stratagene) plasmid (pMOB: Strathmann et til., 1991 ). The introduced transposon elements contained defined priming sites for subsequent manual sequencing of double-stranded plasmid templates using standard dideoxy termination reac- tions with "S-labeled dATP (Sequenase Reaction Kit, USB). Sequencing gels were visualized by autoradiography on X-ray film (Kodak, XAR 5). Nucleotide sequences were entered and edited using the software package MacVector 5.0 (Mac OS: Oxford Molecular Group), and contiguous overlaps between fragments were identified using the soft- ware package AssemblyLign 2.0 (Mac OS; Oxford Molec- ular Group). Both strands of the open reading frame (ORF) were sequenced by overlapping subclones so that most of the contiguous ORF sequence was assembled from three independent sequencing reactions. For phylogenetic com- parisons, nucelotide and putative amino acid sequences from other animal species were structurally analyzed and aligned using the OMIGA 2.0 software package (Oxford Molecular Ltd.). Identity and similarity scores for the de- duced amino acid alignments were calculated from the FASTA routine available in the GCG Wisconsin Package 8.0 (UNIX OS). mRNA analysis: isolation and quantification Total RNA was extracted from each sample by an acid guanidinium-phenol method (after Chomzinsky and Sacchi. 1987) and further purified by sequential precipitations in lithium chloride (4 M LiCl), sodium acetate (3 M NaOAc, pH 4.2) and ethanol (70% EtOH). After each precipitation, the RNA pellets were washed in 70% EtOH and dried under vacuum: before proceeding with the next precipitation, the pellets were resuspended in RNase-free TEN buffer ( 10 mM Tris pH 8.0. 1 mM EDTA and 10 mM NaCl). The final RNA precipitates were resuspended in RNase-free water and quantified by their optical density at 260 nm. From each developmental time point, 10 jug of total RNA was size- separated by formaldehyde gel electrophoresis and blotted overnight via capillary transfer onto nylon membranes. RNA on the nylon membranes was UV cross-linked (Stratalinker), and the membranes were stored dry at room 102 A. G. MARSH ET AL. temperature. A cDNA probe was generated from the 3'- untranslated region (UTR) of clone #020. The terminal 1185 bp were PCR amplified (5'- TGG GAT TGA AGO ACT CAG -3' and T7 oligonucleotide primers) and gel purified for further use in standard Northern hybridizations (see general methods in Ausubel et al., 1992). Membranes were prehybridized for several hours in 40% formamide, 25 mMNa3PO4 (pH 7.2), 5x SSC, 0.1% SDS, 5x Denhardt's, and 50 jug/ml yeast RNA at 45°C in a hybridization oven. The 3'-UTR PCR probe (1185 bp) was radiolabeled by random priming (Promega) with a-32P-dCTP (3000 Ci mmol ' ), added to the hybridization tube with a fresh 10-ml aliquot of hybridization buffer (as above), and incubated overnight at 50°C. The blots were initially washed with 0.1X SSC, 1.0% SDS. and 0.5% Na4P2O7 at 45°C for 1 h. Additional washes at higher temperatures (max. 55°C) were performed as necessary to further reduce the background signal. Autoradiograms (Kodak Biomax X-ray film) were digitized on a high-resolution scanner (1200 dpi), and grain densities for the signal bands were quantified using the image analysis routines in the software program PhotoShop 4.0 (Win95 OS; Adobe). Na+,K+-ATPase enzyme activity Total enzyme activity of Na+,K+-ATPase was measured at short intervals between 20 and 50 h postfertilization, the period during which enzyme activity increases rapidly dur- ing development in S. piirpuratus (Leong and Manahan, 1997). Ouabain-sensitive Na+,K+-ATPase activity (details in Leong and Manahan, 1997) was determined in all sam- ples on the same day with one set of standards to minimize the between-sample assay error. Total Na+,K+-ATPase ac- tivity was measured as the rate of hydrolysis of ATP (Es- mann, 1988). Briefly, embryo tissues were thawed, soni- cated, and resuspended in histidine buffer (10% sucrose, 5 mM EDTA and 5 mM histidine, pH 7.7) at a final protein concentration of 0.5 to 1.0 mg ml"1. In the present study, the total Na+,K"l"-ATPase activity of the sea urchin embryos was measured as the difference in ATPase activity in the presence and absence of 2 mM ouabain at 25°C. A detailed consideration of the inclusion of detergents in the Na+,K + - ATPase assay is presented in Leong and Manahan (1997). In summary, neither deoxycholate (a common detergent used in Na"l",K+-ATPase assays) nor alamethicin (a mem- brane-perrneabilizing agent) had any effect on the total Na+,K+-ATPase activity in homogenates of S. piirpuratus embryos, suggesting that inside-out and right-side-out ves- icles are not a significant problem in assaying Na+,K+- ATPase activity in seu urchin embryos (Leong and Mana- han, 1997). The protein content of the samples was determined by the Bradford assay with the modifications of Jaeckle and Manahan (1989). Table 1 Comparison of nucleolide and deduced ammo acid sequences for different a-subunit Na* . K* -ATPase Amino acid GenBank Nucleotide accession identity Identity Similarity Species number (%) (%) (%) Drosophila AF04494 69.8 73.3 89.2 Caenoi'habditis U 18546 69.7 72.8 89.2 Xenopus U49238 67.6 69.2 89.2 Anemia X56650 65.2 72.3 89.7 Hydra M75140 64.1 67.7 89.2 Identity and .similarity to the Slrongyloc entrains piirpuratus cDNA open reading frame (clone #020) are presented as percentages determined from scoring by the GCG Wisconsin Users Group software program. Scores include only the terminal portion of the sea urchin gene's ORF: 588 base pairs (195 amino acid residues and stop codon). Results A partial Strongylocentrotus piirpuratus cDNA clone (#020: Smith et al., 1996) was characterized in this study and found to contain 1769 bp with the terminal portion of an ORF coding for 195 amino acids (588 bp with the stop codon). The remaining sequence (1181 bp) comprised a putative 3' UTR domain. The clone's ORF was compared to other tt-subunits of Na+,K + -ATPase, and the S. piirpuratus nucelotide sequence ranged from 64% to 70% identity to these terminal ORF domains (Table 1). The deduced amino acid sequences of these organisms were aligned to the putative amino acid sequence of the S. piirpuratus clone and evidenced a high degree of sequence conservation in this terminal domain (Table 2). When compared to the S. piir- puratus sequence, the derived amino acid sequence was 68%-73% identical and 89%-90% similar (Table 1 ). The terminal region of the ORF of known «-Na + ,K + - ATPases is believed to contain several transmembrane do- mains; there is some debate over the exact number of these domains and the extra- vs. intracellular orientation of some of the intervening regions in a-Na+,K+- ATPase (Shull and Greeb, 1988; Takeyasu et al.. 1990; Blanco and Mercer, 1998). The hydropathy of the S. piirpuratus sequence was estimated with Kyte-Doolittle scoring using a grouping of 11 amino acid residues (Fig. 1) and suggests a high proba- bility of four transmembrane domains in the terminal por- tion of this ORF. Overlaying these domains on a structure detailed by Takeyasu et al. ( 1990) indicates that the region between the seventh and eighth transmembrane domains could have an extracellular localization. In the absence of crystallographic data, it is generally believed that most a-subunits of transmembrane ATPases (both Na+ and Ca+ + ) are structurally similar, with 10 transmembrane do- SODIUM PUMPS IN SEA URCHIN EMBRYOS Table 2 103 Alignment of deduced amino acid sequence for the terminal IV5 residues of the a-subunil of Na+. K+-ATPase in the sea urchin Strongylocentrotus purpuratus 1 ! 1 URCHIN SDIMKRRPRD PQNDKLVNER LISVSYGQIG MIQRSAGFFA YFVIMGENGF FLY ADIMKRPPRD PFNDKLVNSR LISMAYGQIG MIQAAAGFFV YFVIMAENGF SHRIMP SDIMKRRPRN PVTDKLVNER LISLAYGQIG MIQASAGFFV YFVIMAECGF NEMATODE SDIMKRQPRD PIRDKLVNER LISLAYGQIG MIQASAGFFT YFWIMADNGF FROG SDIMKRQPRN PKTDKLVNER LISMAYGQIG MIQALGGFFT YFVILAENGF HYDRA SDIMKRHPRN PIRDKLVNER LISLAYGQIG MMQATAGFFT YFIILAENGF 51 1 1 URCHIN LPNDLIMLRS KWDDKAVLNV EDSYGQQWGF YQRKQLEYTC HTAFFASIW FLY LPKKLFGIRK MWDSKAVNDL TDSYGQEWTY RDRKTLEYTC HTAFFISIVV SHRIMP LPWDLFGLRK HWDSRAVNDL TDSYGQEWTY DARKQLESSC HTAYFVSIVI NEMATODE MPWDLYQLRA QWDSRAYNNV LDSYGQEWTY ANRKILEYTC QTAYFVSIVV FROG LPWTLLGIRV NWDDRWTNDV EDSYGQQWTY EQRKIVEFTC HTSFFISIVV HYDRA LPSYLFGLRS QWDDMSNNNL LDSFGSEWTY FQRKEIELTC QTAFFTTIVV •k •k ~k 101 II 1 -"'1150 URCHIN VQWADVIICK TRRNSLIHQG MNNWVLNFGL FFETALAAFL SYCPGLENGL FLY VQWADLIICK TRRNSIFQQG MRNWALNFGL VFETVLAAFL SYCPGMEKGL SHRIMP VQWADLIISK TRRNSVFQQG MRNNILNFAL VFETCLAAFL SYTPGMDKGL NEMATODE VQWADLIISK TRRNSLVQQG MSNWTLNFGL VFETALAWFM CYCPGLDNGL FROG VQWADLIICK TRRNSVFQQG MKNKILIFGL FEETALAAFL SYCPGMDVAL HYDRA VQWADLIISK TRRLSLFQQG MTNWFLNFGL FFETALAAFL QYTPGVNTGL 151 | 1 195 URCHIN RMYPLRIGWW FVAFPFSLLI FVYDECRRFI LRHNPGGWVE LETYYJ FLY RMYPLKLVWW FPAIPFALAI FIYDETRRFY LRRNPGGWLE QETYYJ SHRIMP RMYPLKINWW FPALPFSFLI FVYDEARKFI LRRNPGGWVE QETYYJ NEMATODE RMYGLRFSWW FCALPFSILI FVYDEIRRFL IRRYPGGWVE RETYYJ FROG RMYPLKPTWW FCAFPYSLII FIYDEVRKLI IRRSPGGWVE KESYYJ HYDRA RLRPMNFTWW LPGLPFSLLI FVYDEIRRYL LRKNPGGWVE KETYYJ * * * * * * * * * The shaded blocks and bold lettering indicate the putative transmembrane domains identified by Kyte-Doolittle hydropathy scores in the sea urchin sequence (bold lettering; see Fig. 1). Genus names for the organisms and GenBank accession numbers for the sequences: urchin = Strongylocentrotus (this study), fly = Drosophila (AF04494), shrimp = Anemia (X56650), nematode = Caenorhabditis (U18546), frog = Xenopus (U49238), Hydra = Hydra (M75140); * = amino acid identity for all sequences; J = termination codon. mains and a large extracellular loop between transmem- brane domains 7 and 8 (Canfield and Levenson, 1993; Blanco and Mercer, 1998). Northern blots using the 3' UTR of clone #020 specifi- cally recognized a 4.5 to 4.7 kb transcript under high strin- gency (Fig. 2). In another sea urchin, Hemicentrotus pul- cherriimts, the full-length «-Na+,K+-ATPase cDNA has been cloned and has an mRNA transcript size of 4.6 kb (Mitsunaga-Nakatsubo et a/., 1992a). The a-Na+,K + -ATP- ase gene is differentially expressed during development in 5. purpuratus (Fig. 2). The level of mRNA transcripts is low during early cleavage, then rises rapidly around gastrulation (at 25-36 h postfertilization; Fig. 3). After gastrulation, mRNA returns to a low level comparable to that initially found in the egg (Fig. 3). The rapid disappearance of the a-Na+.K+-ATPase transcript from the total RNA pool after gastrulation closely followed a first-order exponential decay model [/lv)= 98.512 eK)l28xl; r = 0.988; Fig. 3], The decay constant of the regression is equivalent to a degrada- tion rate of 7.8% h"1 of the transcript. At 83 h, a-subunit 104 A. G. MARSH ET AL. extracellular 200 150 100 50 AA residues from 3' ORF terminus TAA Figure 1. Secondary structure characterization for the last 195 amino acids of a putative a-Na+,K*-ATPase cDNA cloned from Stront>vloct'ii- trotus purpuratus. The Kyte-Doolittle hydropathy score suggests the pres- ence of four transmembrane domains, which match the structure of other a-Na+,K+-ATPase subumts (Blanco and Mercer. 1998). transcripts were barely detectable under the conditions we used for Northern blots of total RNA. The rapid increase in a-Na + ,K^-ATPase mRNA tran- scripts during gastrulation in 5. purpuratus was paralleled by a concomitant increase in the total activity of the sodium pump (Fig. 4). Activity levels were very low during early development in S. purpuratus and then increased after 20 h to a maximum level at the pluteus larval stage (Leong and Manahan. 1997). The rapid increase in activity between 20 and 40 h of development (Fig. 4) can be described by the exponential function (fl\) = 1.167(1 + e|lv x""4571)-': r = 0.9664; maximum activity of 1.17 jumol P, h"' mg~' pro- tein]. The present study resolves the increase in enzyme f(x)= 98.51 2e('0128x> r2 = 0.988 I - ' - 1 - ' - 1 20 40 60 Developmental time (hours) 80 Figure 3. Relative transcript levels of the a-Na*,K*-ATPase cDNA during development in Strongylocentrotus purpuratus (quantified from Fig. 2). mRNA levels are presented relative to the maximal expression obtained at 36 h postfertilization. The rapid decline in mRNA abundance after 3f> h his a first-order exponential decay function (r = 0.988; regression line plotted with shaded symbols). activity at a finer time scale (cf. Leong and Manahan. 1997) and reveals the close coordination between a-subunit gene transcription and the assembly of functional sodium pumps in sea urchin embryos between fertilization and gastrula- tion. Discussion It has long been a general goal of physiological ecologists to identify a sensitive biochemical indicator of an animal's physiological state or metabolic activity — for example, the ratio of RNA to DNA (Westerman and Holt, 1994) or the glycolytic enzyme activities (Childress and Somero, 1990). For developmental stages with low biochemical contents, such assays are often not possible. Molecular biological techniques have the necessary sensitivity and potentially offer an alternative for assessing physiological state in lar- vae and small zooplankton. Because the sodium pump con- sumes such a large portion of cellular energy metabolism 9.5- 7.5 — 4.4 — 2.4- 14 — 10 12 14 16 18 20 25 31 36 42 48 60 72 83 <28S <18S Figure 2. Northern blot hybridization of total RNA during development in Strongylocentrotus purpuraius using a radiolabeled probe from the .V-untranslated domain of the «-Na*,K + -ATPase cDNA clone. RNA samples were collected at short time intervals during embryogenesis as shown by the hours post-fertilization at the top of each lane. Molecular size (kilobases) is indicated on the left; ribosomal RNA positions are indicated on the right. The probe recognizes a single transcript that is approximately 4.6 kb in size. SODIUM PUMPS IN SEA URCHIN EMBRYOS 105 Q- Q." fi * E I 1 25 - 1.00 - 075 - 0.50 - 0.25 - 000 -> T - 100% I* - 80% £ 6 oj E h 60% CD g- M - 40% > 1 - 20% a a - 0% 20 40 60 Age (hours) 80 Figure 4. Total enzyme activity of Na+.K+-ATPase during gastrula- tion in Strongylocentrotus purpuratus. Protein-specific enzyme activity (closed circles) is plotted on the left axis; mRNA levels from Fig. 3 are overlaid (dashed line) to illustrate the temporal relationship between a-sub- unit gene expression and the appearance of functional sodium pump proteins. Data from Leong and Manahan (1997) are also plotted (open circles) to show the pattern of relative enzyme activities. (e.g., 40% in sea urchin larvae, Leong and Manahan 1997), it would seem to be a good candidate for such an assay, with the potential to provide sensitive information regarding rates of energy utilization in a single larva. Several lines of evidence strongly support the conclusion that the partial cDNA clone (#020) in Strongylocentrotus purpuratus is the a-subunit of the sodium pump: ( 1 ) the putative amino acid sequences show a high similarity to those of other animals; (2) the 3'-UTR probe recognizes a 4.6-kb transcript, which is the full-length transcript size in other invertebrate species; (3) the ontogenetic increase in expression during gastrulation is similar to the expression pattern in another sea urchin (Mitsunaga-Nakatsubo ct ai, 1992b); (4) total Na+,K+-ATPase enzyme activities show a concomitant increase as mRNA transcripts of clone #020 accumulate during gastrulation. In the sea urchin Hemicentrotus pulcherrimus. the ex- pression of the a-Na+,K+-ATPase gene increases rapidly during gastrulation (Mitsunaga-Nakatsubo et ai, 1992b). In 5. purpuratus, the expression of the a-Na+,K + -ATPase gene evidences a similar pattern of ontogenetic regulation, with a sharp rise during gastrulation followed by a subse- quent decline to much lower levels. In conjunction with the total Na+,K+-ATPase enzyme activity that is present during development (this study. Fig. 4; see also Leong and Mana- han, 1997). temporal changes in both mRNA transcripts and protein activity indicate that the enzyme activity is low during early cleavage. At the point when an embryo ap- proaches gastrulation, a-subunit gene transcription and sub- sequent mRNA translation increase greatly, producing a large increase in sodium pumps (Fig. 3), presumably as a necessary component of the physiological function of pro- liferating cells. Once these pumps have been synthesized, mRNA tran- scripts for the a-Na+,K + -ATPase are rapidly lost. The decrease in mRNA levels over time fits a first-order expo- nential decay model (7.8% h"') so that by 83 h, transcription of the a-subunit gene was barely detectable (Fig. 2). At gastrulation. S. purpuratus appears to have synthesized most of the necessary sodium pumps. Total enzymatic ac- tivities show little increase after 50 h, further supporting this observation that the number of Na+,K+-ATPase ion pumps is set by the rapid transcription during gastrulation. and that once these transcripts are degraded, an early larva's sodium pump complement remains unchanged until further growth occurs, usually after feeding is initiated. In vertebrates, the a-subunit Na+,K+-ATPase has several isoforms (Rossier et at., 1987) that differ in many aspects, including sensitivity to proteases and cross-linking agents (Sweadner, 1979). electrophoretic mobility (Peterson et ai, 1982), and affinity for ouabain (Lytton et al, 1985). In brine shrimp (Anemia salina), the a-Na + ,K+-ATPase is present in two isoforms that are differentially expressed during early development (Peterson et al.. 1982). In the sea urchin Hemicentrotus pulcherrimus, two a-subunit isoforms are expressed during embryogenesis (Yamazaki et al. 1997). However, these two isoforms are encoded by a single gene and have identical sequences except for the 5' leader se- quences (Yamazaki et ai. 1997). If 5. purpuratus, like H. pulcherrimus, has a similar isoform complement, then the cDNA probe we used for the present study (from the 3'- UTR) should hybridize to other a-subunit isoforms ex- pressed during early development. Regardless of the mech- anism, the disparity at 83 h postfertilization between the transcript measurements and the complement of active so- dium pumps indicates the difficulty in isolating a single molecular factor to be used as an index for physiological rate processes. The observation that Na+,K + -ATPase gene transcription and translation events are limited to a brief developmental period is intriguing. The sodium pump is considered to be a "housekeeping" protein. Consequently, for such an impor- tant physiological process, we would have expected the expression of a subunit gene to be constitutive and at a low level so that there would always be some subunit synthesis to replace any turnover in functional pump proteins. Such a continual level of replacement might have offered a sensi- tive assay for assessing the physiological state of individual larvae by providing a molecular index of the activity of one of the most energy-demanding cellular processes. However, this is not the case. The a-subunit expression is develop- mentally regulated so that gene expression is initiated rap- idly at about 20 h. peaks at about 36 h, and is subsequently "turned-off." Such a temporal pattern of regulation high- lights the difficulty of using molecular probes as simple indices of physiological state. Similar difficulties in the interpretation of physiological activity and expression have 106 A. G. MARSH ET AL. been found for other specific housekeeping genes (e.g., Weinstein el al., 1992; Yang and Somero, 1996). For the multiple enzymes in metabolic pathways, the control mech- anisms at the level of genes and proteins are even more complex (Hochachka el al., 1998). Ontogenetic changes in the metabolic rates of embryos have important consequences for subsequent survival be- cause of the finite quantity of energy reserves in an egg. During development, metabolic rates increase in embryos as their cell numbers increase (Marsh et al, 1999), and the activity of the sodium pump can consume a large fraction of total metabolism in some sea urchin embryos and larvae (Leong and Manahan, 1997, 1999). Understanding ontoge- netic changes in sodium pump activities is important for assessing the metabolic energy costs of development. In the pluteus larval stage of S. purpuratus (at 83 h postfertiliza- tion), the in vivo sodium pump activity consumes 40% of total metabolism, with a potential reserve activity that could increase to a maximum of 77% of metabolism (Leong and Manahan, 1997). However, a-Na+,K+-ATPase gene ex- pression is barely detectable at this point in larval develop- ment (Fig. 3). Consequently, molecular assays for expres- sion of this gene would not be informative for assessing sodium pump activity as an index of a larva's physiological state. It is likely that during development and growth many physiological processes have functional rates of protein activity that are not strictly paralleled in time by the expres- sion of their genetic components. A knowledge of the tem- poral relationship between gene and enzyme activities is critical to developing a molecular genetic index of physio- logical state in larval forms. Acknowledgments We thank E. Davidson and C. Smith for providing the clone that we have characterized. D. Pace and M. Moore assisted with the culture sampling. This project was supported by California Sea Grant #R/MP-75C, and NSF #9420803. Literature Cited Ausubel, F. M. . R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Strnlil. 1992. Short Protocols in Molecular Biology. 2nd ed,. John Wiley and Sons. New York. Baker, P. F., and C. M. Connelly. 1966. Some properties of the external activation site of the sodium pump in crab nerve. J. Physiol. 185:270- 297. Blanco, G., and R. W. 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Smith, L. C., L. Chang, R. J. Britten, and E. H. Davidson. 1996. Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. Complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. J. Immunol. 156: 593—602. Strathmann, M., B. A. Hamilton. C. A. Mayeda. M. 1. Simon, E. M. Meyerowitz, and M. J. Palazzolo. 1991. Trunsposon-facilitated DNA sequencing. Proc. Natl. Acad. Sci. USA 88:1247-1250. Sweadner, K. J. 1979. Two molecular forms of (Na+K+)-stimulated ATPase in brain. Separation and difference in affinity for strophanthi- dm. J. Bio/. Chem. 254: 6060-6067. Takeyasu, K., V. Lemas, and D. M. Fambrough. 1990. Stability of Na( + )-K( + )-ATPase alpha-subunit isoforms in evolution. Am. J Physiol. 259:C619-C630. Weinstein, S. P., and R. S. Haber. 1992. Differential regulation of glucose transporter isoforms by thyroid-hormone in rat-heart. Biochim. Biophys. Ada 1136:302-308. Westerman. M., and G. J. Holt. 1994. RNA-DNA ratio during critical period and early larval growth of the red drum Scianeopus ocellatnx. Mar. Bio/. 121:1-9. Yamazaki, K.. C. Okamura, T. Ihara, and I. Yasumasu. 1997. Two types of Na+,K+-ATPase alpha subumt gene transcript in em- bryos of the sea urchin, Hemicentronis pulcherrimus. Zoo/. Sci. 14: 469-473. Yang, T.-H.. and G. N. Somero. 1996. Activity of lactate dehydroge- nase but not its concentration of messenger RNA increases with body size in barred sand bass, Pamlabrax nehulifer (Teleostei). Biol. Bull. 191:155-158. Reference: Biol. Bull. 199: 108-115. (October 2000) Dimethylsulfoniopropionate in Giant Clams (Tridacnidae) RICHARD W. HILL1 *, JOHN W. H. DACEY:. AND AHSER EDWARD3 ' Department of Zoology, Michigan State University. East Lansing. Michigan 48824; 2 Department of Biology, Woods Hole Oceanographic Institution. Woods Hole, Massachusetts 02543; and College of Micronesia-FSM. P. O. Box 159, Kolonia. Pohnpei. Federated States of Micronesia 96941 Abstract. The tridacnid clams maintain symbiotic associ- ations with certain dinoflagellates (termed zooxanthellae). Tridacnids are thus candidates to have high tissue concen- trations of dimethylsulfoniopropionate (DMSP), a tertiary sulfonium compound that is not synthesized by animals but is commonly produced by dinoflagellates. This study estab- lishes that DMSP is about an order of magnitude more concentrated in the light-exposed and shaded mantle and gills of Tridacna maxima and T. squamosa than in any other known animal tissues. The DMSP concentration in the light-exposed, siphonal mantle — the location of most zoo- xanthellae— is an inverse function of body size, paralleling an inverse relation between apparent density of zooxanthel- lae (measured as pheophytin concentration) and body size. The shaded mantle and gills are high in DMSP despite having low densities of zooxanthellae, indicating that high DMSP concentrations occur in molluscan tissue, not just in algal cells. DMSP is almost an order of magnitude less concentrated in the adductor muscle than in other tissues. The high DMSP concentrations found in tridacnids, by providing abundant substrate for formation of volatile di- methylsulfide. probably explain the peculiar tendency of tridacnids to rapidly develop offensive odors and tastes after death: a serious problem for their exploitation as food. Tridacnids are the one group of animals in which DMSP concentrations are high enough in some tissues to be in the range capable of perturbing enzyme function at high phys- iological temperatures. Thus, tridacnids may require en- zyme forms adapted to DMSP. Received I December 1999; accepted 16 June 2000. * To whom correspondence should be addressed. E-mail: hillr@msu.edu Ahhreviations: DMS. dimelhylsulride; DMSP. dimethylsultomopropi- onate. Introduction Some of the most interesting marine animals are those that maintain symbiotic associations with dinoflagellates. The symbiotic dinoflagellates are known as zooxanthellae. Included are the reef-building scleractinian corals, many alcyonarians, all of the about eight species of giant clams of the family Tridacnidae, and a few other bivalves (Ma- ruyama et al.. 1998). Dinoflagellates, as a group, are note- worthy for synthesizing relatively large quantities of di- methylsulfoniopropionate (DMSP), a nonvolatile tertiary sulfonium compound that is the precursor of volatile di- methylsulfide (DMS) (Keller ct al., 1989a,b). DMSP is not synthesized endogenously by animals. However, the wide- spread synthesis of DMSP by dinoflagellates provides rea- son to predict accumulations of DMSP and DMS within the tissues of zooxanthellate animals. This prediction has been assessed heretofore only in reef-building scleractinians. The presence of the reduced-sulfur compounds in reef-building corals was confirmed initially by the observation of DMS release from damaged reefs (Andreae et al.. 1983). Later, two of us quantified DMSP and DMS in the tissues of healthy corals and in free-living dinoflagellates isolated from corals (Hill et al., 1995b). In giant clams, the zooxanthellae, which currently are assigned to two of the major subdivisions of Symbiodiniiim (Rowan, 1998), occur primarily in the siphonal mantle tissue (Norton et al., 1992). This expansive tissue faces upward when the clams are in their natural orientation and is presented to the sun as a light antenna. The part of the mantle that is positioned near the downward-facing byssal opening and hinge, shaded from the sun. contains relatively few zooxanthellae (documented in this study). Similarly, zooxanthellae are sparse or absent from the adductor mus- cle, gills, and other tissues besides the siphonal mantle. IDS DMSP IN GIANT CLAMS 109 Recent molecular evidence confirms that the giant clams are a monophyletic group (Maruyama el ai. 1998). This paper, based on two species, is the first to look for or quantify DMSP and DMS in the group. It is also the first to examine DMSP and DMS in zooxanthellate animals besides reef- building scleractinians. DMSP and DMS are of current interest for several rea- sons. The most prominent is that atmospheric DMS origi- nating from marine organisms affects cloud cover and cli- mate over the oceans (Shaw. 1983; Charlson et ai, 1987; Falkowski et ai, 1992; Andreae and Crutzen. 1997). Coral reefs are sufficiently extensive that DMS from corals could be of local climatic significance (Andreae et al., 1983; Hill et ul.. 1995b). However, DMS from giant clams seems unlikely to be climatically important except as a minor component of reef-community contributions, because the clams are insufficiently abundant, especially in modern times. On the other hand, DMSP and DMS in giant clams are likely important in two major ways. First, DMS is well known to have critical effects on taste whenever it is present in organisms used for food (Mo- tohiro, 1962; Ackman et ai. 1966: Levasseur et ui. 1994). Giant clams have long been important sources of food and protein throughout much of the Indo-Pacific, so much so that many clam populations are decimated (Munro, 1989; Dalzell et ai. 1996). A problem for the indigenous and commercial exploitation of giant clams is that after death, the meat often promptly develops a strong, "unquestionably offensive" odor (Peavey and Riley, 1993, 1994), which is sometimes described as seaweed- or kelp-like (e.g.. Cowan, 1988). The cause has been unknown. Our experience with DMS and with the odors of the clams led us to postulate that the cause is DMS derived from the algal symbionts. If this hypothesis is confirmed, the stage will be set for a rational approach to a problem that seriously detracts from the value of the clams as sources of food in subsistence economies and as aquacultured species. Giant clams are attractive animals for aquaculture (Braley, 1988; Munro, 1989) in part because their symbionts enable them to get most of their energy for maintenance and growth from sunlight (Klumpp and Griffiths, 1994); they have been described as "the only phototrophic, and thus self-feeding, potential farm animals known to humankind" (Munro, 1989). Second, recent research (Nishiguchi and Somero, 1992: Karsten et ai. 1996) has established that DMSP sometimes negatively perturbs enzyme function at high physiological temperatures. The enzyme-perturbing effects of DMSP have heretofore been considered relevant only to plants and algae, because no animals have been known to have DMSP concentrations sufficiently high to be influential. We hy- pothesized that the giant clams might have tissue DMSP concentrations high enough that they could potentially re- quire biochemical adaptations to DMSP. Materials and Methods Tridacmi maxima and T. sc/namosa were collected near Pohnpci in the Federated States of Micronesia. Two collec- tions were made: one of six specimens of T. maxima (1 1-14 cm shell length) and four of T. squamosa ( 10-14 cm) on 13 July 1995. on the northern barrier reef of the main island of Pohnpei; the second of three specimens of T. maxima (16-18 cm) and nine of T. si/iuunosa (14-22 cm) on 17 August 1995, in the lagoon of Ant atoll. 27 km from the first location. Collected animals were taken by boat to Kolonia. Pohnpei. where they were promptly dissected (3-4 h after collection). Samples (1-2 g) were cut from four tissues of each clam: light-exposed, siphonul mantle: shaded mantle from near the byssal opening: adductor muscle; and gill (sometimes both right and left gill sets combined). Each sample of tissue was weighed and placed into 20 ml of HPLC-grade absolute methanol in a 37-ml glass vial sealed with a Teflon-faced butyl-rubber septum (Regis Technol- ogy) secured with a crimped aluminum ring. Samples prepared in Pohnpei were shipped in light-tight containers to Woods Hole, Massachusetts, for assay. Ship- ments required 6-8 days to reach Woods Hole, and assays were completed 8-10 days after collection. Vials had been filled with methanol and weighed prior to shipment to Pohnpei, and they were reweighed on return to Woods Hole as a check for leakage (none occurred). To measure DMSP and DMS, 1.0 ml of methanol was drawn by syringe from each tissue-sample vial and placed in 25 ml of 2 N KOH in a sealed vial. Incubation in cold base quantitatively converts DMSP to DMS (Dacey and Blough, 1987). Thus, after incubation (20 h. 2°C), DMSP (plus any DMS present in the initial samples) could be measured by assaying DMS. For assay, the vials of KOH were brought to room temperature (ca. 22° C), and DMS was measured in head-space samples by gas chromatography, using a Chro- mosil 330 (Supelco) column at 54°C for separation, Sievers 350B sulfur chemiluminescence detector, and Hewlett Packard 3390A integrator. Standards were prepared in 25 ml of 2 N KOH plus 1 ml methanol using reagent grade DMS (Fluka). All measures were duplicated. Chlorophyll in each tissue sample was measured as an index of the density of zooxanthellae. In fact, because some chlorophyll could have degraded to pheophytin during sam- ple preparation and shipment, and because our interest was not in chlorophyll itself but in an index of relative levels of zooxanthellae, we degraded all chlorophyll by acidification (3 niA/ HC1) and used the resulting total pheopigment levels as our index (Hill et al.. 1995b). Chlorophyll a and pheopig- ment a were measured using a calibrated Turner model 10 fluorometer and standards of chlorophyll a from spinach (Sigma) in methanol following procedures recommended by Holm-Hansen and Reimann (1978). Aliquots of methanol drawn by syringe from tissue-sample vials were diluted in 110 R. W. HILL ET AL. 600 500 400 05 § 200 I g 100 .c o^ 0 V=-17.6X+ 568 10 12 14 16 18 20 22 Length of Shell (cm) Figure 1. Pheophytin a per gram of siphonal mantle (wet weight) as a function of shell length in Tridacna maxima (filled symbols) and 7". squamosa (open symbols). The line and equation are results of linear, least-squares regression, all data taken together. Two data points for 1 7-cm clams are shifted laterally for clarity. absolute methanol to place concentrations on the linear parts of calibration curves. In Pohnpei. tissue samples were prepared, weighed, and inserted in vials as solid blocks of tissue to minimize po- tential loss of DMS. To assure that extractions of DMSP, DMS, and chlorophyll from samples into methanol were complete, tissue samples were removed briefly from sample vials after completion of the measurements described above and minced with scissors into small pieces (ca. 1 mm greatest thickness) that fell back into the vials (the proce- dure required about 1 min per sample). After 24-48 h, all measurements were repeated. Chlorophyll concentrations were not altered by mincing, and concentrations of pooled DMSP and DMS (measured as earlier described) were al- tered little, if at all (possibly 3%-5% in the case of mantle samples). The mincing test demonstrated that extraction from whole tissue was complete or virtually complete, and all assays for a sample were averaged to obtain the results reported. A second check on our technique was to test whether the high tissue DMSP concentrations we encoun- tered might be so high as to saturate the methanol. The highest concentration of DMSP in the methanol in any tissue-sample vial was 4.3 mA/. Without attempting to de- termine the absolute solubility of DMSP in methanol, we ascertained that pure DMSP sufficient to make a solution three times as concentrated dissolved rapidly in methanol. Thus, saturation of the methanol in the sample vials did not occur. Total amounts of pooled DMSP and DMS (in micro- moles) and of pheopigment a (in micrograms) in tissue samples were calculated from concentrations in sample-vial methanol by multiplying by the volume of methanol (20 ml), then expressed per unit wet-weight of tissue. Results For simplicity of language, we express results in terms of DMSP, although we do not know what proportions of the DMS analyzed were initially in the form of DMS or DMSP. Most was probably DMSP (the nonvolatile form known to be the principal chemical species in algae; see also com- parative data on molluscs presented later). Results from Tridacna maxima and 7". squamosa were not statistically distinguishable and thus are generally pooled. Pheophytin a per gram of siphonal (light-exposed) mantle was an inverse function of body size (analyzed by linear regression, P = 0.008, r = 0.30), as shown in Figure 1. DMSP per gram of siphonal mantle was likewise an inverse function of body size (P = 0.015. r = 0.26). as shown in Figure 2. The concentrations of DMSP and pheophytin in the siphonal mantle were strongly correlated (P < 0.001 ), as shown in Figure 3. The ratio of DMSP concentration to pheophytin concentration in siphonal mantle was quite con- sistent, the mean and standard error being 0.107 ± 0.0048 (range: 0.070-0.158) /j,mol//u,g. Pheophytin per gram did not show a systematic relation to body size in the byssal (shaded) mantle, adductor, or gill. Table 1 summarizes the pheophytin concentrations in these tissues. Comparison to Figure 1 shows that the concentra- tions were far lower than in the siphonal mantle. The simple mean concentration in the siphonal mantle, 303 ju.g/g, is of uncertain utility because of the regular relation between siphonal-mantle concentration and body size, but it helps bring to light that pheophytin concentrations in the byssal mantle and gill were only about 7% and 3% as high as those in siphonal mantle. Concentrations of pheophytin in the adductor approached zero. 50 40 30 . Q. t/5 C 57 CO 20 10 X=-1.43X+52.7 10 12 14 16 18 Length of Shell (cm) 20 22 Figure 2. Concentration of dimethylsulfomopropionate (DMSP) in siphonal mantle (wet weight) as a function of shell length in giant clams. The line and equation are results of linear, least-squares regression, all data taken together. Filled symbols. Tridacna maxima; open symbols, T. squa- DMSP IN GIANT CLAMS Ill o 3_ = c 50 40 30 f en | 20 r=0.74 o i 00 100 200 300 400 500 [Pheophytm] in Siphonal Mantle (|ig/g) Figure 3. Correlation between concentrations of dimethylsulfoniopro- pionate (DMSP) and pheophvtin a in siphonal mantle of giant clams. Filled symbols. Tridacna maxima; open symbols, T. squamosa. r = correlation coefficient. 70 r=0.68 « 0> 60 15 0 E 0 -= 50 • . c 0 5 40 0 0 • ~ca 0 W w >, CD 30 • 0 ° • *> o • c & 20 0 Q_ 0 10 20 30 40 [DMSP] in Siphonal Mantle (umol/g) 50 Figure 4. Correlation between concentrations of dimethylsulfoniopro- pionate (DMSP) in byssal and siphonal mantle in giant clams. Filled symbols. Tridacna maxima: open symbols. T. si/iuimosa. r = correlation coefficient. As shown in Figure 4. the concentration of DMSP in the byssal mantle tissue was strongly correlated with (P < 0.001 ) and similar to that in the siphonal mantle tissue, even though densities of zooxanthellae in the byssal tissue, as inferred from byssal pheophytin concentrations, were a small fraction of those in the siphonal tissue. Presumably because of the correlation between siphonal and byssal concentrations, the byssal DMSP concentration exhibited a regular relation to body size, similar to that in Figure 2 [linear regression: Kl/imol/g) = — 2.46X(cm) + 74.4; P = 0.001]. Byssal DMSP concentration showed no correlation with byssal pheophytin concentration. The DMSP concentrations in gill and adductor were Table 1 Pheophytm a per gram of tissue of giant clams (wet weight, species combined) in the three tissues that showed no relation benveen concentration and body size Pheophytin a (/ng/g) Tissue Mean Range Byssal mantle* Gill Adductor muscle 22.10 10.00 0.74 1.34-86.90 0.35-31.30 0.05-3.32 Data for siphonal mantle are omitted because they are presented else- where (Fig. 1) and because the mean is a possibly misleading statistic for a parameter that varies systematically with body size. * The four highest values for byssal mantle occurred in four of the smallest clams (two of each species), suggesting that the effort to keep tissue-sample size consistent might have led to the inclusion of other types of tissue in byssal-mantle samples of some small clams. If the four highest values are excluded, the mean and range for byssal mantle are 12.8 and 1.34-33.1 unrelated to body size and uncorrelated with pheophytin concentrations in the respective tissues. The DMSP concen- tration in gill was positively correlated with that in mantle (/• = 0.61, P < 0.01 for siphonal mantle; r = 0.45. P < 0.05 for byssal mantle), but the DMSP concentration in adductor was not correlated with that in mantle. Table 2 presents DMSP concentrations in gill and adductor. For comparison, the means in siphonal and byssal mantle were 31.2 and 37.4 iiunoi/g (see Fig. 4). Note that gill exhibits DMSP concen- trations similar to those of mantle. In adductor, however. DMSP is almost an order of magnitude less concentrated than in mantle. Discussion DMSP in the mantle and gill tissues of Tridacna maxima and T. squamosa is far more concentrated than in any animal tissue heretofore known. Most comparative data in the literature represent pooled concentrations of DMSP and DMS (similar to the data we collected). In discussing the literature, we distinguish DMSP and DMS only if the orig- Table 2 Dimethylsulfoniopropionate (DMSP) per gram of tissue of giant clams (wet weight, species combined) in the nro tissues that showed no relation between concentration and bod\ size DMSP (/imol/g) Tissue Mean Range Gill Adductor muscle 33.3 4.4 20.3-46.1 1.8-7.2 Data for mantle are omitted for reasons stated in note to Table 1. 112 R. W. HILL ET AL. inal investigators did. We also exclude from consideration tissues (e.g., stomach) that could contain unassimilated food. Two surveys of DMSP concentrations in molluscs have been carried out. lida and Tokunaga ( 1986) measured both DMS and DMSP in 1 1 species of bivalves and 5 of gastropods from Japanese waters. An average of 8% of the total molar amount of the two compounds was DMS and 92% was DMSP in the mantle, gill, and adductor tissues of the bivalves. The sum of the two concentrations was usually less than 0.2 jumol/g. The single highest sum was 0.9 ^mol/g in adductor muscle of oysters (Crassostrea gigas). Ackman and Hingley (1968) reported 0-1.8 /nmol/g in the tissues of 10 species of bivalves and gastropods from Ca- nadian waters. Most values were toward the low end of the range; the highest concentrations were in adductor muscles of scallops (Placopecten megullciniciis) and oysters (C. vir- ginica). The highest concentrations observed in populations of mussels (Mytilux editlis) at Cape Cod, Massachusetts, were 2-4 /imol/g (Hill et al., 1995a). In wild-caught fish, muscle or liver concentrations of 0.2-1.0 /nmol/g (predom- inantly DMSP in fresh tissue) are high and commercially problematic because they cause off-flavors (see later) (Mo- tohiro, 1962; Ackman ft al., 1967; lida et al., 1986; Dacey ct al.. 1994). Even the highest tissue concentrations in fish fed DMSP supplements were only 4-8 /imol/g (Ackman et al., 1966). A survey of DMS and DMSP in several species of shrimp and krill indicated that tissue concentrations are very low in most species, although pooled concentrations (predominantly DMSP) as high as 3 /j,mol/g are sometimes observed in muscle of Eiiplun/sia superba (Tokunaga ct al., 1977). In the context of these comparative data, the con- centrations in the mantle and gill tissues of tridacnids, averaging 31-37 jumol/g, are extraordinary. The only pub- lished animal data that are at all in the same range come from a single report on pteropods (Levasseur et al., 1994) in which extremes of 30-40 /Ainol/g were observed (calcu- lated form published data on tissue dry weights assuming the animals to be 70% water). Such concentrations, how- ever, are exceptional in the literature on pteropods; other reports are 0.2-3.7 ju.mol/g (Motohiro. 1962; Ackman and Hingley, 1968) or lower (Ackman ct al., 1972). Further- more, the pteropod data are for whole animals, including digestive-tract contents. In terms of documented evidence on tissues of animals collected in the wild, the concentra- tions in tridacnid mantle and gill are extreme: an order of magnitude higher than the highest concentrations observed in other bivalve or gastropod molluscs, fish, crustaceans, and other animals. Other than the tridacnids, the animals mentioned acquire DMSP strictly from ingested foods. We hypothesize that the high concentrations in tridacnids are a consequence of the production of DMSP by their algal symbionts. Three pieces of evidence support this hypothe- sis. First, at least in T. M/iianinsa, symbiont photosynthesis supplies over 90% of all organic carbon acquired by indi- viduals of the body sizes we studied (Klumpp and Griffiths, 1994). Thus, although the clams ingest food to some extent and could acquire DMSP from their food, the known par- titioning between phototrophic and heterotrophic nutrition makes phototrophic production of DMSP the more likely source of most DMSP. especially because symbiotic dinofiagellates are documented to produce DMSP (Hill et al., 1995b). Second, the sheer magnitude of the DMSP concentrations in the tridacnids points to their algal symbi- onts as the source, because in all the many species of studied molluscs that lack algal symbionts, even the most extreme DMSP concentrations seen do not come close to the routine concentrations seen in the tridacnids. Third, there are sev- eral reports that the density of zooxanthellae in tridacnid tissues is an inverse function of body size (e.g.. Fisher et al., 1985; Griffiths and Klumpp. 1996). and we found evidence of this same relation in siphonal mantle (Fig. 1 ). If the zooxanthellae are the primary source of DMSP and if the zooxanthellae become less dense in the siphonal mantle as individuals grow, one would predict that the DMSP con- centration would be an inverse function of body size. This is what we found (Fig. 2). The DMSP and pheophytin concentrations in the siphonal mantle were well correlated (Fig. 3). and the ratio of the concentrations was consistent at 0.107 ± 0.0048 (SE) /nmol/^g. If our hypothesis is correct that the zooxanthellae are the main source of DMSP in giant clams, then tissues other than the siphonal mantle must receive DMSP by internal trans- port from the siphonal mantle because, as our pheophytin data confirm, the zooxanthellae occur at high densities only in siphonal mantle (Table 1 ). Internal transport could occur by circulation of hemolymph or by transport in the zoo- xanthellal tubular system (Norton et al., 1992). Molluscan tissues are well known to accumulate DMSP (Ackman and Hingley, 1968; Hill et al., 1995a). The high concentrations of DMSP in siphonal mantle could possibly be explained by high concentrations in the algal cells only. However, the byssal mantle and gills are so low in algal cells that the high concentrations of DMSP there almost certainly demonstrate that the animal cells of giant clams can experience very high DMSP concentrations. The physiological basis for the dra- matic difference in concentration between the adductor muscle and other tissues (Fig. 5) awaits study. Whatever the cause of the high DMSP concentrations in tridacnids, the concentrations are likely to be important to the biology of the clams in two major ways. The first is taste. One of the principal conclusions of research on com- mercialization of giant clams is that their meat is exception- ally perishable because of the rapid development of a "par- ticularly offensive and pervasive odor" (Peavey and Riley. 1994). Refrigeration (Peavey and Riley, 1994) or freezing in a domestic free/er (Peavey and Riley, 1993) does little to prevent this problem, even if the viscera have been re- moved. Comparing mantle and adductor meal, a disagree- DMSP IN GIANT CLAMS 113 Kidney Siphonal Mantle Pericardium Adductor 'Muscle Hinge Region Cilli Umbo Byssal Mantle Figure 5. A giant clam in the natural orientation showing average pooled concentrations 1/u.mol/g) of dimethylsulfoniopropionate (DMSP) and dimethylsulhde (DMS) in the four tissues studied (drawing by Jayne Doucette). able "seaweed-like" odor has been particularly associated with the mantle (Cowan, 1988), and the mantle is far less commercially valuable than the adductor (Braley, 1988; Tisdell and Tacconi, 1993). These are serious matters in many parts of the world where giant clams occur. The clams are sources of food and protein in subsistence economies, and a number of cash-poor governments are making sub- stantial investments in the development of giant clam aquaculture (Lucas, 1994). Partly because of preservation problems, the mantle meat may actually have a negative monetary value in commercial aquaculture (Hambrey and Gervis. 1993). We believe we have discovered the cause of the unattrac- tive odor and taste, thereby setting the stage for a rational approach to improvement. Although DMS has not been mentioned previously as a likely component of triducnid tissues, it is well recognized as an important taste constitu- ent in other seafoods. DMS produced from dietary DMSP is a negative taste factor in fish. The DMS generated during certain sorts of processing offish meat containing as little as 0.1-1 ju.mol DMSP/g can cause the meat to smell "like petroleum" or taste like turnip or radish (Motohiro, 1962; Ackman et al., 1966) and force catches to be discarded. On the other hand, very low concentrations of DMS are part of the valued flavor of some clams and oysters (Ackman and Hingley. 1968: Brooke et ai, 1968; lida and Tokunaga. 1986). Brooke et al. (1968), for example, found that about 0.02 /xmol/g of DMS helps impart a desirable "clamlike" odor to A/vfl arenaria, whereas more than 0.3 jumol/g of DMS is excessive. Our data reveal that the potential for DMS formation in tridacnid tissues, particularly mantle, is enormous. We as- sume that in the fresh clams we studied, most of the sulfur we measured was in the form of DMSP (lida and Tokunaga, 1986), which has unknown taste effects. After the death of the clam. DMSP is likely under many circumstances (Mo- tohiro. 1962: Ackman et ai. 1966) to be broken down to DMS enzymatically (e.g., by bacterial DMSP lyases; Led- yard el al., 1993) or nonen/ymatically (e.g.. Dancey and Blough. 1987). With over 30 jumol/g of DMSP present, the concentrations of DMS that could readily be formed are far in excess of ones known to make all other foods inedible. It is probably no accident that the methods devised by indig- enous people to preserve tridacnid meat include acidic washes and drying (Munro, 1989; Hambrey and Gervis, 1993). Acid pHs inhibit the nonenzymatic breakdown of DMSP to DMS (Motohiro. 1962; Dacey and Blough, 1987), and because DMS is very volatile, drying would remove it from tissue. Looking to the future, it might be possible to inhibit DMS formation in a chemically specific manner or even to develop strains of zooxanthellae that produce little DMSP. The second major way in which DMSP concentrations are potentially important to tridacnid biology is their bio- chemical significance. Recognizing that DMSP is employed by some organisms to help set the colligative properties of cellular solutions. Nishiguchi and Somero (1992) studied the effects of DMSP on cellular proteins. They found evi- dence of temperature dependence. Whereas DMSP exhib- ited stabilizing effects on proteins at low temperatures, it could perturb protein function at high physiological temper- atures. In particular. Nishiguchi and Somero found that DMSP promotes the denaturation of glutamate dehydroge- nase at 37°C in a concentration-dependent manner, with effects evident at the lowest concentration tested, 100 mM DMSP. Similarly. Karsten et al. ( 1996) observed a concen- tration-dependent suppression of activity of malate dehy- drogenase at 30°C: the suppression became evident at con- centrations between 19 and 75 mM DMSP. Such effects of DMSP have been tacitly assumed to be relevant just to plants and algae because only plants and algae have hitherto been thought to have native DMSP concentrations high enough to be in the effective range. Our results make clear that alone among animals, tridacnids can have DMSP con- centrations within the range shown to have enzyme-perturb- ing effects. In T. maxima and T. squamosa of the sizes we studied, the mantle averages about 0.83 ml water per gram wet weight (our unpublished data). Thus, if we assume that the DMSP in mantle is entirely dissolved and distributed evenly in tissue water, the mean concentration of DMSP in the tissue water is over 40 mM. On the basis of the chemical structural properties of DMSP and the preferential hydration model. Nishiguchi and Somero (1992) argue that DMSP. like dimethylsulfoxide. may be toxic to cells and may de- nature proteins at high physiological temperatures. In warm tropical waters and especially in shallows where solar heat- 114 R. W. HILL ET AL. ing can occur, the high DMSP concentrations of tridacnids may be stressful by-products of extreme exploitation of phototrophic nutrition (Klumpp and Griffiths, 1994). Tri- dacnids may require specializations of metabolic chemistry to reduce or tolerate enzyme-perturbing and other toxic effects of their high DMSP concentrations. 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EVAN WARD2 ^Department of Ecolog\ and Evolution. State University of New York. Stony Brook. New York 11794; and 'Department of Marine Sciences. University of Connecticut. Groton, Connecticut 06340 Abstract. The capture, transport, and sorting of particles by the gills and labial palps of the freshwater mussel Dreissena pohmorpha were examined by endoscopy and video image analysis. More specifically, the morphology of the feeding organs in living zebra mussels was described; the mode and speeds of particle transport on the feeding organs was mea- sured; and the sites of particle selection in the pallial cavity were identified. Particle velocities (outer demibranch lamellae. 90 jam s~ '; inner demibranch lamellae. 129 jam s~ '; marginal food groove of inner demibranchs. 1 56 ju,m s '; dorsal ciliated tracts, 152 /xm s"1). as well as the movement of particles on the ctenidia and labial palps of D. polymorpha. are consistent with mucociliary, rather than hydrodynamic. transport. Parti- cles can be sorted on the ctenidia of zebra mussels, resulting in a two-layer transport at the marginal food groove of the inner demibranch. That is: preferred particles are transported inside the marginal groove proper, whereas particles destined for rejection are carried superficially in a string of mucus. Sorting also occurs at the ventral margin of the outer demibranch; desirable particles are retained on the outer demibranch. whereas unacceptable particles are transferred to the inner demibranch and ultimately excluded from ingestion. We sug- gest that the structure of homorhabdic ctenidia does not pre- clude particle sorting, and that some ecosystem modifications attributed to zebra mussels may ultimately be due to ctenidial sorting mechanisms. Introduction Many suspension-feeding organisms, including bivalves, sort food particles on the basis of size ( Vahl, 1972; Stenton- Received 27 October 1999: accepted 14 July 2000. * Present address: Department of Fisheries and Aquatic Sciences. Uni- versity of Florida. 7922 NW 71st Street. Gainesville. Florida 32653-3071. E-mail: smbaker@mail.ifas.url.edu •\hhrc\-uitioits: OIT, optical insertion tube of an endoscope. Dozey and Brown, 1992; Defossez and Hawkins, 1997) and quality (MacDonald and Ward, 1994; Arifin and Bendell- Young. 1997; Ward et al.. 1997). Moreover, endoscopie examination and video image analysis are now frequently used to directly observe particle capture, transport, and sorting in marine bivalves (Ward et ai. 1991, 1998; Beninger et ai. 1992; Ward. 1996). With this method, pallial structures can be observed in vivo in relatively un- disturbed specimens, so that the direction, velocity, fate, and hydrodynamic mechanisms of ciliary transport of particles within the pallial cavity can be determined directly (Ward et al.. 1993). The endoscope does not disturb the morpholog- ical and hydrodynamic relationships of pallial organs, an advantage over the usual techniques of excision and surgical alteration of living bivalves (Nelson, 1960; Galtsoff, 1964; J0rgensen, 1966). In previous studies. Ward et al. ( 1998) found that particle sorting in oysters (subclass Pteriomorphia; psuedolamelli- branch, heterorhabdic. plicate gills) takes place on the ctenidia; particles of differing food qualities are partitioned between the marginal groove and the dorsal ciliated tract. In contrast, the ctenidia of marine mussels (subclass Pteri- omorphia; filibranch. homorhabdic, nonplicate gills) play little role in particle selection. Ward et al. (1998) suggested that selection by the oyster ctenidia reflects the greater complexity of those organs. (For review of bivalve gill anatomy, see Atkins, 1937a,b; Ruppert and Barnes, 1994.) Zebra mussels [Dreissena polymorpha (Pallas. 1771 )] are freshwater suspension-feeding bivalves in the subclass Het- erodonta. Like marine mussels, zebra mussels have homo- rhabdic. nonplicate ctenidia. But. unlike marine mussels, they are eulamellibranchs; ctenidial filaments are connected by interfilamentous tissue junctions. This ctenidial condi- tion is more similar to that of the pseudolamellibranch oysters in which the filaments are connected by some. 116 PARTICLE TRANSPORT IN THE ZEBRA MUSSEL 117 although not extensive, interfilamentous tissue junctions. An examination of zebra mussel ctenidia. therefore, might indicate whether ctenidial sorting is related to morphology or to phylogeny. Indeed, recent flow-cytometry studies by Baker et al. ( 1998) demonstrate that the zebra mussel pallial organs effectively sort particles. In this investigation, we use endoscopic examination to observe ( 1 ) the morphology of feeding organs in living zebra mussels. (2) the mode and speeds of particle transport on the feeding organs, and (3) sites of particle selection in the pallial cavity. We compare our endoscopic examination of zebra mussels with previous reports of feeding processes in both zebra mussels and marine bivalves. Zebra mussels have invaded many freshwater localities in Europe and North America. In systems where large popu- lations of these mussels have become established, not only has phytoplankton biomass declined (Holland, 1993; Fah- nenstiel et al., 1995; Caraco et ul., 1997), but seston com- position has changed as well (Vanderploeg et al,, 1996; Smith et al., 1998; Strayer et al.. 1999). Understanding the form and function of feeding structures in zebra mussels and their mechanisms of particle selection will contribute to a better understanding of these effects on ecosystems. Materials and Methods Phytoplankton cultures were obtained from the Univer- sity of Texas Culture Collection and grown in a freshwater enrichment medium WCL1 (Guillard, 1983: Guillard and Hargraves, 1993). Cultures were grown at room tempera- ture, under a 16:8 h light:dark regime. Species of phytoplankton that were cultured included Cvclotellu »ie- neghiniana (LB 2455; barrel-shaped, 18x6 jam) (Bacilla- riophyceae). Crucigenia tetrapedia (63; disk-shaped, 5 X 11 /nm), Scenedesmus quadricauda (LB 614; four cells stacked, total 25 X 10 ;am) (Chlorophyceae), and Micro- cystis aeruginosa (LB 2386; spherical, 4 ju,m) (Cyanophy- ceae). Cell dimensions were measured with a compound microscope and calibrated ocular micrometer. Nonliving particles were also used in endoscopic obser- vations. Polystyrene beads (Polysciences. Inc., Warrington, PA) of 1, 10, or 22 /xm were often used as tracer particles. Dead cattail (Typha sp.) leaves from the previous growing season were collected for use as detrital material. The leaves were washed of debris and processed in a blender with distilled water for 5 min. The resulting suspension was sieved through a 20-ju.m nylon screen, and the retained particles (>20 ju,m) were discarded; 90% of the particles in the remaining suspension were <3.5 jam, as measured by an electronic particle counter (Coulter Electronics, Multisizer). Specimens of Dreissena polymorpha, about 20 mm in length, were collected from the Hudson River at Tivoli, New York, or from the Huron River, Ann Arbor, Michigan. Mussels were maintained in 40-1 aquaria at 16°C and fed a daily ration of cultured phytoplankton plus a mixture of preserved diatoms (Diet C, Coast Seafoods, Co.. Quilcene. WA). Partial water changes (ca. 20%) were performed on alternating days; freshwater was prepared according to Sprung (1987). We prepared zebra mussels for endoscopy by drilling a small hole (<2 mm in diameter) in one valve with a rotary tool (Dreinel. Racine, WI) and cauterizing the underlying mantle tissue. The hook side of a piece of hook and loop fastener (Velcro brand) was cemented with epoxy to the valve opposite the drilled hole for later use in positioning the animal for examination. The mussels were allowed to recover for at least one day. This treatment caused no apparent adverse change in the behavior of the mussels, and shell and mantle repair at the site of the drilled hole often began within several days. Endoscopic examinations were performed according to Ward et al. (1991, 1993. 1994). An endoscope (K12-09-15- 53, Olympus Corp., Lake Success, NY), with an optical insertion tube (OIT) of 1.2 mm diameter, was connected to an optical zoom-adaptor (Scholly Fiberoptic, Denzlinger, Germany) and attached to a monochrome or color CCD camera (4990 or 8280. Cohu Electronics. San Diego, CA). A halogen (HLS24-0, Welch Allyn. Skaneateles Falls. NY) or xenon lamp (ALS-6250U, Olympus High Intensity He- lioid Light Source) provided light to the OIT. The camera and endoscope were mounted on a macro-focusing rail, allowing hue adjustments of the OIT. Video was recorded at 30 frames s~' on an sVHS videocassette recorder (VCR) (AG-1960, Panasonic Industrial Company, Secaucus, NJ). For endoscopic examination, mussels were placed in a 500-ml plastic container set in a 15-1 water bath with its temperature maintained between 16° and 18°C. A dome inside the plastic container had been covered with the loop side of Velcro-brand fastener, allowing rapid mounting and precise positioning of mussels. The OIT was inserted into the pallial cavity of the mussel through the inhalent siphon, the pedal gape, or the drilled and cauterized hole. Record- ings were made after the mussel showed active feeding behavior, as indicated by extension of the mantle and si- phons and by the intake of particles. Mussels were exposed to suspensions of one or two particle types at concentrations of 104, 105, and 106 particles ml~'. Particle suspensions were delivered to the plastic container by gravity from a 4-1 carboy. The container was frequently flushed to maintain particle concentration, which was also monitored with a Coulter Multisizer. We observed and recorded the positions and movements of the ctenidia and labial palps, as well as the movement of particles on these organs. The best observations were made when the OIT was inserted into the pallial cavity through the drilled hole in the shell and mantle. In this position, the mussels fed normally, uninterrupted by movements of the OIT. Although the pallial cavity could be entered through 118 S. M. BAKER ET AL the inhalent siphon, any movement of the OIT resulted in the cessation of feeding. And when the pallial cavity was entered through the pedal gape, the foot usually touched the OIT, coating it with mucus. Results were based on the examination of 21 mussels. Particle velocities on feeding structures were determined from the number of video frames required for a particle to traverse a known distance. Distances were calibrated ac- cording to Ward (1996): i.e.. the pallial organs were dis- sected from several mussels, and the widths of the ctenidial filaments, palp ridges, and marginal grooves were measured with a compound microscope equipped with a calibrated Ant iPL ocular micrometer. Velocities means ± 1 standard deviation. Results s~') are presented as When observed by endoscopy, the positions of the ctenidia within the pallial cavity are different from those that might be expected from dissected specimens (Fig. 1). The demibranchs are held curved towards the visceral mass, and the ventral margin of the outer demibranchs is partic- ularly bent inward (Fig. 2). These gill postures are main- tained despite variation in the overall orientation of the mussels. Through the relatively transparent ciliated epithelia of the ctenidia. we observed internal bands of muscular cross- struts (Medler and Silverman, 1997) that are perpendicular to the ctenidial filaments and 60-80 /xm apart. Ostia, lo- cated in the epithelium of the interfilamentary spaces, are lacking directly above the struts (Medler and Silverman. 1997). The inhalent flow of suspended particles sometimes stops, or even reverses momentarily, especially under high particle concentrations. In addition, the ctenidia often con- tract during active feeding; the interfilamentary spaces. where the ostia are located, alternately flare and close at a rate of 1 cycle s '. The extension of the mantle and siphons. often used as an indication of steady feeding, does not change during flow cessation and reversals, or during pul- sation of the ctenidia. Particles captured by the ctenidia move smoothly along the frontal surfaces of the ctenidial filaments, and particles of different types and sizes maintain their distance from each other. Mucous strings were observed on the frontal surfaces of the filaments only when the particle concentra- tion was extremely high. Outer demibranchs and their ventral imirt>iii!i The outer demibranchs and their ventral margins were observed in seven specimens, on 42 occasions, for 15.5 h of total observation time and 1.7 h of video recording. The outer demibranchs are held relatively straight, but with an inward bend, especially of the ascending lamella, near the Ventral dL of oD and iD oDCT "aL of iDand oD Dorsal Figure 1. Diagram of labial palps and anterior portions of the inner and outer demibranchs of Dreissena polymorpha as observed through the endoscope (looking dorso-anteriorly). Palp lamellae are shown slightly spread apart, with the outer Palp lamella (oPL) curled back. Solid arrows indicate particle paths after capture. The white arrow indicates movement of the outer demibranch (oD). Panicles in the medial dorsal ciliated tract (niDCT) are transported in mucous clumps and strings. Particles are transported at the ventral margin of the inner demibranch (iD) as both a groove mucous string (gMS) in the marginal groove proper (mGp) and as a superficial mucous string (sMS). The palps (iPL and oPL) enclose the inner demibranch only, drawing in the superficial mucous string (sMSl from the inner demibranch (iD) (see text for details). (aL = ascending lamella. Ant = anterior, dL = descending lamella. gMS = groove mucous string, iD = inner demibranch. iDCT = inner dorsal ciliated tract. iPL = inner palp lamella. mDCT = medial dorsal ciliated tract, mGp = marginal groove proper. oD = outer demibranch, oDCT = outer dorsal ciliated tract. oPL = outer palp lamella, scS = smooth ciliated surface, sG = superficial groove, sMS = superficial mucous string. VM = ventral margin.) See Video Note. p. 124. ventral margin (Fig. 2). The position of the outer demi- branch changes with pumping activity: the outer demi- branch is positioned near the inner demibranch when inhal- ent flow speeds are low; as flow speeds increase, the outer demibranch moves laterally away from the inner demi- branch (Fig. 1). Particles captured on the descending la- mella of the outer demibranch are transported dorsally to the PARTICLE TRANSPORT IN THE ZEBRA MUSSEL 119 VM Ventral dL of oO and ID aL of ID and oD Dorsal Figure 2. Diagram of the ventral margin (VM) of the outer demi- branch (oD) ot Dreissena polymorpha as observed through the endoscope (looking dorso-anteriorlyl. Solid arrows indicate particle paths after cap- ture. Particles on the ascending lamella (aL) are transported either dorsally or ventrally, depending on whether they are above or below a divergence area (DA) when captured. Upon reaching the ventral margin (VM), parti- cles traveling ventrally either ( 1 ) move over the ventral margin and proceed dorsally on the descending lamella (dL): (2) continue moving ventrally. leaving the surface of the ctenidia and becoming resuspended; or (3) make a right angle turn and begin moving anteriorly on the ventral margin (VM) (see text for details). (aL = ascending lamella. Ant = anterior. DA = divergence area, dL = descending lamella. mDCT = medial dorsal ciliated tract. oDCT = outer dorsal ciliated tract, VM = ventral margin.) See Video Note, p. 124. space between the two demibranchs is small (Fig. 1). Par- ticles captured ventral to the divergence line on the ascend- ing lamella of the outer demibranch move ventrally. Upon reaching the ventral margin, one of the following three behaviors occurs (Fig. 2): ( 1 ) The particles move over the ventral margin and proceed dorsally on the descending lamella of the demibranch. (2) The particles continue mov- ing ventrally, leave the surface of the ctenidia. and become resuspended. Most often, these resuspended particles are then recaptured by the descending lamella of the inner demibranch and continue moving ventrally. The majority of particles that leave the ventral margin of the outer demi- branch are large, like Scenede sinus. (3) The particles make a right angle turn and begin moving anteriorly on the ventral margin. Individual particles bounce along the ventral mar- gin from filament to filament at a mean velocity of 65 p.m s"' (Table 1). At high concentrations Old6 ml"1), parti- cles are sometimes earned along the ventral margin in clumps of mucus. As a result of these three particle trajec- tories, desirable particles are retained on the outer demi- branch, while unacceptable particles are transferred to the inner demibranch and ultimately excluded from ingestion. Inner demibranchs and their marginal grooves The inner demibranchs and their marginal grooves were observed in seven specimens, on 60 occasions, for 29.7 h of total observation time and 2.9 h of video. Particles captured on either descending or ascending lamellae of the inner demibranch are transported toward the ventral margin, whose mean width is 276 /j,m (n = 7; Fig. 1 ). Mean particle velocities on the ascending and descending lamellae of the inner demibranch are 129 /xm s"1 (Table 1). Material at the ventral margin of the inner demibranch is transported anteriorly in one of two channels, one deep and one superficial (Figs. 1. 3). The deep channel, the marginal groove proper, is nearly enclosed by the projection of the ventral tips of the filaments over the groove. The superficial medial dorsal ciliated tract (Fig. 2). On the ascending la- mella of the outer demibranch, particles are transported either dorsally or ventrally, depending on whether they were captured above or below a divergence area located near the bend of the demibranch (Figs. 1,2). When particles are captured directly at the divergence area, they oscillate in place for several seconds before proceeding either ventrally or dorsally. The position of the divergence area can shift ventrally or dorsally by about 1 mm. This shift does not appear to be correlated with any particular stimulus, such as particle type or concentration. Mean particle velocity on the frontal surfaces of the ascending lamella of the outer demi- branch is 90 jam s"1 (Table 1). Due to the bend of the outer demibranch, the ventral margin is pointed towards the inner demibranch, and the Table 1 Particle velocities (22-jiim polystyrene beads) on the riullial organs of -ebru mussels. Dreissena polymorpha, at 18°C Location Mean velocity ± SD Range l^m s ' ) (/itn s"' Outer demibranch Frontal surface 90 ± 22 60-123 9 Ventral margin 65 ± 23 24-104 19 Inner demibranch Frontal surface 129 ± 54 42-25 1 33 Ventral food groove 156 ± 53 45-354 106 Dorsal ciliated tract 152 ± 62 41-305 50 Labial palps Frontal surfaces 94 ± 34 54-150 10 Ventral margin 54 ± 21 16-1 13 33 120 S. M. BAKER ET AL channel or groove is a depression at the center of the ventral margin, with openings between adjacent and opposite fila- ment tips leading to the marginal groove proper (Fig. 3). The superficial groove is entirely exposed to the inhalent flow. Particles transported in the superficial groove are embed- ded in a string of mucus, up to 80 /xm thick (Figs. 1, 3), which moves at a mean velocity of 156 /xm s~' (Table 1). The presence of a superficial mucous string, as opposed to a particle slurry, was confirmed by observing the dislodg- ment of strings from the ventral margin when the valves were rapidly adducted, or when a jet of water was pipetted into the hole through which the OIT was inserted. After dislodgment, the superficial mucous string remains unbro- ken and returns to the ventral margin of the demibranch. Polystyrene spheres (1 /LUII) and the large green alga Scenedesmns are incorporated into the superficial mucous string and are eventually rejected as pseudofeces. Particles transported anteriorly inside the marginal groove proper are also embedded in a string of mucus ( Figs. 1, 3). These particles appear to be those that are eventually ingested. This observation was confirmed when specimens were fed a combination of Microcystis, which is preferen- tially ingested, and Scenedesmns or Typha detritus, which are both rejected (Baker el at., 1998). The resulting colors of the mucous strings, as well as the relative particle sizes, indicate that Microcystis is incoiporated into the groove mucous string, whereas Scenedesmns or Typha is incorpo- rated into the superficial mucous string. Two other obser- vations suggest that particles moving inside the marginal groove proper are also embedded in mucus. First, particles inside the marginal groove proper move at the same velocity as particles transported in the superficial mucous string. Second, particles both inside and outside the marginal groove maintain positions relative to each other as they move anteriorly. Additional observations suggest that the deep and superficial mucous strings are not continuous with each other, but are physically separate. For example, when the superficial mucous string is dislodged from the superfi- cial groove, the groove mucous string is not disturbed and remains within the marginal groove proper. Particles approaching the ventral margin of the inner demibranch have three potential fates (Fig. 3): ( 1 ) As par- ticles round the crest of the ventral margin, some move anteriorly and diagonally, bouncing from filament to fila- ment, before being entrained in the superficial mucous string. These particles join the more ventral portion of the superficial mucous string, and move in a uniform, smooth manner (Fig. 3, path 1 ). (2) Other particles move laterally prior to rounding the crest of the ventral margin, and enter the interfilamentary space between two adjacent filaments. Some of these particles stall, oscillate for several seconds, and then disappear from view, possibly lost to an underlying water tube through an ostial opening. Many particles, how- Ventral dL of oD and ID . of ID and oD Dorsal Figure 3. Diagram of the ventral margin of the inner demibranch of Dreissena pohmorpha as observed through the endoscope (looking dorso- anteriorly). The groove mucous string (gMS) and a portion of a superficial mucous string (sMS) are shown. Solid arrows indicate particle paths after capture. Shaded arrows indicate particle paths behind a filament. Particles approaching the ventral margin (VM) of the inner demibranch (iD) enter the mucous strings in three ways: ( 1 ) As particles round the crest of the ventral margin (VM), they move anteriorly and diagonally, bouncing from filament to filament. These particles are entrained in the top (ventral side) of the superficial mucous string, where they move smoothly towards the anterior of the ctenidium. (2) Particles move laterally prior to rounding the crest of the ventral margin and enter the furrow between two adjacent filaments (dashed arrow). These particles become entrained in the bottom (dorsal side) of the superficial mucous string and follow the contours of the filament tips as they move anteriorly. (3) Particles remain on the frontal MII laces of the filaments until reaching the tips, where they move laterally and down (dorsally) into the marginal groove proper (see text for details). These particles move smoothly towards the anterior in the groove string. (aL = ascending lamella. Ant = anterior, dL = descending lamella. gMS = groove mucous string. mGp = marginal groove proper. sG = superficial groove, sMS = superficial mucous string, VM = ventral mar- gin.) See Video Note. p. 124. ever, continue moving toward the superficial groove and became entrained in the more dorsal portion of the super- ficial mucous string. These particles do not move smoothly but follow the contours of the filament tips, bouncing as they move anteriorly (Fig. 3, path 2). (3) Still other particles appear to remain on the frontal surfaces of the filaments until reaching the tips, where they move laterally into the PARTICLE TRANSPORT IN THE ZEBRA MUSSEL 121 marginal groove proper through gaps between adjacent and opposing filament tips (Fig. 3, path 3). Those particles transported in the groove proper are alternately seen through the gaps between adjacent filament tips and, faintly, as they pass behind the relatively transparent filament tips. Because the superficial mucous string is sometimes opaque, we were unable to observe the marginal groove proper at all particle concentrations and types. Therefore, it is unclear whether the filament tips forming the marginal groove proper flare "open" and "closed." Although the gaps through which particles enter into the marginal groove proper appear absent at times and large at others, the overall width of the superficial groove does not change markedly, ranging from 17 to 38 jam wide (/; = 10). At high particle concentrations, the ventral margin of the inner demibranch occasionally presses against the visceral mass for several seconds. In these cases, the superficial mucous string is transferred to ciliated tracts on the visceral mass and is transported posteriorly, presumably to the in- halent siphon for rejection. Movement of the mucous string inside the marginal groove proper does not appear to be interrupted. Dorsal ciliated tracts The dorsal ciliated tracts were observed in three speci- mens, on 12 occasions, for 1 1.4 h of total observation time and 47 min of video. There are three dorsal tracts on each side of the visceral mass: at the junction of the viscera and inner demibranch (inner dorsal ciliated tract), between the two demibranchs (medial dorsal ciliated tract), and at the junction of the outer demibranch and the mantle (outer dorsal ciliated tract) (Fig. 1). Particles enter the medial dorsal ciliated tract from the descending lamella of the outer demibranch (Figs. 1.2); they are carried anteriorly as indi- viduals at low particle concentrations, or in mucous clumps and discrete strings at higher concentrations (Fig. 1 ). Parti- cles moving in the medial dorsal ciliated tract sometimes stop or reverse for several seconds, and this behavior is associated with extreme flaring of the interfilamentary spaces on the adjacent demibranchs. In addition, quick successive contractions by the adjacent demibranchs seem to make the mucous clumps less cohesive. At high particle concentrations, the two demibranchs occasionally contract strongly, and a slurry of particles becomes resuspended in the pallial cavity. It was not possible to determine whether these particles are recaptured. Particles in the medial dorsal ciliated tract move at a mean velocity of 152 /u.rn s~' (Table 1). A few particles are also transported anteriorly in the inner and outer dorsal ciliated tracts (Fig. 1 ). Particles enter these tracts not only from the demibranchs, but also from the mantle or body, suggesting that cilia on these surfaces can trap some particles. Labial palps The labial palps were observed in four specimens, on 14 occasions, for 1 1 h of total observation time and 2.2 h of video. Two pair of palp lamellae lie at the anterior end of the ctenidia, one pair on each side of the mouth. A pair of palps forms a functional unit consisting of one inner and one outer palp lamella (Figs. 1, 4). The apposing surfaces of each pair of palp lamellae are highly ciliated and folded into deep grooves and ridges (see Galtsoff, 1964; Ward et ai. 1994). The labial palps transport material from the ctenidia to the mouth, control the volume of food ingested, and may also sMS pMS iPL Ant mDCT oDCT Ventral dl_ of oO and ID aL of ID and oD Dorsal Figure 4. Diagram of the labial palps and the anterior portions of the inner and outer demibranchs of Dreissena polvmorpha as observed through the endoscope (looking medially). Solid arrows indicate particle paths after capture. Dashed arrows indicate particle paths behind the outer demibranch (oD) and outer palp lamella (oPL). At the anterior termination of the outer demibranch (oD). mucous clumps and strings from the medial dorsal ciliated tract (mDCT) move ventrally onto the inner demibranch (iD) and enter between the palp lamellae (PL). The superficial mucous string (sMS) is drawn from the ventral margin (VM) of the inner demibranch (iD) and between the palp lamellae (PL). The material disperses on the palp lamellae (PL), and rejected particles move ventrally from the palp grooves (pG). Rejected particles ire incorporated into a mucous string (pMS) that moves posteriorly and forms an irregular ball at the palp apices. This hall is expelled as pseudofeces (PF) (see text for details). (Ant = anterior, DA = divergence area. Dor = dorsal, iD = inner demibranch. iPL = inner palp lamella, mDCT = medial dorsal ciliated tract, oD = outer demibranch. oDCT = outer dorsal ciliary tract, oPL = outer palp lamella, pA = palp apex, PF = pseudofeces, pG = palp groove, pMS = palp mucous string. sMS = superficial mucous string. VM = ventral margin.) See Video Note, p. 124. 122 S. M. BAKER ET AL. sort particles before ingestion (Yonge, 1926; Menzel, 1955; Newell and Jordan, 1983). In zebra mussels, the labial palps enclose the inner demi- branch only. Along the entire free dorsal edge of each palp lamella is a smooth ciliated surface that rests against the demibranch lamellae. These smooth ciliated surfaces are narrow at the distal apices of the palp lamellae and increase in width anteriorly, up to four palp ridges wide. The distal apex of the inner palp lamella often sweeps from the as- cending lamella of the associated demibranch, across the superficial groove, and back, drawing in the superficial mucous string (Fig. 1). Although the superficial mucous string is drawn between the palp lamellae at their distal apices, the superficial groove extends the entire length of the inner demibranch, ending at the oral groove between the palp pair. It was unclear whether, or at what point, the mucous string is removed from the marginal groove proper. This material may remain within the marginal groove proper to be deposited directly into the oral groove, without processing by the palps. At the anterior terminus of the outer demibranch, mucous clumps and discrete strings from the medial dorsal ciliated tract move ventrally on the filaments of the descending lamella of the inner demibranch (Figs. 1. 4). The superficial mucous string is drawn between the palp lamellae at a point posterior to this location (Fig. 1). Rather than proceeding anteriorly in the superficial groove, the mucous clumps from the dorsal tract continue moving in a ventral direction, entering between the labial palp lamellae. Mucous strings or clumps from the superficial groove or the dorsal ciliated tract disperse on the palp lamellae. Indi- vidual particles bounce anteriorly over the ridges of the palps, slowing in the grooves and moving more quickly over the ridges to the next groove (Fig. 1 ); the mean velocity is 94 /j,m s~ ' (Table 1 ). The palp lamellae alternate between being spread slightly apart and being closely appressed. When the palp lamellae part slightly, particles remain close to one or the other lamella and continue their bouncing motion. Occasionally, the palp lamellae spread wide apart, and particles can be seen suspended between the apposing palp surfaces and moving posteriorly. This material may include particles that are moving ventrally from the dorsal ciliated tract. When appressed, the lamellae rub together with an anterior-posterior displacement of the width of one to two palp ridges (one palp ridge == 125 /xm), and the smooth outer surfaces of the palp lamellae undulate in waves from dorsal to ventral. Rejected particles move ventrally from the palp grooves (Figs. 1; 4). At the ventral margins of the palp lamellae, these particles form a mucous string which then moves posteriorly at a mean velocity of 54 jam s~" (Table 1 ). The palp mucous string moves toward the distal apices of the palps, where it forms an irregular ball (Fig. 4). Once the ball of mucus and particles reaches a particular size, the palps push or "clap" the ball away. In this manner, the ball is transferred to ciliated tracts on the mantle, presumably to be expelled from the inhalent siphon or pedal gape as pseudo- feces. Sometimes the palp mucous strings are transferred to the mantle before reaching the palp apices. During exposure to high particle concentrations ( 1 06 ml"'), the processing of particles by the labial palps changes according to the acceptability of the particles. When mussels were fed high concentrations of a combina- tion of both desirable and unacceptable particles (Microcys- tis and Scenedesmus), the ball of mucus that forms near the palp apices was drawn back between the palp lamellae and once again dispersed. We observed mucous balls being reprocessed by the palps up to four times before finally being rejected. In contrast, when mussels were fed high concentrations of primarily unacceptable particles (Scenedesmus alone), the superficial mucous string from the inner demibranch sometimes by-passed processing by the labial palps. In this case, the superficial mucous string does not disperse on the ridged surfaces of the palp lamellae but is transferred from the marginal groove of the inner demi- branch directly to the palp apices by the extreme posterior section of the smooth ciliated surface (see Fig 1 ). At the palp apices, the material is formed into a mucous ball and rejected. Discussion The observations reported here explain the efficient se- lection of particles measured in our previous work (Baker et al, 1998) with Dreissena polymorpha. Particles are sorted on the ctenidia of zebra mussels, and more specifically, at the marginal food groove of the inner demibranch. We observed a two-layer transport at the marginal food groove: desirable particles appear to be transported inside the groove proper, while unacceptable particles are carried su- perficially. We also observed sorting at the ventral margin of the outer demibranch: desirable particles are retained on the outer demibranch, while unacceptable particles are transferred to the inner demibranch and ultimately rejected. Here, we compare and contrast our observations with pre- vious reports of feeding processes in both zebra mussels and marine bivalves. We suggest that the structure of homo- rhabdic ctenidia does not preclude particle sorting, and that the changes in seston composition attributed to /ebra mus- sels may ultimately be due to the ctenidial sorting mecha- nisms observed in this study. Foster-Smith ( 1975) proposed that three conditions must be met for particle selection to take place at the marginal groove of bivalve ctenidia (i.e., in Mytilus edulis, Cerasto- denna edule. Venerupis pullastra): (1) some particles must be able to enter the deep area of the marginal groove; (2) particles in the deep area of the marginal groove must be physically separate from the superficial material: and (3) the PARTICLE TRANSPORT IN THE ZEBRA MUSSEL 123 superficial material must be rejected, while the material in the deeper area of the marginal groove is accepted. The two-layer transport that we observed at the marginal groove of zebra mussels meets these requirements for particle se- lection. Two-layer transport has previously been described for filibranchs and pseudolamellibranchs, but does not neces- sarily indicate the capacity for particle selection. Foster- Smith (1975) reported two-layer transport in M. edidis (fili- branch), with the particles in the deep region of the marginal groove tending to be small, and those in the superficial material tending to be larger. But the two layers are con- tiguous, precluding particle selection. In M. editlis. parti- tioning between the two layers may be temporal, rather than physical. J0rgensen ( 1975) reported that particles arriving at the marginal groove might either enter the groove between the bases of the filament tips or pass outside, depending on whether the groove is open or closed. We never observed the marginal groove in zebra mussels to be "open" with filament tips flared, as J0rgensen (1975) illustrated for M. edulis, although our observations suggest that there may be some regulation of the amount of material allowed to enter the marginal groove proper. Two-layer transport at the marginal groove, in combina- tion with particle sorting, has previously been reported only for pseudolamellibranchs. Atkins (1937a) described both two-layer transport and the potential for size sorting at the marginal groove in Pinna fmgilis and several Pinna-like species (pseudolamellibranchs). In these species, which have plicate heterorhabdic ctenidia, fine particles trans- ported by the principal filaments are deposited into the depth of the marginal groove proper, while coarse particles transported by the ordinary filaments are deposited outside the groove and are usually rejected (Atkins, 1937a). Al- though the mode of particle introduction to the marginal groove of D. polymorpha differs from that observed in Pinna sp. due to the nonplicate nature of the zebra mussel ctenidia, the marginal groove appears to function similarly in both species. Previous feeding studies have indicated that, in addition to selection by particle size in D. polymorpha, a chemical mechanism of selection is also present (Ten Winkel and Davids. 1982; Baker et ai, 1998). In the present study, the disparate sizes of particle types embedded in the superficial mucous string in D. polymorpha suggest that some factor other than size is important in the shunting of particles either to the marginal groove proper or to the superficial groove. The superficial mucous string is picked up by the apices of the palps, and much of the material is rejected. The arrangement of the ctenidium/palp junction suggests that material within the marginal groove proper may be trans- ported to the anterior portion of the labial palps or directly to the oral groove. The differing degree to which the two mucous strings are processed by the palps suggests that the material in the superficial mucous string is of lower quality than that in the groove string. This two-layer transport at the marginal groove could potentially increase the rate of pro- cessing and decrease the possibility of sorting mistakes at the palps. Microscopic examination of the structure and function of cilia at the marginal groove may help elucidate the sorting mechanisms. The labial palps of zebra mussels function very similarly to those of other bivalves, despite differences in demibranch structure and function. Zebra mussels have a smooth ciliated surface along the free dorso-posterior edge of the labial palp lamellae, similar to that of oysters (Ward et at., 1994). Our observations of mucous ball formation near the apices of the labial palps are similar to those described for both oysters (Menzel, 1955; Galtsoff, 1964; Ward et ai, 1994) and marine mussels (Beninger and St-Jean, 1997a). As in oys- ters, D. polymorpha palp lamellae alternate between being appressed and being slightly separated. When separated, we observed off-surface posterior movements of particles like those reported by Galtsoff (1964) and Ward et al. (1994) for oysters. Ward et al. (1994) speculated that the posterior movement allows the particles to be cycled through the palps several times before being rejected or ingested. In addition to this type of reprocessing, we observed a second recycling method: the mucous ball forming near the palp apices is sometimes re-engulfed by the palps up to four times before finally being rejected. Video endoscopy allowed us to observe. /// situ, the position of the feeding organs within the pallial cavity of living zebra mussels. These observations build on previous reports of feeding organ functioning based on dissected specimens of zebra mussels (Atkins. 1937b; Morton, 1969). For example, like Atkins (1937b) and Morton (1993). we observed particles passing off the outer demibranch at the ventral margin and being transferred to the inner demi- branch. Dissected preparations, however, did not allow the authors of previous studies to observe the bend in the outer demibranch and the curvature of the inner demibranch that occurs under natural feeding conditions. Our observations suggest that maintenance of the ctenidia in these positions may facilitate particle recapture; this natural ctenidial mor- phology enhances the transfer of some particles from the outer demibranch to the inner demibranch. Our observations of particle transport in zebra mussels contradict some previous observations and corroborate oth- ers. For example, Atkins ( 1937b) described rare filaments of the descending lamella of the outer demibranch that trans- port particles ventrally; these particles are then passed to normal filaments that transport them dorsally. During our observations of this area (five specimens on 14 occasions, for 8.2 h total observation time), all filaments of the de- scending lamella of the outer demibranch transported par- ticles dorsally. In addition, Atkins (1937b) did not report any anteriorly directed movement on the ventral margin of 124 S. M. BAKER ET AL the outer demibranch, such as we occasionally observed. That anterior movement is. however, similar to that of mucous-particle masses on the ventral bend of Placopecten magellanicus ctenidia, which also lack a ventral groove (Beninger et ai. 1992). In greater contrast, both Atkins (1937b) and Morton (1969) reported ventral movement of particles on the as- cending lamella of the outer demibranch. whereas we ob- served dorsally directed movement, above a divergence area. Atkins (1937b) reported dorsally directed currents on the ascending lamella of the outer demibranch of the Union- idae, another unrelated group of freshwater bivalves. The dorsally directed movement on the ascending lamella of the outer demibranch allows some proportion of material to be directed to the outer dorsal tract, rather than to the medial dorsal ciliated tract between the two demibranchs, perhaps preventing overloading of the latter tract. Both Atkins (1937b) and Morton (1993) described the anterior movement in the dorsal tract at the junction of the mantle and ascending lamella of the outer demibranch as well; Atkins (1937b) noted that anterior movement in this outer tract usually occurs only in bivalves with heterorhabdic ctenidia. Partitioning material between two dorsal tracts may increase the rate of total particle transport. Particle velocities, as well as the movement of particles on the ctenidia and labial palps of D. polymorphic are consistent with mucociliary, rather than hydrodynamic, transport (Ward el a!.. 1993; Beninger and St-Jean, 1997b). The velocities of particles transported on the frontal sur- faces of the demibranchs overlap the ranges reported for M. echtlis and for the plical crests of C. virginica (Ward a ai, 1993). In addition, the superficial mucous string at the marginal groove moves at a rate similar to the mucous strings observed in C. virginica, M. edulis, M\u arenaria, and Placopecten magellanicus (Ward et al., 1993, 1994). Material in the dorsal ciliated tract, however, travels at a rate many times slower than it does in C. virginica or P. magellanicus. In D. polymorpha, material at the dorsal ciliated tract is embedded in mucous clumps and trains, but in the oyster and scallop, the material is in a slurry (Ward et al., 1993). Transport rate is generally inversely correlated with the viscosity of the mucus (Menzel, 1955; Winet and Blake, 1980), and therefore, material in a slurry moves at a faster rate than material in more cohesive mucous clumps. The lack of hydrodynamic transport in zebra mussels may reflect a dorsal tract that is smaller and less well developed than that in oysters and scallops. Zebra mussels have had major impacts on the freshwater systems in which they have become established. Because of the high clearance rates of these mussels, phytoplankton biomass has decreased by more than 609J- in many of the invaded systems (Morton, 1971; Kryger and Riisgard. 1988; Holland. 1993; Fahnensteil et al.. 1995). In addition, seston composition has changed in some systems, including the Hudson River, New York, where the phytoplankton com- munity has shifted from a prevalence of cyanobacteria to diatoms (Vanderploeg et al., 1996; Smith et al., 1998). Recent studies using flow cytometry (Baker et al., 1998) have shown that zebra mussels can very effectively sort particles and preferentially accept the cyanobacterium Mi- crocystis. In the present study we found that accepted par- ticles were directed to the inside of the marginal groove of the inner demibranch and appear to be transported directly to the mouth for ingestion. In summary, we observed pallial organ morphology, par- ticle transport, and particle sorting in zebra mussels by using video endoscopy. These observations contribute to a grow- ing body of information on the feeding dynamics of bi- valves and suspension-feeding invertebrates. More impor- tantly, our results suggest that particle sorting occurs on zebra mussel ctenidia, despite their homorhabdic nature and their lack of adjacent tracks of frontal cilia beating in opposing directions. Our direct observations of zebra mus- sel ctenidia provide an explanation for the efficient selection of particles measured by Baker et al. ( 1998) and, ultimately, for the role of zebra mussels in ecosystem modification. The role of ctenidial morphology in particle selection by zebra mussels exemplifies the direct link between the functioning of individual bivalves and ecosystem-level processes. Acknowledgments This paper is dedicated to the memory of Dr. James H. Crichton. uncle and mentor (SMB). We thank the following persons: C. Nieder, New York State Department of Envi- ronmental Conservation, and H. Roditi, SUNY at Stony Brook, provided zebra mussels. Amy Dunham. SUNY at Stony Brook, and Heather Herb, University of Florida, drew the illustrations. J. Kurdziel and P. Baker, SUNY at Stony Brook, provided assistance during all aspects of this study. Comments from two reviewers improved this manuscript. This study was supported by grants from the Hudson River Foundation (01096A and 00798A) and the National Science Foundation (OCE981 1380). 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Reference: Binl. Bull. 199: 126-134. (October 2000) Biogeography of Two Species of Symbiodinium (Freudenthal) Inhabiting the Intertidal Sea Anemone Anthopleura elegantissima (Brandt) T. c. LAJEUNESSE* AND R. K. TRENCH Department of Ecologv, Evolution and Marine Biology, University of California nt Santa Barbara, Santa Barbara, California 93106 Abstract. We have analyzed the genetic profiles of dinoflagellate populations obtained from the Pacific coast sea anemone Anthopleura elegantissima (Brandt) at collec- tion sites from Washington to California. Genetic dif- ferences within the symbiont populations of California anemones have been uncovered by restriction length polymorphism (RFLP) analysis of the small subunit (SSU) and large subunit (LSU) ribosomal RNA genes, and by denaturing gradient gel electrophoresis (DGGE) of the in- ternal transcribed spacer region 2 (ITS 2). The existence of two Symbiodinium species is substantiated by sequence analysis of the variable regions VI, V2. and V3 of the SSUrDNA. which also establishes their phylogenetic relat- edness to other members of the genus Symbiodinium. Anemones from Washington and Oregon harbor a single dinoflagellaie species, for which we propose the name S. muscatinei sp. nov. At these northern locations, S. muscati- nei either exists alone or co-occurs with the Chlorella-\\ke green algal symbiont. Our results indicate that S. muscatinei co-occurs with a second dinoflagellate, 5. californium, in mixed populations in central and southern California. We suggest that the geographic distribution of these dinoflagel- lates is related to the temperature cline created by latitude. Introduction Intertidal anemones of the genus Anthopleura are abundant along the Pacific coast of North America (Hand, 1955). A. Received 2 February 2000; accepted 17 July 2000. * To whom correspondence should be addressed. E-mail: lajeunes@ lifesci.ucsb.edu Abbreviations: DGGE, denaturing gradient gel electrophoresis; ITS 2, internal transcribed spacer region 2; LSU. large subunit; RFLP, restriction fragment length polymorphism; SSU. small subunit. elegantissima. the most common and wide ranging species, is distributed along the rocky intertidal from Alaska to central Baja California (Hand. 1955; Francis, 1979; McFadden et al., 1997). Throughout its geographical range this species harbors intracellular dinoflagellates of the genus Symbiodinium. Anem- ones in regions north of California may also host a Chlorella- like (Chlorophyta) alga alone or in mixed populations with the dinoflagellate (Muscatine, 1971). Whether a particular algal symbiont occurs in an anemone, and whether it is present alone or in mixed populations are phenomena believed to be influ- enced by physical parameters. Relative sensitivities to light and temperature appear to be most significant in regulating the distribution of algal populations. Anemones in warm, bright habitats typically possess the dinoflagellate symbiont, whereas anemones in cool, shadier habitats contain the green alga (Secord. 1995; Saunders and Muller-Parker, 1997). Environmental parameters have also been implicated in modulating Svmbiodinium populations in some tropical symbioses. The Caribbean reef-building corals Montastraea annularis and M. faveolata either host a single algal taxon or have mixed algal populations with patterns of distribution and relative abundance that may be in response to irradiance and temperature (Rowan and Knowlton, 1995; Rowan et al., 1997). Continued analyses of Symbiodinium populations in scleractinians are resolving greater diversity within individ- ual hosts than previously recognized (Baker and Rowan, 1997; Baker et al., 1997; Baker, 1999). Restriction fragment length polymorphism (RFLP) analysis of the large subunit (LSU) ribosomal DNA from 107 Pacific and Caribbean corals (Baker, 1999) identified 69 species that harbored a single algal taxon; 13 other species harbored more than one Svmbiodinium taxon, but not in the same colony, and 25 species sometimes harbored more than one algal taxon within the same colony. Depth (and thus reduced light) was 126 SYMBIODINIUM SPECIES IN ANTHOPLEURA 127 regarded as the most significant factor regulating the distri- bution of a particular alga. Previous studies conducted on algae isolated from A. elegantissima collected in Washington and California showed marked differences in mitotic indices (Wilkerson el ill.. 1983) and carbon translocation (Shick and Dykens, 1984; Verde and McCloskey, 1996). The possibility that these differences reflect different Symbiodinium species has been suggested (Verde and McCloskey, 1996), but has not been empirically tested. A limited genetic study was con- ducted by Rowan and Powers (1991) on symbiont popula- tions collected from Pacific Grove, California. Their RFLP and partial small subunit (SSU) ribosomal DNA sequence analyses from cloned amplification products detected only one dinoflagellate taxon belonging to lineage B Symbio- dinium (sensii Rowan and Powers, 1991). It is now recog- nized that analyses based on the conserved SSUrRNA gene underestimate genetic diversity (Rowan, 1998). Further- more, sampling from a single collection site would not necessarily identify symbiont diversity in a host with a range of thousands of kilometers. The extensive geographic range of A. elegantissima, its occupancy of diverse intertidal habitats, its occurrence in two different growth forms — solitary and clonal — which may represent two distinct species (McFadden el ai, 1997). and its ability to harbor symbionts from different algal divisions suggest that A. elegantissima may harbor more than one taxon of symbiotic dinoflagellate. This possibility is strengthened by the finding that different Symbiodinium spp. are adapted to different regimes of light (Iglesias-Prieto and Trench. 1994, 1997b) and temperature (Warner el al.. 1996). To test the hypothesis that different dinoflagellate taxa may co-occur in Anthopleura, anemones were col- lected from intertidal habitats along a latitudinal gradient ranging from Puget Sound in Washington to San Diego in Southern California. A genetic examination, using tradi- tional RFLP analyses of the SSUrDNA and LSUrDNA, and analyses of partial SSUrDNA sequences were conducted on Symbiodinium populations isolated from these anemones. Denaturing gradient gel electrophoresis (DGGE; Myers el al., 1985; Abrams and Stanton, 1992) is a technique fre- quently used to characterize and compare genetic diversity in complex microbial assemblages from samples collected over spatial and temporal scales (Muyzer et al.. 1993; Muyzer and Smalla, 1998). This technique was used here to analyze the variable internal transcribed spacer region 2 (ITS 2), and thus to visualize and identify the occurrence of more than one algal taxon within a host. Materials and Methods Anemone collections Specimens of symbiotic Anthopleura elegantissima were collected from rocky intertidal locations along the Wash- ington, Oregon, and California coastlines. Between Febru- ary 1997 and August 1998, collections were conducted at Anaco Beach, Fidalgo Island. Washington (48°29'; 122°42'); Coos Bay, Charleston, Oregon (43°34': 124°33'); Carmel, California (36°55'; 121°92'); Cayucos, California (35°44'; 120°88'); Ellwood Beach and Campus Point. Santa Barbara, California (34°43'; 119°83'); and Swami's Beach, Cardiff-by-the-Sea (Encinitas). California (33°04'; 117°29'). Symbiotic A. xanthogrammica were collected only from Cayucos. Individual anemones were collected from a range of habitats, from exposed upper intertidal to shaded lower intertidal locations. Aposymbiotic A. elegan- tissima were obtained from drainage sluice-ways beneath the wet lab facilities of the Marine Science Institute of the University of California, Santa Barbara, California. Isolation of algal cells from tissues of host anemones Oral discs and tentacles from anemones were macerated in a Tenbroek tissue grinder. This step was followed by a 5-min centrifugation at about 800 X g in a Dynac II bench- top centrifuge. The pellets containing the algal cells were resuspended and further homogenized with a Tissue Tearor (Model 985-370) to dissociate most of the remaining animal cellular debris. After a second centrifugation, the algal pellets were resuspended in Millipore-filtered (porosity 0.22 /urn) seawater, centrifuged, and resuspended two or three times to remove most of the animal debris from the algal cell preparation. The cultured Symbiodinium californium (#383. Banaszak et al.. 1993) was originally isolated, in 1989, from a solitary form of A. elegantissima by using a technique developed by Polne-Fuller (1991). The isolate was grown in 1 1 of ASP-8A (Blank, 1987) for 2 months at 17°C, illuminated by banks of VitaLite fluorescent tubes delivering 80 /zmol quanta m~2s~' photosynthetically active radiation on a 14:10 (light:dark) photoperiod. Algae were harvested by centrifugation at 9000 X g in a Sorvall RC-5B centrifuge. DNA extraction, amplification. RFLP Symbiodinium populations were isolated from 64 speci- mens of A. elegantissima (37 clonal and 27 solitary) and three of A. .\anthogrammica. DNA was extracted from about 25 mg of algal material by using a proteinase diges- tion and spin-column separation protocol described in the QIAamp Tissue kit (Qiagen Corporation, Santa Clarita, CA). From the spin-column eluate, 1 p,\ of product was used as a template to amplify the small-subunit ribosomal RNA gene (SSUrDNA) and part of the large subunit ribosomal RNA gene (LSUrDNA) (Lenaers et al., 1989). SSUrDNA was amplified on a Perkin-Elmer thermal cycler 2400 using primers of Rowan and Powers (1991), and under the fol- lowing conditions: an initial denaturing step of 3 min at 92°C followed by 35 cycles of 30 s at 92°C. 40 s at 52°C, 128 T. c. LAJEUNESSE AND R K. TRENCH and 30 s at 72°C, followed by a single cycle of 5 min at 72°C. A fragment corresponding to a region between 28 bp and 929 bp of the Prorocentnun micans LSU rRNA gene containing the variable portions D1-D3 (Lenaers el ai, 1989; Wilcox, 1998) was amplified using primers described by Wilcox (1998) with the same protocol used for the SSUrDNA, but with an optimal annealing temperature of 48°C. Restriction digests were performed by incubating ampli- fied products with 1-5 units of Taq I (New England Bio- Labs, Beverly, MA) at 65°C for 3 to 5 h or with 1-5 units of Dpn II (New England BioLabs) for 4 to 5 h at 37°C. Products of the digests were separated by electrophoresis in a 2.5% high-melt agarose gel at a constant 70 V for 3 h. Denaturing gradient gel electrophoresis (DGGE) Primers for polymerase chain reaction (PCR)-DGGE analyses were designed to amplify the variable internal transcribed spacer region II (ITS 2), producing a fragment size of 330-360 bp. ITS and 5.8S rDNA sequence data (unpublished) from cultured Symbiodinium spp. isolated from various cnidarian and molluscan hosts were compared to identify conserved regions. An internal primer "ITSint- for2" (5'GAATTGCAGAACTCCGTG-3'( was designed from this analysis, and it anneals to a conserved region of the 5.8S rDNA. Primer ITS-Reverse (Coleman et ul.. 1994) was modified with a 40-bp GC clamp (Sheffield et ai, 1989) and is referred to as "ITS2CLAMP" (5'CGCCCGCCG- CGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGGGA- TCCATATGCTTAAGTTCAGCGGGT-3'). A "touch- down" amplification (Don et ul., 1991 ) protocol was used to ensure specificity. Initial annealing conditions began 10°C above the final annealing temperature of 52°C. Every two cycles, the annealing temperature was decreased one de- gree. After 20 cycles the annealing temperature was held, and remained at 52°C for another 15 cycles. Reaction prod- ucts were loaded onto an 8% acrylamide denaturing gradi- ent gel (a 40% to 75% gradient, 100% consists of 7 M urea and 40% deionized formamide). PCR products were loaded on the gel with a 2% Ficoll loading buffer (2% Ficoll-400, 10 mM Tris-HCl pH 7.8, 1 mA/ EDTA, 1% bromophenol blue) and separated by electrophoresis for 22 h at 90 V at a constant temperature of 60°C. The gel was then stained in a IX TAB and 5 jug/ml ethidium bromide solution for 15 min, washed in deionized water for 15 min and photographed. DNA sequencing, alignment, and anal\sis Partial sequences of the SSUrDNA and the ITS spacer (ITS 1 and 2 and 5.8 rRNA gene) were obtained from direct cycle sequencing of amplified products. Cycle sequencing of amplified SSUrDNA products was accomplished using Rowan and Power's ( 1991 ) forward primer and an internal primer (Anderson et ai, 1993), which permitted sequence reads spanning the VI, V2, and part of the V3 variable regions (Sogin and Gunderson, 1987; as described in Rowan and Knowlton. 1995). Reagents were supplied and reaction conditions specified in the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Ap- plied Biosystems, Foster City, CA). Reaction products were analyzed on an Applied Biosystems 310 Genetic Analyzer (Division of Perkin Elmer, Foster City, CA). The resulting chromatograms were checked and edited using Sequence Navigator, version 1.0, software (ABI, Division of Perkin Elmer, Foster City. CA). Sequence composites of the SSUrDNA were assembled and, including gaps, totaled 375 nucleotides. Partial sequences from the two algal types identified in our RFLP and DGGE analyses were then aligned by eye with published S\mbiodinium sequences obtained from Genbank; they included S. corciilomm, S. microadriaticum, S. pilosum, S. pulchrorum, Gymnodinum varians, G. simplex, Porocentrum micans, and four Svmbio- diniuni spp. symbiotic with the coral Montastraea annu- laris. Cladistic analyses, by the method of parsimony, were conducted on the aligned data set using PAUP 4.0b4 soft- ware under default settings (Swofford, 1993). A bootstrap analysis was conducted on the most parsimonious tree to assess relative support for each branch (Felsenstein, 1985). Results Anemones collected from high and low intertidal habitats at each study site contained the same Symbiodinium popu- lations. We found no indication, based on our molecular analyses, that the Symbiodinium populations in A. elegan- tissima are influenced by the local environmental differ- ences within the littoral zone. However, we found signifi- cant differences between the algal populations in anemones collected from Washington and Oregon, and those collected at lower latitudes in California. Figure 1 summarizes the findings from our RFLP and DGGE analyses that detected a single genetic entity in northern anemones and at least two genetic entities in southern anemones. Taq I digests of the SSUrDNA amplified from northern populations of Symbiodinium exhibited a characteristic "clade B" restriction pattern (Rowan and Power. 1991 ) (Fig. 1 A, lane 1 ). The same analysis conducted on southern populations produced a mixed pattern consisting of the "clade B" type with a second undescribed pattern (Fig. 1C, lane 1 ). We have identified this latter pattern as diagnostic of a second algal type. It is identical to the restriction pattern of cultured S. californium, whose SSUrDNA does not yield a classical "clade A. B, or C" pattern (Fig. 1C. lane 2) (cf. RFLP type "T6" in Darius et ul., 1998). This new pattern results from the loss of a restriction site at approximately position 1500 and the gain of a restriction site at position 870 of the amplified 1785 bp product, as determined from the entire SSUrDNA sequence of 5. californium (GenBank SYMBIODINIUM SPECIES IN ANTHOPLEURA 129 NORTHERN SYMBIODINIUM POPULATIONS (WA, OR) Fidalgo Island, WA 2 3 B 1018 - 516 - 220 = • Charleston, OR 220 Taq 1 (SSU) Dpn II (LSU) DGGE (ITS 2! SOUTHERN SYMBIODINIUM POPULATIONS (CA) C 1 2 34 D 1 2 3 4 220 1018 - 1018 - 516 - Z 516 220 Taq 1 (SSU) Dpn II (LSU) DGGE (ITS 2) (Carmel, CA JCayucos, CA (Santa Barbara, CA Figure 1. Pacific coastline of North America depicting collection sites and genetic analyses of northern and southern Symbiodinium populations. ITS 2, internal transcribed spacer region 2; LSU, large subunit; SSU, small subunit. (A) RFLP analysis of the SSUrDNA and LSUrDNA from northern algal populations. Lane 1. Taq I digest of SSUrDNA indicative of lineage B Svmhioilinium (Rowan and Powers, 1991 ); lane 2, Dpn II digest of the D1-D3 variable region of the LSUrRNA gene: and tor comparison, in lane 3. Dpn II digest of the same region from 5. californium (#383). (B) DGGE gel of the ITS 2 depicting a single ITS signature (black arrow). (C) RFLP analysis of the ribosomal repeat from southern algal populations; lane 1, Taq 1 digest of SSUrDNA showing the lineage B Symbiodinium co-occurring with a second pattern not belonging to any of the described "clades" (sensu Rowan and Powers, 1991 ); lane 2, Taq 1 digest of SSUrDNA of #383, a pattern identical to the undescribed RFLP profile in lane 1. Lanes 3 and 4 are Dpn II digest on LSUrRNA gene amplified from natural populations and the cultured clonal isolate #383 respectively; indicates more than one algal taxon in southern populations. (D) DGGE gel showing three representative profiles of ITS 2 signatures from natural populations, lanes 1. 2, and 4; and for comparison, lane 3 depicts the ITS signature for #383 (white arrow). (See text for further explanation.) accession #AF225965). The poor amplification of the SSUrDNA from S. californium in mixed populations may explain why the smallest fragment (130 bp) appears to be absent from RFLPs on natural samples (Fig. 1C, lane 1). Dpn II restriction digests of SSUrDNA from both freshly isolated northern and southern algal samples and cultured 5. 130 T. c. LAJEUNESSE AND R. K. TRENCH californium produced a single restriction pattern character- istic of "clade B" Symbiodinium (Rowan and Powers, 1991 ) (data not shown). The utility of RFLP analyses of the SSUrDNA in distinguishing sequence variation, especially among congeneric members, has limitations. The more vari- able gene regions and more informative techniques were therefore employed to achieve a better resolution of the variation detected by the Taq 1 enzyme. An 850-bp fragment of the LSUrRNA gene was exam- ined by restriction analysis in an attempt to measure the extent of the genetic varitation observed in our RFLP anal- ysis of the small subunit and to uncover possible variation not resolved by the SSUrRNA gene (Baker el al.. 1997; Wilcox, 1998). Dpn II restriction digests of LSUrDNA amplified from all northern Symbiodinium populations pro- duced the fragment pattern in Figure 1A, lane 2. A restric- tion site exists at one end of the amplified product and produces two bands, one 740 bp, the other about 70-80 bp. A Dpn II digest of the LSUrDNA from S. californium lacks a restriction site for this enzyme, and a single band is depicted (Fig. 1A. lane 3, and also 1C. lane 4). RFLPs conducted on algal populations from southern anemones always contained a nondigested fragment, as observed for S. californium, as well as two fragments identical to those observed in restriction patterns from the northern algal populations. This indicates the presence of two different gene sequences in the same amplification product — one with and one without a restriction site for Dpn II. These data are consistent with our results from the SSUrDNA digests. Taq 1 digests of the LSUrDNA (data not shown) also showed the presence of a single type in northern populations and two types in southern populations of A. elegantissima. The same two algal types were also found in A. .\antho- grammica from Cayucos. The ITS region has historically been useful in resolving species-level differences (Gonzalez et ai, 1990; Coleman el til.. 1994; Goff et ai, 1994; Larsen and Medlin, 1997). Among dinoflagellates. it has been used to resolve differ- ences between closely related species within the genus Alexandrhtm (Adachi el al.. 1996) and to assess intraspe- cific variation in Gymnodinium catenatiim (Adachi et al., 1997). Hunter et al. (1997), in their preliminary study. reported that the ITS is potentially a good marker for interspecific comparisons between Symbiodinium taxa. In this study, a region encompassing the ITS 2 and a portion of the 5.8S was chosen for DGGE analyses. DGGE separates amplification products by differences in sequence composition and nucleotide order. The results are therefore a qualitative assessment of the entire sequence. Application of DGGE has permitted the rapid assessment of complex microbial populations and identification of specific microbes from marine, aquatic, and terrestrial samples (Muyzer and Smalla, 1998). This analysis, applied to Sym- hiodiniuin populations, has verified the existence of a single Symbiodin'nun species in anemones collected from northern locations (Washington and Oregon) (Fig. IB, black arrow). In contrast, at least two algal species were identified in populations of Antliopleura from California. In southern populations, the algal ITS "signature" found in northern anemone populations (black arrow) is often associated with an ITS signature consistent with S. califor- nium (white arrow; Fig. ID, lanes 1 and 3 respectively). An unidentified ITS type was observed (light gray arrow; Fig. ID, lane 2) but is believed to be a variant of S. californium based on our findings from the RFLP data on the LSUrDNA. In our analyses of southern populations, a fourth ITS type (hatched arrow), although sometimes ap- pearing faint, is always associated with the most common ITS type (black arrow). This is believed to either be a DGGE artifact or to represent fixed variation within the ribosomal repeat. An inferred phylogeny (Fig. 2) reconstructed from partial SSUrDNA sequences shows the relationships between the algae identified in A. elegantissima and those from tropical hosts. The algal species found throughout northern and southern anemone populations is a member of the B lineage found by Rowan and Powers ( 1991 ). Here, we propose the name Symbiodinium muscatinei sp. nov. to refer to the dinoflagellate symbiotic with A. elegantissima from Wash- ington to California. (A morphological description must Symbiodinium microadriaticum S . corculonttn Symbiodinium sp. (M . annularis) S . pilosurn S . californium 1_4 I (southern A . elegantissima) 100) L Gymnodinium variant Symbiodinium sp.(Marginopora) Symbiodinium sp.(M. annularis) Symbiodinium sp. (M. annularis) _ Symbiodinium sp. (M . annularis) S. pit Ichror urn — S. muscatineii (A. elegantissima) - Gymnodinium .simplex Figure 2. Phylogenetic reconstruction of the Symbiodinium lineage by the method of parsimony using a 375-bp composite of the SSUrRNA gene spanning the VI, V2, and V3 variable region. S. californium is closely related to Gymnodinium varians and forms a separate lineage. 5. nniscan- nei groups with members of the B lineage and is probably the same taxa identilied in the study by Rowan and Powers (19911. Numerals above branch segments indicate number of base pair changes; numbers in paren- theses below show bootstrap consistency based on 1000 replicates. For simplicity, the out group, Prorocentrum inicans, is not shown. SYMBIOD1NIUM SPECIES IN ANTHOPLEURA 131 await achieving axenic culture, but the partial sequence of the SSUrDNA has been given the GenBank accession num- bers AF228362 and AF228363 for the VI. and V2 and V3 regions of the SSUrDNA. respectively.) S. californium, identified in anemones from California only, is closely related to Gymnodinium various; together they form a lin- eage separate from the described "clades A, B, and C" (bracketed). Sequence differences between the SSUrDNA from S. mnscatinei and 5. californium are significantly greater than differences observed between described spe- cies. The entire ITS region was sequenced from each alga and compared (data not shown). No reliable alignment was possible due to extreme sequence divergence. The ITS sequence of S. mnscatinei was aligned and compared with sequences from several other lineage B Syinbiodinium. Re- sults indicated a level of divergence many times greater (12%) than interspecific variation observed among other dinoflagellates (Adachi el al.. 1997). These data collectively indicate the presence of two distinct dinoflagellate species. Animal DNA isolated from aposymbiotic A. elegan- tissimu was used to determine whether host material would be a source of contamination in freshly isolated algal sam- ples. Amplification was never achieved with the primers and reaction conditions used for the amplification of LSUrDNA and SSUrDNA. However, the ITS 2 primers designed for DGGE amplified host DNA. but only rarely when algal DNA was present. Cloned S. californium (#383) served as a control, so we could ascertain the presence of pseudogenes or natural variation in ribosomal repeats within a single genome. Ri- bosomal pseudogenes have been observed in some dinoflagellates (Scholin et al., 1993: Adachi et a!.. 1996). Although none have been reported in Symbiodinium, their presence cannot be discounted. The DGGE analysis on S. californium (#383) repeatedly identified a single type with no obvious intragenomic variation. Discussion The molecular methods employed in this study demon- strate that there are two distantly related species of S\mbio- dinium in populations of Anthopleura elegantissima along the Pacific coast of the United States. Evidence based on RFLP. DGGE, and sequence analysis indicates the presence of a single Symbiodinium species, designated here as S. muscatinei, in northern populations. This is consistent with previous reports that many hosts harbor a single population of algae (Schoenberg and Trench, 1980a; Baker and Rowan, 1997;Bythell ff«/.. 1997: Billinghurst et al., 1997; Stochaj and Grossman, 1997). Our analyses also show that anemo- nes from southern latitudes in California harbor a mixed dinoflagellate population consisting of two congeneric spe- cies: S. mnscatinei identified from northern anemones, and S. californium. It has been recognized for some time that some inverte- brate taxa may simultaneously harbor more than one algal taxon (Muscatine. 1971; Trench and Winsor. 1987). The coexistence of two or more Symbiodinium taxa in the same host was first described by Rowan and Knowlton (1995) in the Caribbean reef building corals Montastrea unmiluris and M. faveolata. Since then, more than one Symbiodinium taxon has been identified in populations of certain other coral species. Furthermore, some individual colonies have been shown to harbor mixed symbiont taxa (Baker and Rowan, 1997; Darius et al.. 1998; Baker, 1999; Carlos et al.. 1999: Banaszak et al., 2000). From the data of Baker ( 1999), about 23% of the total coral taxa sampled may have mixed symbiont populations, indicating that the presence of more than one symbiont simultaneously is not uncommon. Environmental parameters have been hypothesized to regulate the distribution and population dynamics of each symbiont in hosts that harbor more than one algal species (Rowan et al.. 1997; Baker, 1999). The patterns of these distributions are specific and correlate closely with environ- mental gradients. Symbiodinium species examined in cul- ture and /';; hospite show species-specific physiological adaptations to photosynthetically active radiation and tem- perature (Chang et al.. 1983. Iglesias-Prieto and Trench. 1994, 1997a, b; Warner et al.. 1996, 1999). Algal species better adapted for a particular environment will out-compete those less suited (Schoenberg and Trench, 1980b; Rowan et al., 1997; Saunders and Muller-Parker. 1997). Previous studies have focused primarily on the reef-wide distribution of symbioses involving more than one algal taxon. but very little is known about the biogeography of algal symbionts and about how the distribution of algal species in geographically widespread hosts may be influ- enced by differences in environment. Ultimately, the prob- lem revolves around the determination of the species of algae involved in the associations. The difficulty in delin- eating species has been a long-standing problem for ocean- ographers studying phytoplankton biogeography (Round, 1981). With regard to S\mbiodiniitm, one example is the coral Plesiastrea versipora, which has an unusually broad latitudinal distribution along the east coast of Australia. On the tropical Great Barrier Reef it forms a symbiosis with S\mbiodinium sp. of the C lineage, while in cooler temper- ate waters off Sydney it harbors a Symbiodinium sp. from the B lineage (Baker. 1999). Temperature and irradiance are the most significant en- vironmental variables that change predictably with latitude. Within the tropics, among coral species with more than one algal taxon. the algae exhibit ecological zonation that cor- relates with irradiance (Rowan and Knowlton, 1995; Baker, 1999). Irradiance in temperate regions is less consistent and may not be an important parameter regulating the distribu- tion of S. californium. When compared to California. Wash- ington and Oregon have longer periods of daylight during 132 T. c. LAJEUNESSE AND R K. TRENCH the summer, but experience shorter periods in the winter. Assessment of the influence of light as an environmental factor that regulates algal distribution is further complicated because anemones act to control irradiance levels impinging on the algae by covering their surfaces with fragments of rock and shell (Dykens and Shick, 1984). Temperature is an environmental factor that regulates species distributions along the coast of California (New- man, 1979), and it probably governs the distribution of S. californium. Collection sites from Oregon and Washington routinely experience colder temperatures than locations in California (Fig. 3), particularly in winter. In addition, north- ern anemones experience greater annual temperature fluc- tuations, which may also be of selective importance. Low temperature extremes, like high temperatures, may result in the loss of symbionts from hosts (Muscatine et al.. 1991). Temperature as a selective force is supported by the obser- vation that, in northern anemone populations that harbor S. muscatinei and the green Chlorella-like alga, symbiont growth rates are more strongly affected by temperature than by irradiance (Saunders and Muller-Parker, 1997). Temper- ature changes associated with increased latitude may also influence the competitive balance between S. californium KU G" 18 , ! , HI rr Z> 16 * A A • • 1 1 I — o: | * ' * a LU Q. 14 LU • * • ; • • T a A • A Q LU 12 O A ° D LU ^ 10 A * A A D CO : LU o CO 8 6 a a a o i i i i i i i i i i c -° « ^ S~? ^oS.*- ><. foQ)™ Q-5D"5 30)ooc _n "^ -^ *=Z. ^? — **• rr\ r\ —r t- Figure 3. Average coastal sea surface temperature data from the Comprehensive Ocean-Atmosphere Data Set (COADS) monthly climatol- ogy records (1946-1989). Graph compares the seasonal fluctuation of sea surface temperature in degrees Celsius at collection sites in Washington (open square). Oregon (open triangle), and from central and southern California (Carmel, solid circle; Cayucos. solid triangle; Santa Barbara, solid square; Cardiff-by-the-Sea (Encinitas), solid diamond). The locations are shown on the map in Figure 1. The southern locations in California have similar seasonal temperatures and are always 1° to 6°C warmer than northern locations depending on the time of year. The range in temperature fluctuation between March (lowest yearly temperature) and September (highest) is greater for Washington and Oregon (ca. 7°C) than for locations in California (ca. 4°-5°C). and 5. muscatinei by affecting their relative growth rates. We also suggest the possibility that low temperatures found in the north exclude S. californium; in the south. S. califor- nium and 5. muscatinei coexist because the latter species may have a wider temperature tolerance. Because attempts to culture S. muscatinei were unsuccessful, controlled phys- iological comparisons were not possible. The nonrandom sorting or specificity of algal-inverte- brate symbioses is contingent on the interplay of multiple factors (Trench et al.. 1981; Trench, 1988. 1997), and is not expressed as one alga for one host (Schoenberg and Trench. 198()b). Cnidarian hosts are symbiotic with selectively few microalgal taxa (Trench, 1997), yet they are exposed to hundreds and possibly thousands of "free-living" microalgal species. The "complementariness of the dynamically inter- acting attributes" (Dubos and Kessler, 1963) of both sym- biont and host substantially limits possible symbiotic com- binations. The extent to which a host shows specificity for one or more algal species depends ultimately on the poorly understood cellular and molecular processes that may take place during initial intracellular contact, and possibly also after the association is established (Colley and Trench. 1983; Trench. 1993). In those hosts harboring more than one algal taxon, symbiont distributions are strongly influ- enced by changes in the physical environment over both local (Rowan and Knowlton, 1995; Secord, 1995) and geo- graphic sales (Baker. 1999). Because the influence of envi- ronment on host-symbiont dynamics can be variously inter- preted, further experimental analyses are needed to explain the mechanisms that result in these observed patterns (Saun- ders and Muller-Parker, 1997). For example, it is unclear whether changes in the physical environment have an in- trinsic or extrinsic effect on the biology of the symbiosis. Changes in the environment might modify the physiological integration of the symbiotic partners so that they are no longer compatible. Finally, differential changes in symbiont growth rates may, and can, cause competitive exclusion of one algal species over another (Provasoli et al., 1968). Progress in elucidating these mechanisms should provide a more accurate description of host symbiont interactions and specificity. Acknowledgments We thank Drs. Ed Delong and Scott Hodges for use of their lab facilities. We thank Dr. Gisele Muller-Parker for collecting specimens from Anacortes. Washington; Dr. Jeff Goddard for collecting anemones in Coos Bay, Oregon, and Dr. Shana Goffriedi and Victoria Orphan for collections at Cardiff-by-the-Sea, near Encinitas. California. Dr. Robert A. Andersen and Dr. Craig Bailey at the Center for Culture of Marine Phytoplankton. Bigelow Laboratory for Ocean Sciences, and Dr. Lynda Goff and Dr. Deb Moon at the University of California, Santa Cruz, are greatly appreciated SYMH10DIN1UM SPECIES IN ANTHOPLEURA 133 for their assistance in sequencing. We especially thank Dr. Alison Murray for her advice, invaluable technical guidance on DGGE analysis, and comments on an early draft of this paper. Literature Cited Abrams, E. S., and V. P. Stanton, Jr. 1992. Use of denaturing gradient gel electrophoresis to study conformational transitions in nucleic acids. Methods Enzymol. 212: 71-104. Adachi, M., Y. Sako, and V. Ishida. 1996. 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ROSENTHAL AND FRANCISCO BEZANILLA* Departments of Physiology and Anesthesiology, UCLA School of Medicine, Los Angeles, California 90095 Abstract. To determine whether the electrical properties of the squid giant axon are seasonally acclimated, action potentials, recorded at different temperatures, were com- pared between giant axons isolated from Loligo pealei caught in May, from relatively cold waters (-10°-12°C), and in August, from relatively warm waters (-20°C). Pa- rameters relating to the duration of the action potential (e.g., maximum rate of rise, maximum rate of fall, and duration at half-peak) did not change seasonally. The relationship be- tween conduction velocity and temperature remained con- stant between seasons as well, in spite of the fact that May axons were significantly larger than August axons. When normalized to the fiber diameter, mean May conduction velocities were 83% of the August values at all temperatures tested, and analysis of the rise time of the action potential foot suggested that a change in the axoplasmic resistivity was responsible for this difference. Direct measurements of axoplasmic resistance further supported this hypothesis. Thus seasonal changes in the giant axon's size and resistiv- ity are not consistent with compensatory thermal acclima- tion, but instead serve to maintain a constant relationship between conduction velocity and temperature. Introduction Signal transduction in the nervous system is profoundly temperature sensitive. Acclimation of higher order nervous function to variation in environmental temperature has been the subject of many investigations (see Prosser and Nelson, 1981); however, the precise mechanisms of such acclima- Received 3 March 2000; accepted 7 July 2000. E-mail: fbezanil® UCLA.edu * To whom correspondence should he addressed. Abbreviations: Cm, membrane capacitance: MRF. maximum rate of fall: MRR. maximum rate of rise; /?,., external resistivity; /?,, internal resistiv- ity; i\.. external resistance; r,. internal resistance. tions are not well understood. Do they involve changes at the level of the action potential, the synapse, or both? Action potential duration and propagation are strongly in- fluenced by acute temperature changes (Hodgkin and Katz, 1949), largely due to temperature sensitivity of the under- lying ion channels (Hodgkin et ai, 1952). If action poten- tials themselves are a common target for thermal acclima- tion, which properties are affected? In the giant nerve fibers of earthworms, cold acclimation speeds the action potential duration, conduction velocity, and refractory period vs. tem- perature relationship, but not to the extent that, when mea- sured at rearing temperatures, the kinetics of cold- and warm-acclimated worms are equivalent (Lagerspetz and Talo, 1967; Talo and Lagerspetz, 1967). In goldfish cardiac muscle cells, the action potential's duration is reduced in cold-acclimated fish (Ganim et ai, 1998). In certain Aplysia neurons, however, early potassium current kinetics are not affected by rearing temperature (Treistman and Grant. 1993). The squid giant axon, long a model for understanding the basic physiology of voltage-dependent ion channels, is, for a variety of reasons, an excellent system for examining temperature-dependent acclimation of the action potential. First, its output participates in a known function — the jet- propelled escape response (Prosser and Young, 1937; Young, 1938; Otis and Gilly, 1990) — and presumably it is advantageous for this response to be rapid. Second, the dimensions of the giant axon permit action potentials, mac- roscopic ionic currents, gating currents, and single-channel currents to be measured from the same preparation (Hodgkin and Huxley, 1952; Armstrong and Bezanilla, 1973; Conti and Neher, 1980; Bezanilla, 1987). Third. Na and K currents, which underlie the action potential, have been extensively characterized in this system (see Gilbert et 0.78) except colcemid (P = 0.032). * — indicates significant difference. 13). As more of the axoneme entered the cytoplasm, the axoneme extended deeper into the egg cortex relative to the sperm head, which remained near the egg cortex (Fig. 14 A, A'). Once the bulk of the axoneme passed through the oolemma, the sperm head frequently moved rapidly through the egg cytoplasm. This movement did not occur in the sinusoidal pattern typical of sperm swimming. Rather, a portion of the axoneme extended ahead of the trailing sperm head along the direction of movement. Although they did not impact entry of the sperm head into the egg cortex. MT inhibitors suppressed the rotation and lateral movement of the sperm head along the cortex. Fur- thermore, MT inhibitors prevented the incorporation of the sperm axoneme into the egg cortex (Figs. 12A-C; 14B, B'; 15; 16). Under these conditions, the sperm heads exhibited rapid, sporadic oscillations immediately subjacent to the insemination cone for several minutes after passage through the cone. This sporadic oscillation seen in the presence of MT inhibitors corresponded temporally to the period of expected axoneme incorporation in control fertilizations. In most inhibitor observations, the sperm remained immedi- ately subjacent to the cone up to 7 min PI and the axoneme ultimately fractured, allowing the sperm nucleus to move deeper into the ooplasm — although more slowly than in controls (Fig. 12). During this period the sperm flagellum remained outside the egg. extending out through the insem- ination cone. On occasion, the unincorporated portion of the flagellum detached from the egg surface and remained ac- tive for several minutes. In contrast to control observations, probing MT-inhibited eggs with the flagellar monoclonal antibody failed to label an axoneme associated with decon- densing sperm nuclei (Fig. 14B, B'). Once sperm passed through the egg cortex, inhibition of MF polymerization did not affect sperm head or axoneme incorporation or subsequent nuclear decondensation. Expo- sure of eggs to 12.6 p.M CB at various times pre- and postinsemination showed a temporal effect of CB on insem- ination (P < 0.001 ) (Fig. 17). Eggs incubated in CB 10 min prior to insemination showed no sperm incorporation or polar body formation (see Fig. 8). Eggs exposed immedi- ately prior to insemination (0 min) showed almost complete 4 min Figure 11. DIC microscopy Time series, in differential interference contrast (DIC) microscopy, of captured video images showing the incor- poration of the sperm head during a normal (control) fertilization. The arrowhead parallels the left side of the sperm and illustrates the orientation of the sperm head with the apex of the arrowhead corresponding to the anterior end of the sperm nucleus. (A) Sperm head immediately subjacent to insemination cone. (B-D) Sperm head rotated and was laterally dis- placed along the egg cortex. Notice the rotation and lateral displacement of the sperm nucleus along the egg cortex relative to the insemination cone. Times indicate elapsed time from initial frame (A). Bar = 20 /xm. Figure 12. DIC microscopy time series of captured video images showing the incorporation of the sperm head into eggs exposed to 100 nA/ nocodazole. The arrowhead parallels the left side of the sperm and illus- trates the orientation of the sperm head, with the apex of the arrowhead corresponding to the anterior end of the sperm nucleus. (A) Sperm head immediately subjacent to insemination cone ( * ). ( B ) Sperm head remained subjacent to insemination cone, did not undergo rotation or lateral dis- placement. (C) Sperm head separated from the attached flagellum and moved deeper into the egg. The arrow marks a particle adhering to the flagellum. Notice that the flagellum remains external to the egg. Times indicate elapsed time from initial frame (A). Bar = 20 ;um. 150 M. J. MISAMORE AND J. W. LYNN blockage of sperm entry and were not significantly different from the preincubution trial (P < 0.05). Addition of CB at 2 min PI significantly reduced sperm entries (P < 0.05) relative to controls but did not cause complete inhibition (Fig. 17). Sperm entry was not significantly different from controls when CB was added 4 min PI. Sperm entry and rotation in the egg cytoplasm occurred in eggs treated at 4 min PI with CB. At about 3 min PI, sperm-head transloca- tion and axoneme incorporation were observed. The pres- ence of the axoneme in the egg cytoplasm was verified using the monoclonal antibody to acetylated a-tubulin. At about 8 min PI, sperm nuclei began to decondense in control (Fig. 18) and MF and MT inhibitor trials. Sperm mitochondria separated from the nucleus and moved away from the nucleus concurrent with the decondensation of the sperm chromatin. At the same time, or immediately afterward, a small sperm aster started to develop (Fig. 19). The aster formed at the base of the decondensing sperm head and was only detectable with the monoclonal yeast a-tubulin antibody, visualized with confocal microscopy. In contrast, the female meiotic spindle and astral array were clearly discernible with light microscopy. In control fertilizations, the sperm aster was detectable as a diminutive structure as late as 40 min PI. During this period the female aster and associated bundle were substantially larger (Fig. 19) (Walker, 1996). Colchicine- treated eggs exhibited no obvious sperm aster, and the female meiotic spindle was not observed. Since egg activation was determined based on the resumption of meiosis as visualized by movement of chromosomes into anaphase I, disruption of the meiotic spindle made the status of egg activation in MT inhibitor treatments difficult to ascertain. Flow of cytoplasmic particles Concurrent with axoneme entry, sporadic "vibrations" were observed in the egg cytoplasm between the sperm head Figure 13. A diagrammatic representation of the oscillations exhibited by a single sperm head following its passage through the insemination cone. Once the sperm head had entered the egg cortex there was a coordinated flexing of the internal and external portions of the flagellum. This movement resulted in oscillation of the sperm head 1 1 • • 4) within the egg cortex. Figure 14. Complementary epifluorescent micrographs of the incor- porated sperm dual-labeled for flagella (A, B — a monoclonal antibody to acetylated tubulin) and sperm nucleus (A'. B' — Hoechst 33342). (A, A') Control fertilization — The sperm axoneme (arrowhead) extended deeper into the egg cytoplasm relative to the peripherally positioned sperm nu- cleus (m). (B, B') Eggs pretreated with colchicine prior to insemination. No incorporated axoneme (B) was observed in association with fertilizing sperm nuclei (m) seen in (B'l. These findings support real-time observa- tions that the sperm axoneme was not incorporated in the presence of MT inhibitors. 10 min PI. Bar = 25 jam. Figure 15. Epifluorescent micrograph of an colchicine-treated egg fertilized with a sperm dual-labeled with FITC-WGA (arrow) and Hoechst 33342 (arrowhead) prior to insemination. The sperm nucleus (arrowhead) was incorporated into the egg cytoplasm and began decondensing. Colchi- cine inhibited the incorporation of the sperm flagellum. which was visu- alized by the FITC-WGA labeling of the sperm plasma membrane (arrow). Bar = 25 /im. Figure 16. Epifluorescent micrograph of Hoechst-stained eggs insem- inated and exposed to 12.4 iiM cytochalasin B 4 min PI. By 10 min PI, sperm entered into the egg cortex and began decondensing. The incorpo- rated sperm nucleus (m) was clearly identified by the less intensely fluo- rescing halo formed by the dispersing chromatin. Polar body formation was inhibited, f — female chromalin. and the sperm entry site near the expected position of the axoneme. A directed flow of cytoplasmic particles was similarly observed originating near the base of the sperm CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION 151 100% 80% 60% 40% 20% 0% a a.c c JL DMSO Pcelnc O1 CB Z CB Treatment Figure 17. Temporal effects of cytochalasin B (CB) on sperm entry following the addition of the microfilament inhibitor at various time points pre- and postinsemination (PI). CB was added to sodium periodate-treated eggs at 12.4 jiA/. Treatments were (DMSO). Control of 0.6% DMSO at 10 min preinsemination; (Prelnc). CB added 10 min preinsemination; (()' CB). immediately following insemination; (2' CB). 2 min PI; and (4' CB), 4 min PI. Eggs were fixed at 10 min PI and scored for the number of eggs with incorporated sperm. Values are mean ± standard error (n = 5). Letters indicate statistically significant differences based on a two-way ANOVA and Tukey multiple comparisons (P < 0.05). head and penetrating as deep as 10 ju.m into the ooplasm (http://www.mbl.edu/BiologicalBulletin/VIDEO/BB.video.hrml). The vibrations in the egg cytoplasm began shortly after Figure 18. Phase-micrograph time series of video-captured images showing the sperm decondensation and pronuclear formation. Sperm nu- cleus (arrowhead) was visible in the egg cortex by 4 mm PI. (B) Sperm chromatin (arrowhead) decondensation occurred by 10 min PI. (C) Sperm pronuclear (arrowhead) formation began by 30 min PI. Similar results were observed in eggs incubated in 100 \iM colchicine prior to insemination (D-G)orin 12.4 \iM cytochalasin B at 4 min PI (not shown). Note multiple decondensing sperm pronuclei. indicating polyspermic eggs. Bar = 25 ,um. rotation and lateral displacement of the sperm head, lasted from 1-3 min. and ceased shortly before the sperm nucleus began to decondense. Although the extent varied between fertilizations, this cytoplasmic flow was evident in greater than 70% of the 20-30 similar filmed observations and on about 80 more unri lined occasions. These observations are the norm rather than the exception in the hundreds of fertilizations observed during several reproductive seasons. Furthermore, polyspermic eggs exhibited multiple currents associated with the polynumery sperm. When flagellar in- corporation was inhibited, no flow of cytoplasmic particles was observed in eggs. Similarly, the flow of cytoplasmic particles was also observed in eggs exposed to MT inhibi- tors followed by washing prior to insemination. Discussion Initial sperm entry The incorporation of the sperm components through the oolemma into the egg cortex in Dreissena polymorpha occurs in two morphologically distinct steps (Misamore et ai, 1996). During the initial incorporation, the sperm head and midpiece gradually enter into the egg cortex at a rate of 1 jum/min (Misamore et ai. 1996). A distinct, cylindrical insemination cone encompasses the sperm as it passes through the oolemma. The insemination cone of D. pol\- morpha consisted of many 6-nm-thick filaments (Fig. 5) and labeled with FITC-phalloidin (Fig. 6), suggesting the pres- ence of microfilaments. The cone assumed a more pyrami- dal configuration once the sperm entered the egg cortex. This is similar to morphological changes in the insemination cone of sea urchins, in which an initially rounded cone becomes a "spike-like" cone following sperm entry (Tilney and Jaffe. 1980: Cline and Schatten. 1986). When treated Figure 19. Laser scanning confocal image of a diminutive sperm aster in an egg. The 30-min PI egg was labeled with a monoclonal antibody to yeast a-tubulin. Notice the relatively small size of the male aster (small arrow) in relation the large microtubule array (large arrow) associated with the female pronucleus and polar body. The asterisk (*) indicates the relative position of the sperm nucleus dual-labeled with Hoechst 33342 (not shown). Bar = 25 /urn. 152 M. J. MISAMORE AND J. W. LYNN with the inhibitor to MF polymerization, cytochalasin B, this initial sperm entry was blocked (Fig. 9C. C"). and no fertilization cone formed. These findings suggest that the initial incorporation of the sperm into the egg cortex was dependent on the polymerization of MFs. Although the critical involvement of microfilaments in sperm incorporation has been reported for many marine invertebrates (Gould-Somero et ai, 1977; Longo, 1978a, 1980; Byrd and Perry, 1980; Schatten and Schatten, 1980; Cline and Schatten, 1986; Schatten el ai. 1986) and the present freshwater model, exactly how sperm pass through the oolemma is not well understood. Microfilaments are associated with several processes during fertilization in D. pol\morpha that could potentially account for the inhibition of initial sperm entry. The major sites of polymerized MFs include the sperm acrosome, the egg microvilli. and the fertilization cone. Furthermore, MFs are critical for cytoki- nesis during polar body formation (Longo, 1972; Longo et ai, 1993). The sperm of several marine bivalves including Spisula and Mytilus possess preformed acrosomal processes that do not undergo a polymerization-driven elongation (Hylander and Summers, 1977; Longo, 1978a). Following activation, Mytilus sperm extend a preformed acrosomal process with- out the polymerization of new MFs (Dan, 1967; Longo. 1977, 1983). Like these marine bivalves, D. polymorpha has sperm that possess a preformed acrosomal process (Fig. 2) that does not elongate and is apparently insensitive to CB treatments at the dosages tested. Although it was not pos- sible to expose only sperm to CB because washing disrupted the fragile acrosomes, polymerization of sperm MFs is not believed to be the critical component in sperm entry. This conclusion is based on several pieces of evidence. First, no elongation of the acrosomal process was observed during fertilization in D. polymorphu. Second, several studies in which washing of sperm was possible have shown that CB does not affect the fertilization capability of sperm (Sanger and Sanger, 1975; Longo. 1978a; Byrd and Perry, 1980). Third, the relative polarity of MFs in sperm acrosomes and egg microvilli is inappropriate to allow a myosin-actin ratcheting mechanism to draw the sperm into the egg in urchins (Tilney, 1978), and this is presumably the case for D. polymorpha as well. Finally, the addition of CB at 2 min PI allowed sufficient time for the sperm binding to occur prior to inhibition; however, sperm entry was still sup- pressed, suggesting MF involvement at a stage later than sperm binding. The involvement of microvilli in sperm entry varies greatly between species, but a role has been suggested in hamsters (Yanagimachi and Noda, 1970), urchins (Tilney and Jaffe, 1980), annelids (Anderson and Eckberg, 1983), and bivalves (Longo, 1987). Furthermore, Wilson and Snell (1998) propose that microvillus-like structures may be es- sential for most types of cell-cell fusion events. Hylander and Summers (1977) proposed a generalized model of fer- tilization in Mollusca. According to their model, sperm binding occurs between an inner acrosomal region of the sperm and microvilli tufts. Similar microvillar tufts were observed in D. polymorpha: however, no obvious associa- tion between these tufts and the inner acrosomal membrane was observed. Furthermore, microvilli appeared to remain perpendicular to the egg surface and did not reorient toward bound sperm as reported for Spisula (Longo and Anderson, 1970; Hylander and Summers, 1977). Misamore et ai (1996) reported extracellular fibers extending toward at- tached sperm; however, these fibers are substantially smaller than the egg microvilli. Microfilament presence in insemination cones has been well documented. In urchins, insemination cones may form from the fusion of microvilli (Schatten and Schatten, 1980), and MFs in the cones are polymerized into discrete bundles (Tilney and Jaffe, 1980) from monomeric actin in the egg cortex (Spudich and Amos. 1979). Molluscan insemination cones are markedly smaller than urchin cones and MFs are not consistently reported in the cones (Longo, 1983). As in Mytilus and Spisula (Longo and Anderson, 1970: Longo, 1983), in D. polymorpha MFs in fertilization cones run the length of the ooplasmic projection, but not in discrete bun- dles as observed in urchins (Fig. 5). While the exact mech- anisms involved remain unclear, insemination cones are implicated in sperm entry (Longo, 1980). The inhibition of sperm entry by CB also suppresses the formation of insem- ination cones (Longo, 1980; Schatten and Schatten, 1980. 1981). Cytochalasin B was shown to have a reversible, dose- dependent effect on fertilization: partial inhibition occurred at 6.2 juA/CB and complete inhibition at 12.4 /u,A/. Byrd and Perry (1980) reported similar dose-dependent findings in two urchin species. Strongylocentrotus purpuratiis and L\techinus pictus. Sperm entry was decreased at 2.5 jug/ml (5 p.M) CB in the former species and at 5 /Ag/ml (10 ^.M) in the latter; inhibition was complete at 5 /ng/ml ( 10 |U.A/) and 10 /Ltg/ml (20 H,M) respectively. Gould-Somero et ai ( 1977) found that slightly lower levels of CB partially (1 /aAf) or completely (2 ju,A/) blocked sperm entry. That the dose- dependent responses are similar is somewhat remarkable considering the variability in the gametes, extracellular coats, and fertilization mechanisms between these diverse species. Sperm entry was effectively blocked when CB addition preceded or was concomitant with insemination (Fig. 17). Addition of CB at 2 min PI resulted in fewer eggs exhibiting sperm penetration, but at 4 min PI sperm entry was not significantly affected. These findings suggest that CB was able to rapidly (within 1-2 min) block sperm entry, and that the period of susceptibility to CB inhibition was completed by 4 min PI. The first 4 min PI during D. polymorpha fertilization corresponds to the 1 ju.ni/min gradual-incorpo- CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION 153 ration phase into the egg cortex. After 4 min PI, CB was unable to inhibit sperm-axoneme incorporation, mitochon- dria detachment, or male-chromatin decondensation and pronuclear formation. CB impact on fertilization is limited to the first 6 min PI in several urchin species (Longo, 1980; Byrd and Perry, 1980: Schatten and Schatten, 1980), and echiuroid worms (Gould-Somero ft ai, 1977). The restric- tion of MF involvement in sperm incorporation to the first 6 min following insemination applies across a wide taxo- nomic range. Unlike MF polymerization, MT polymerization was not required for the initial incorporation of the sperm nucleus into the egg cortex in D. polymorpha. Initial entry of sperm into eggs in marine invertebrates (Schatten and Schatten, 1981; Schatten er ai, 1982. 1989) and algae (Swope and Kropf. 1993) also does not require MTs. Sperm were able to enter the egg cortex in MT inhibitors at a rate ( 1 /xm/min) similar to that observed in normal fertilizations. MT inhib- itors were able to penetrate the egg and were effective at disrupting the meiotic spindle, thereby preventing polar body formation. Furthermore, no MT array was observed to be associated with entering sperm nuclei when a-tubulin monoclonal antibody was used to label fertilized eggs. Sperm nuclear translocation and flagellar incorporation After passing through the fertilization cone and entering the egg cortex, D. polymorpha sperm rotated 180°, posi- tioning the basal end of the nucleus centrad (Fig. 1 1 ). The first fluorochrome-detectable MTs associated with entering sperm were the diminutive sperm asters adjacent to decon- densing sperm chromatin (Fig. 19). Small sperm asters have been reported for other bivalve species (Longo and Ander- son, 1969, 1970; Longo et ai, 1993). In contrast, the sperm aster is significantly larger in most invertebrate and mam- malian systems and is believed to be responsible for the migration of the male and female pronuclei during syngamy. For example, in sea urchins the sperm aster ex- tends toward the female pronucleus and is thought to effect the migration of the two pronuclei (Zimmerman and Zim- merman, 1967; Longo and Anderson, 1968; Longo, 1976; Schatten, 1981; Bestor and Schatten, 1981; Sluder et ai. 1985). The role of the sperm aster in D. polymorpha is not fully understood as it does not extend toward the female pronucleus. A markedly larger MT array is associated with the female chromatin in D. polymorpha (Walker, 1996). A dense bun- dle of MTs is observed immediately subjacent to the polar bodies. Emanating from the MT bundle toward and sur- rounding the female pronucleus is a prominent cone-shaped array of MTs. This MT bundle is believed to anchor the female pronucleus and guide its centrad movement into the egg (Walker, 1996). The large female aster is also believed to play an important role in movement of the male pronu- cleus. An analogous structure may also be present in both Spisula and Mytilus (Longo, 1973a). Furthermore, as has been found in urchins (Zimmerman and Zimmerman, 1967), colcemid prevents pronuclear migration in D. polymorpha (Walker, 1996). Following sperm rotation, most of the sperm axoneme was incorporated into the egg cytoplasm and the sperm head often was rapidly (1 /im/s) translocated through the egg cytoplasm (Misamore ct til., 1996). Microfilament polymer- ization appeared to play little or no role in the second stage of sperm entry. Addition of CB after initial sperm entry (4 min PI trials) failed to prevent the rapid translocation of the sperm nucleus or the incorporation of the flagella. Further- more, no obvious association of MFs and incorporated sperm was observed using epifluorescence or electron mi- croscopy. Conversely, MT polymerization played a prominent, yet somewhat unconventional, role in sperm nuclear transloca- tion and flagellar incorporation. Following entry of the sperm head into the cortex, the bulk of the sperm axoneme was incorporated into the egg cytoplasm in D. polymorpha. During this incorporation, dramatic movements of the sperm head were observed, as well as a lateral migration of the sperm head along the egg cortex. These movements may be analogous to "jerking" movements exhibited by urchin sperm during axoneme incorporation (Schatten, 1981). Schatten (1981) suggests that the continued movement of the sperm tail may be involved in its movement through the fertilization cone and into the cytoplasm proper. In D. polymorpha, there is an obvious correlation between move- ments of the flagellum as it enters the egg cytoplasm and movements exhibited by the sperm head in the egg cortex. The exact mechanisms involved in flagellar incorporation are not known. Video microscopic observations of both urchins (Schatten, 1981) and zebra mussels (this study) suggest that flagellar movement may be involved. Further- more, flagellar incorporation in D. polymorpha was blocked by MT inhibitors. Microtubule polymerization appears to be essential for flagellar incorporation in D. polymorpha. This finding is in contrast to the results of studies with urchins, in which nocodazole did not inhibit axoneme incorporation (Schatten and Schatten, 1981; Fechter et «/., 1996). Further- more, the exaggerated movement of the sperm head imme- diately subjacent to the insemination cone during the inhib- ited axoneme incorporation further supports the concept that flagellar movement takes part in axoneme incorporation. In contrast, Epel et at. (1977) reported that deflagellated sperm heads were able to bind and enter sea urchin eggs. Attempts to duplicate those experiments with D. polymor- pha in the present investigations were unsuccessful. Finally, Schatten and Schatten (1981) reported that MT inhibitors increased the lateral displacement of the sperm head along the cortex and that the formation of the sperm aster may signal the end of this lateral movement (Schatten, 1982). In 154 M. J. MISAMORE AND J. W. LYNN D. polymorpha, lateral displacement of the sperm head was restrained by the attached yet unincorporated flagellum. Once the flagellum was severed, however, lateral displace- ment and decondensation were observed. Following axonemal incorporation and quiescence, the sperm mitochondria separated from the nucleus as it began to decondense. As in other invertebrate species (Schatten and Schatten. 1981), sperm decondensation in D. polymor- pha was not affected by either MF or MT inhibitors. In zebra mussels, nuclear decondensution and mitochondria! separation are apparently unaffected by MT inhibitors, sug- gesting that flagellar detachment is also unaffected. In sea urchins, microtubules appear to be essential for detachment of the sperm tail, its migration toward the female pronu- cleus, and its disassembly (Schatten and Schatten, 1981; Fechter et al., 1996). Similarly, activation of D. polymorpha eggs by sperm was unaffected by the presence of either MF or MT inhibitors. Several studies have shown that early egg activation occurs during fertilization even in the presence of CB (Byrd and Perry, 1980: Schatten and Schatten, 1980; Dale and DeSantis, 1981; Lynn, 1989). In these studies, initiation of the cortical granule release or an electrophysi- ological response were used as indicators of egg activation. Like those of most molluscs (Humphreys, 1967; Longo, 1983), the eggs of D. pol\morpha do not release cortical granules immediately following egg activation. Since D. polymorpha eggs are inseminated at metaphase I arrest, the resumption of meiosis can serve as an indicator of egg activation (Longo, 1972, 1978a: Longo et al., 1993). In this study, D. polymorpha sperm readily bound to the egg sur- face in the presence of MF or MT inhibitors and apparently induced the resumption of meiosis, since eggs devoid of bound sperm remained in metaphase arrest. Flow of cytoplasmic particles During axoneme incorporation, a significant cytoplasmic movement was noted in the region of the axoneme. Flows of cytoplasmic particles were observed in numerous regions near the site of sperm entry and conspicuously originating at the basal region of the sperm head. The impetus or func- tional significance of this flow remains in question. Two possible mechanisms for generating these currents include beating by a functional axoneme displacing the particles or plus-end-directed transport along axonemal MTs by motor proteins associated with either the cytoplasmic particles or the sperm axoneme. There appear to be few. if any. reported instances where flagella retain dynamic function once incorporated into the egg cytoplasm (Schatten, 1981; 1982). The last movement typically associated with flagellar bending occurs shortly after sperm binding, and movement of the sperm nucleus once inside the egg is typically associated with cytoskeletal elements, specifically the sperm aster (Schatten. 1982; Longo, 1987). Technical limitations make it difficult to isolate movements attributed to incorporated axonemes from egg-derived events; nevertheless, several pieces of evidence support the concept of an active axoneme inside the egg. First, the sperm axoneme retains the ability to generate movement following demembranation during incorporation. Active movement by isolated, demembraned sperm axon- emes has been demonstrated in other species (Bray, 1992). Second, video microscopy of mechanically-ruptured, fertil- ized D. polymorpha eggs revealed incorporated sperm vig- orously moving within the collapsing egg membrane (Mis- amore, pers. obs.). Third, there is an apparent alteration of the flagellar beat pattern in incorporated sperm. During the rapid translocation of the sperm through the egg cytoplasm (Misamore et al., 1996). the sperm head trails the proximal portion of the axoneme. The proximal third of the axoneme becomes the leading portion of the moving sperm cell. Similar types of flagella-driven movement can be seen in other systems. For example, the single-celled flagellate Eu- glena moves via a singular flagellum that extends slightly more anterior than the cell body before bending posteriorly (Bray, 1992). Helical waves running the length of the fla- gellum propel the cell forward, resulting in a rotational movement to the cell body. Hamster sperm exhibit a pro- nounced change in beat pattern upon entry into the dense cumulus oophorous surrounding the egg. Penetrating sperm frequently progress with the proximal portion of the flagel- lum extending slightly forward, with the head ratcheting through the dense cumulus oophorous (Yanagimachi, 1994). The change in flagellar beating seen in D. polymor- pha may be attributable to the greater viscosity of the egg cytoplasm relative to the external milieu. A second potential source of the observed cytoplasmic flow could result from active movement of particles down the exposed axoneme MTs by molecular motors. The plus- end-directed flow of particles suggests the presence of a kinesin or kinesin-like motor. Initial attempts to label in- corporated axonemes with kinesin antibodies have been unsuccessful (data not shown); however, support for this hypothesis is as follows. Microtubule motors are relatively abundant in the egg cytoplasm. Gilksman and Salmon (1993) reported substantial MT gliding along surfaces coated with an ooplasm extract. Scholey et al. ( 1985) have isolated a kinesin from urchin eggs. Porter et al. (1987) showed that this egg kinesin exhibited plus-end movement along isolated axonemes, and kinesin-coated beads translo- cate along centrosome MTs. Furthermore, Kozminski et al. (1995) found that a flagellar kinesin. FLAK), facilitated movement of intraflagellar particles, or rafts, along the length of the axoneme of Chlamydomonas flagella. Clearly, more detailed testing of this hypothesis is needed. Finally, no flow of cytoplasmic particles was observed in D. p(il\morpha zygotes when axoneme incorporation was CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION 155 blocked with MT inhibitors. The observed flow is either directly, or at least indirectly, related to the incorporated flagellum. The significance of this particle flow down the axoneme remains an intriguing question. In summary, initial sperm entry into the egg cortex is a gradual. MF-dependent process, while subsequent flagellar incorporation is MT dependent. Dynamic movement of the incorporated sperm head and flagella is observed inside the egg cytoplasm, and a flow of cytoplasmic particles associ- ated with the incorporated axoneme was observed. D. poly- morpha serves as a good model for studying fertilization and exhibits many similarities to other fertilization models. 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Reports of Papers Presented at THE GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY, Woods Hole, Massachusetts 14 to 16 August 2000 Program Chairs: BARBARA BOYER, Union College WILLIAM ECKBERG, Howard University CHARLES HOPKINSON, Ecosystems Center, MBL ROBERT PAUL MALCHOW, University of Illinois at Chicago Each of these reports was reviewed by two members of a special editorial board drawn from the research community of Woods Hole, Massachusetts. Reviewers included scientists from THE MARINE BIOLOGICAL LABORATORY, THE WOODS HOLE OCEANOGRAPHIC INSTITUTION, AND THE NATIONAL MARINE FISHERIES SERVICE. SHORT REPORTS FROM THE 2000 GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY FEATURED REPORT The Editors Introduction to the featured report. On mapping odor quality 161 Wachowiak, Matt, Michal Zochowski, Lawrence B. Cohen, and Chun X. Falk The spatial representation of odors by olfactory re- ceptor neuron input to the olfactory bulb is concen- tration invariant. . 162 Hale, Melina E. Startle responses of fish without Mauthner neurons: escape behavior of the lumpfish (C.-yrlnfilmts lumpus) 18(1 Epstein, David A., Herman T. Epstein, Frank M. Child, and Alan M. Kuzirian Memory consolidation in llrniiissi'mla crassicornis . . . 182 Abenavoli, A., L. Forti, and A. Malgaroli Mechanisms of spontaneous miniature activity at CA3-CA1 synapses: evidence for a divergence from a random Poisson process 184 NEUROBIOLOGY Landowne, David Heavy water (D.,0) alters the sodium channel gating current in squid giant axons 164 White, Thomas H., Harris Ripps, Miduturu Srinivas, and Roberto Bruzzone \'oltage gating properties of channels formed by a skate retinal connexin 165 Molina, AnthonyJ.A., Peter J.S. Smith, and Robert Paul Malchow Hydrogen ion fluxes from isolated retinal horizontal cells: modulation bv glutamate 168 Wang, Jing W. Odor-induced oscillatory activity in Dmsitphila CNS ... 17(1 Hitt, James M., Frederick A. Dodge, Ehud Kaplan, and Robert B. Barlow C.ircadian rhythms in the receptive fields of the Limu- lu\ lateral eve 171 Fay, Richard R., and Peggy L. Edds-Walton Frequency response of auditory brainstem units in toadfish (O/>siinus Ian) 173 Yamaguchi, Ayako, Leonard K. Kaczmarek, and Darcy B. Kelley Intrinsic membrane properties of laryngeal mo- toneurons that control sexually differentiated vocal behavior in .African clawed frogs, Xena/ius lamis .... 175 Atherton, Jillian L., Matthew A. Krutky, James M. Hitt, Frederick A. Dodge, and Robert B. Barlow Optic nerve responses of Limulus in its natural habi- tat at night 1 76 Krutky, Matthew A., Jillian L. Atherton, Spence Smith, Frederick A. Dodge, and Robert B. Barlow Do the properties of underwater lighting influence the visually guided behavior of Limulus? 178 PHYSIOLOGY A\D BIOCHEMISTRY Novales Flamarique, Inigo. Kristiina Ovaska, and Theodore M. Davis UV-B induced damage to the skin and ocular system of amphibians 187 Harrington, John M., and Peter B. Armstrong Initial characterization of a potential anti-fouling sys- tem in the American horseshoe crab, Limulus polyphemus 189 Asokan, Rengasamy. Margaret T. Armstrong, and Peter B. Armstrong Association of a.,-macroglobulin with the coagulin clot in the American horseshoe crab, Limulus pohphemus: a potential role in stabilization from pro- teolysis 190 Kuhns, William J., Max M. Burger, Mohan Sarker, Xavier Fernandez-Busquets, and Tracy Simpson Enzymatic biosynthesis of N-linked glycan by the ma- rine sponge Microciniia fmiUJmi 192 Armstrong, Peter B., and Rengasamy Asokan A Ca + J-independent cytolytic system from the blood of the marine snail Busyron canaliculum 194 Heck, Diane E., Lydia Louis, Michael A. Gallo, and Jeffrey D. Laskin Modulation of the development of plutei by nitric oxide in the sea urchin Arbaria punctulata 195 Jung, Sung-Kwon, Katherine Hammar, and Peter J.S. Smith Development of self-referencing oxygen microsensor and its application to single pancreatic HIT cells: effects of adenyiate cyclase activator forskolin on ox- vgen consumption 197 Hanselmann, Rhea, Roxanna Smolowitz, and Daniel G. Gibson Identification of proliferating cells in hard clams. . . 199 159 160 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Brothers, Christine, Ernest Marks III, and Roxanna Smolowitz Conditions affecting the growth and zoospomlation of the protistan parasite QPX in culture 200 CELL BIOLOGY Sandberg, Leslie, Phillip Stafford, and George M. Langford Effects of myosin-II antibody on actin-dependent ves- icle transport in extracts of clam oocytes 202 Stafford, Phillip, Jeremiah Brown, and George M. Langford Interaction of actin- and microtubnle-based motors in squid axoplasm probed with antibodies to myosin V and kinesin 203 Tran, P.T., V. Doye, F. Chang, and S. Inoue Microtubule-dependem nuclear positioning and un- clear-dependent septum positioning in the fission yeast, Saccharomyces pombe 205 Crawford, Karen The role of microtubules during blastodisc forma- tion of the squid Loliga pealei 207 Weidner, Earl Cytoplasmic proteins on the surface of discharged microsporidian sporoplasms 208 MacKenzie, Roger, David Newman, Max M. Burger, Rene Roy, and William J. Kuhns Adhesion of a viral envelope protein to a non-self- binding domain of the aggregation factor in the marine sponge Microdona prolifera 209 Goda, Makoto, Mario H. Burgos, and Shinya Inoue Fertilization-induced changes in tin- fine structure of stratified Arbacia eggs. I. Observations on live cells with the centrifuge polarizing microscope 212 Burgos, Mario H., Makoto Goda, and Shinya Inoue Fertilization-induced changes in the fine structure of stratified Arbnria eggs. II. Observations with election microscopy 213 Gould, Robert M., Concetta M. Freund, John Engler, and Hilary G. Morrison Optimization of homogenization conditions used to isolate mRNAs in processes of myelinating oligoden- drocytes 215 ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY Kirkby, Ryan, Luc Claessens, Charles Hopkinson, Jr., Edward Rastetter, and Joseph Vallino Modeling the effects of land-use change on nitrogen biogeochemistry in the Ipswich watershed, Massachu- setts 218 Perring, Anne, Michael Williams, Charles Hopkinson, Jr., Edward Rastetter, and Joseph Vallino Solute dynamics in storm flow of the Ipswich River Basin: effects of land use 219 Westgate, Elizabeth J., Kevin D. Kroeger, Wendy J. Pabich, and Ivan Valiela Fate of anthropogenic nitrogen in a nearshore Cape Cod aquifer 221 Denault, Michelle, Erica Stieve, and Ivan Valiela Effects of nitrogen load and irradiance on photosyn- thetic pigment concentrations in Clndoplwrn vaga- bunda and Gniciliiria tikvalriae'm estuaries of Waquoit Bav. . 223 Greenbaum, Adena, and Anne Giblin Differences in properties of salt marsh sediment be- tween hayed and reference sites 225 Chikarmane, Hemant M., Alan M. Kuzirian, Robbin Kozlowski, Mark Kuzirian, and Tony Lee Population genetic structure of the goosefish, Lo- phius ammcanus 227 ORAL PRESENTATIONS Published bv title onlv. . Reference: Biol. Bull. 199: 161. (October 2000) Introduction to the Featured Report On Mapping Odor Quality In vertebrate olfactory systems, the coding of odor quality by the brain is constrained by four considerations. Three of these suggest that the code is specific: i.e.. about 1000 genes encode olfactory receptors; every primary olfactory receptor neuron expresses only one of those genes; and the axons of all of the neurons expressing a particular gene project to the same glomerulus (a synaptic exchange site in the olfactory bulb). The fourth consideration suggests that coding lacks specificity, for single receptor neurons respond, though not always with the same potency, to a variety of odor molecules. Thus, the recognition of a chemically pure odor cannot be restricted to its effect on a single specific set of receptor neurons and their common glomerulus. Rather, odor quality must be identified by some array or pattern of inputs induced in diverse neurons and glomeruli, Many previous experiments show that these patterns of response or input vary with concentration. On the other hand, psychophysical experimentation — as well as common experience — tells us that recognition of an odor is not confounded by even enormous differences in concentration. Matt Wachowiak and his colleagues point, in their brief report, to a resolution of this paradox. They fill the olfactory nerve terminals of the three-toed box turtle with a fluorescent dye, apply pulses of odor, and produce a map representing the location of the glomeruli responding to the odor, as well as the amplitude of the response. With this preparation, the variation in the pattern of olfactory receptor neuron inputs to the olfactory bulb can be measured as a function of concentration. In fact, the experiments confirm that the absolute size of the response to an odor increases with concentration. But if the responses are normalized, the resulting maps are virtually invariant with concentration. Wachowiak et al. conclude that — if higher olfactory centers receive and can analyze normalized maps of the input to the olfactory bulb — then odor recognition, independent of concentration, would be possible. — The Editors August 2000 161 162 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull. 199: 162-163. (October 2000) The Spatial Representation of Odors by Olfactory Receptor Neuron Input to the Olfactory Bulb is Concentration Invariant Matt Wachowiak, Michal Zochowski, Lawrence B. Cohen, and Chun X. Falk {Department of Cellular ami Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520) We wish to understand how odorants are distinguished and how one odorant is recognized as the same across a concentration range of several orders of magnitude. To this end we have measured the spatial pattern of the olfactory receptor neuron input to the olfac- tory bulb in the three-toed box turtle (Terepene triunguis). To monitor the input to the bulb we labeled the nerve terminals of the olfactory receptor neurons with Calcium Green- 1 dextran 10 kD (Molecular Probes) following the method developed by Friedrich and Korsching (1). We then formed a magnified (4X) image of the bulb on an 80 X 80 CCD camera (NeuroCCD; RedShirtlmaging. LLC, Fairfield. CT) and recorded the changes in fluorescence that resulted from a 2-s odorant pulse delivered to the nose. The signals we measured had approximately the same time- course everywhere in the bulb, and we therefore characterized the response by the amplitude of the signal as a function of its position on the bulb. Figure 1 shows three pseudocolor representations of activity in response to the odorant, hexanone. Red represents a large signal in each measurement and blue represents a signal 30% as large. The left-hand image shows the response to hexanone at a concentration that was 0.3% of saturation. The largest signal in the response was colored red (normalized scaling). Both right-hand images show the Concentration-dependence: normalized vs. absolute maps 10% hexanone normalized scaling 6.2 4.7 — % dF/F 1.4 0.3% hexanone normalized scaling % dF/F 1.9 — to max=4.7% dF/F 4.7 % dF/F 1.4 — max=6.2% dF/F absolute scalinc max=6.2°b dF/F Figure 1. Normalized maps of receptor neuron input t(> the turtle olfactory bulh are concentration-invariant. The left panel shows a pseudocolor map of the response Jo a 0.3f/f dilution of saturated vapor of 2-he\anone. The map is normalised to the maximum signal amplitude for this trial. The right panels show pseudocolor maps of the response to a 10% dilution of 2-hexanone. The map on the toft is normalised to its maximum signal amplitude. The map on the bottom (absolute scaling) shows the same data using the same scaling as for the 0.3% hexanone trial. The figure shows a concentration-dependent increase in the number ofglomeruli activated above a given absolute level, but sho\vs concentration-invartancc in the relative levels of input to all glomeruli activated b\ an odorani. 4 • image magnification. The field of vie\v i\ approximately 4 mm • 4 mm. FEATURED ARTICLE 163 response to 10% hexanone using two different scaling procedures. lion could be achieved if higher olfactory centers "read" the The bottom image shows the response using the same scale as that normalized maps of the input to the olfactory bulb, used for the response to 0.3% hexanone (absolute scaling). This Supported by NINDS Grant, NS08437 and an NRSA fellow- image is qualitatively different from the 0.3% image. In contrast, ship, DC 00378. the top image shows the response using normalized scaling. Again. the largest signal was colored red. This image is essentially iden- tical to the image on the left, even though the concentration of odorant differed by a factor of 30. Thus, normalized maps of input . „. , to the olfactory bulb appear to be concentration invariant. We hypothesize that concentration invariant odorant identifica- I Friedrich, R., and S. Korsching. 1997. Neuron 18: 737-752. 164 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull. 199: 164-165. (October 2000) Heavy Water (D2O) Alters the Sodium Channel Gating Current in Squid Giant Axons David Landowne (University of Miami, Miami, Florida 33101 ) When nerve axon membranes are abruptly depolarized, a small outward displacement current precedes the sodium current that underlies the propagated nerve impulse. This displacement current is asymmetric; it does not appear in a symmetrical hyperpolariza- tion. It was named "gating current" by earlier workers because of its association with the opening of the activation gates of the sodium channels. The gating currents were isolated by replacing permeant ions with impermeant ones, thus reducing ionic currents, subtracting away symmetrical currents and. in most cases, block- ing the ionic sodium current with tetrodotoxin (1, 2). Replacing the H,O in solutions with D^O slows many chemical and biological reactions including the squid axon action potential (3), ionic currents (4), and the sodium pump (5). However. Meves (6) reported that D2O had no significant effect on the asymmetry currents of squid axons. This result was confirmed in Myxicalu (7) and crayfish (8) axons. Most measurements were made at voltages less than +20 mV, which would not be expected to open all of the channels, and in the presence of tetrodotoxin. Both the ionic and gating currents can be recorded if the experiments are carried out in solutions with low sodium content (9). When this is done, D-.O can be seen to reduce the amplitude of the gating currents at more positive potentials. Segments of squid axons were bathed in an artificial seawater containing 44 mM NaCl, 396 mM tetramethylammonium (TMA) chloride, and 2 mM TMA Hepes. pH 7.4; and the internal Cs perfusion fluid contained 150 mM Cs glutamate, 50 mM CsF, 750 mM sucrose, and 40 mM Cs Hepes. pH 7.4. Solutions were made up with either H-.O or 99.8% D^O. The axons were voltage- clamped at a —70 mV holding potential. Gating currents were recorded with a p/8 protocol, as follows: the holding potential was shifted to - 140 mV. and 8 small "subtraction" pulses, the amplitude of the test pulse, were applied and their currents summed. Then the potential was shifted back to —70 mV, and a single test pulse was applied. This procedure was repeated every two seconds. Currents were filtered at 40 kHz and sampled at 100 kHz. The records presented are the difference between the test current and the summed subtraction currents averaged over 64 cycles. Experiments were performed at 3°-4°C. The figure shows records made with pulses to +25 and +50 mV. The effect of D2O (filled symbols) is to reduce the initial outward gating current by about 30%, to increase the time to peak inward current to about 1 .4 times its value in H,O, and to slow the decline of inward current associated with inactivation of the so- dium channels. The sodium conductance was reduced by about 35%. The changes in ionic currents are similar to those previously described (3, 6, 7). In 56 measurements at 0 to +75 mV on 1 1 axons in the absence of tetrodotoxin, D,O reduced the peak of the LOO 1 6 50 LI A/cm 250 LIS 2ms Figure 1. D:O alters gating and ionic currents. Open symbols are currents in H:O-based solutions; filled symbol.i indicate D:O-based solutions. The records are for a steps from a — 70 mV holding potential. The upper records are to +25 mV; the lower, to +50 mV. The records on the right are shown at the e.\panded timchase. NEUROBIOLOGY 165 initial outward gating current to 0.70 ± 0.02 times its value in H,O. The simplest interpretation of these results is that D2O slowed the rate of the conformational change by 30%, thus reducing the amplitude of the gating current and increasing the time required to open the channels. This could occur by changing the channels or changing the environment in which they operate. The viscosity of D,O is larger than that of H,O. and in fact, the reduction of gating and the slowing of ionic currents described above are qualitatively similar to those seen in solutions with a viscosity that has been increased with non-electrolytes (10). On the other hand, the D2O effect seems larger than predicted by viscosity alone. Perhaps D,O alters the gating machinery. About 40% of the amide protons of the Streptomyces lividans K+ channel exchange within 3 minutes of D,O exposure accompanied by subtle structural changes (11). To test between these two possibilities, currents were recorded during the transition from 0 mM sodium H,O seawater into 44 mM sodium D^O seawater. In the 0 mM Na H-.O solution, there was no inward sodium current. By 90 s after beginning the switch into the 44 mM Na D2O solution, the inward current appeared — but in the H2O pattern, similar to the open symbols in Figure 1 . Over the next 3 min the current pattern switched to the D-.O pattern, similar to the filled symbols. This suggests that the D2O effect involves changes in channel structure. I thank Dr. R. J. Lipicky and the Howard Oilman Foundation for encouragement and support. Literature Cited I Armstrong, C. M., and F. Bezanilla. 1973. Nature {Land.} 242: 459-461. 2. Keynes, R. D., and E. Rojas. 1973. ./. Physiol. {Lund) 233: 28P- 30P. 3. Thies, R. E., and F. I). Carlson. 1955. Biol. Bull. Ill: 295. 4. Conti, F., and G. Palmier!. 1968. Biophvsik. 5: 71-77. 5. Landowne, D. 1987. ./. Mcmhr. Biol. 96: 277-281. 6. Meves, H. 1974. J. Pliyxiol. ll.oihl) 243: 847-867. 7 Schauf, C. L., and J. (). Bullock. 1979. Biophys. J. 21: 193-208. 8. Alicata, D. A., M. D. Rayner, and J. G. Starkus. 1990. Biophys. J. 57: 745-758. 9. Bezanilla, F., and C. M. Armstrong. 1974. Science 183: 753-754. 10. Kukita, F. 1997. J. Physiul. tLtwd.) 498: 109-133. 1 1 Tatulian, S. A., D. M. Cortes, and E. Perozo. 1998. FEBS Leu. 423: 205-212. Reference: Biol. Bull. 199: 165-168. (October 2000) Voltage Gating Properties of Channels Formed by a Skate Retinal Connexin Thomas W. White1, Harris Ripps~, Miduliiru Srinivas , and Roberto Bruzzone (Marine Biological Laboratory, Woods Hole, Massachusetts 0254:!) Gap junctions provide pathways for electrical and chemical communication between networks of coupled cells. They act as simple electrical synapses, and also synchronize and regulate a broad range of cellular activities. The structural proteins constitut- ing gap-junctional channels in vertebrates are the connexins, mem- bers of a multigene family that exhibit a common topology: four transmembrane domains separating two well-conserved extracel- lular loops and three cytoplasmic domains. Six connexin polypep- tides oligomerize to form a membrane hemichannel or connexon. clusters of which join with the connexons of adjacent cells to create the gap junction. Once assembled, the gap-junctional chan- nel consists of an aqueous pore that allows the cell-to-cell diffu- sion of ions, second-messenger molecules, and small metabolites. Different cell types contain connexins that are unique to their special needs, and variations in the molecular structure of the individual connexins determine the gating properties, voltage dependence and cellular interactions of their gap-junctional chan- nels ( 1 ). The vertebrate retina is a useful model with which to studv the 1 Department of Neurobiology. Harvard Medical School. Boston. MA 02115. 2 Department of Ophthalmology and Visual Sciences, UIC College of Medicine, Chicago, IL 60612. 3 Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461. 4 Department de Virologie. Institut Pasteur, 75015 Paris, France. diversity of electrical coupling in nervous tissue. Diverse experi- mental approaches have shown that virtually every class of retinal neuron and glial cell makes gap junctions with neighboring cells of similar type, and in some cases with cells of another type (2, 3). Moreover, coupling between different cell types appears to be mediated by gap junctions that exhibit asymmetric dye transfer, as well as distinct pharmacological properties (4. 5). Although there is abundant evidence that the electrical synapses formed by gap junctions affect every aspect of retinal function, relatively little is known about the connexins mediating these effects. The situation has been changed by the identification of a distinct subgroup (y) of the connexin family that shows a pattern of expression restricted to the retina and the central nervous system (6-1 1 ). The first member to have been discovered, Cx35, was cloned from a skate retinal cDNA library (6), and some of its functional characteristics were examined in the Xenopits oocyte expression system (12). In the present study, we have extended these observations to analyze more fully the voltage sensitivity and kinetics of the gap junctions formed by Cx35 in paired oocytes. In addition, we have investi- gated the properties of the non-junctional hemichannels formed in single oocytes. and have compared the effects of quinine on the kinetics of the tail currents evoked at the termination of voltage pulses. The procedures for preparing cRNA, and its analysis in Xenopus oocytes have been described previously (12). Oocytes were iso- lated by enzymatic digestion and injected with either an antisense oligonucleotide (3 ng/cell) to suppress the endogenous Xenopus 166 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 40 nA 2 sec 75 70 65 60 50 45 40 35 V. = 100 mV = 0.448 sec 0.0 0.4 0.8 1.2 1.6 2.0 time [sec] control quinine 1.2- 1.0- 0~ 0.8- 0) N "oj E O 0.4 0.2- 0.0-J-r B -120 -80 -40 0 40 80 120 0.8-, 0.6- 0.4- 0.2- 0.0 025- 0.20- V 0.15^ 0 to M 0.10-1 0.05- 0.00 V [mV] 80 90 100 110 120 V, [mV] — • — control n— 100 nM quinine 40 50 60 70 80 Figure 1. Voltage gutinx <>) 'channels in Xenopus oocytes expressing skate Cx35. (A) Gap junctions! currents (lt) were elicited h\ transjunetional voltage (V :) steps, 4 s in duration, applied in ±20 mV increments from a holding potential of —40 mV. (B) Plotting steady state junctional conductance (GJS,, normali-eit in the values measured at ±20 mV) vs. VjShowsthat. even at the extremes of &Vj(± 120 mV), the residual conductance is equal to about half the initial value: data are the means (±SD) of 4 cell pairs. Cunvs drawn through the data were derived from the Bolt-maun equation in which the parameters for positive values ofV: were A = 0.06; V,, = 95; G,,,,,,, = /; and GJmin = 0.40. For negative values ofVf the corresponding mines were 0.05, NEUROBIOLOGY 167 Cx38 (13), or a combination of antisense (as above) plus Cx35 RNA (5 ng/cell). To study intercellular channels, oocytes were stripped of their vitelline membranes and brought into contact at their vegetal poles for 48 h before electrophysiological analysis. This step was omitted to explore hemichannel activity, which was recorded 48-72 h after RNA injection. Intercellular communication was quantified by dual cell voltage clamp ( 12, 14). To determine the voltage-gating properties of the intercellular channels, transjunctional potentials (V,) of opposite polarity were generated by hyperpolarizing or depolarizing one cell in 20 mV steps (over a range of ±120 mV). while clamping the second cell at -40 mV. Currents were measured 4 s after the onset of the voltage pulse, at which time they approached steady state (IJSV), and the macroscopic conductance (G|ss) was calculated by dividing Ilss by Vj. GJSS was then normali/ed to the values determined at ±20 mV, and plotted against Vr Data describing the relationship of GJSS as a function of Vj were fit to a Bolt/.mann relation ( 14) of the form: G^ = { 80 mV (see figure legend) confirm- ing the relatively weak Vj gating described earlier. Thus, voltage is not likely to be a primary modulator of Cx35-mediated intercel- lular communication in retinal neurons. The voltage gating characteristics of Cx35 were further explored by analyzing the kinetics of channel closure for values of Vj > 80 mV. i.e., sufficient to consistently induce current decay. Figure 1C illustrates results obtained from one cell pair in response to a transjunctional voltage step of +100 mV. The time-dependent decline in Ij was well fit by a single exponential function with a time constant (ri of 0.448 s. Interestingly, the mean values of r, obtained both for different values of Vr as well as for positive and negative voltage steps, hovered about 0.4 s and showed no signif- icant change as a function of either the polarity or the magnitude of V, (Fig. ID). These data are in sharp contrast to kinetic analyses of many other connexins, where T values decreased with increasing driving force (15, 16). This feature is shared by another y con- nexin, mouse Cx36 (data not shown), and illustrates further the unique properties of this subgroup. The ability of connexins to form hemichannels in Xenopus oocytes. a property reminiscent of membrane currents observed in some retinal neurons (17. 18), prompted us to investigate the kinetics of C.\35 hemichannel closure by analyzing tail currents. As we showed previously, quinine-sensitive hemichannel currents can be recorded from oocytes expressing skate Cx35 (12). This is confirmed in Figure IE. which shows the increase in the outward (non-junctional) current recorded from a single oocyte in response to depolarizing voltage increments z 40 mV, and the current enhancement produced by the addition of 100 juM quinine to the normal bath solution. To determine whether quinine exerted an effect on the gating properties of the hemichannels, we measured the kinetics of the tail currents recorded at the termination of the voltage step. Figure IF shows that the mean of the time constants of the single exponential decay functions describing the data for the return of Vm to -40 mV from values of +40 to +80 mV were unaffected by quinine. However, the hemichannel time constants are not directly comparable to the intercellular channel r values, as the ionic strength of control bath solution is greatly reduced in comparison to ooplasm, and K+ is replaced by Na+ as the prin- cipal cation. Further studies are required to determine the precise relationship between hemichannel and intercellular channel gating, and to clarify the mechanism whereby quinine markedly increases Cx35 mediated hemichannel currents. The authors thank Jane Zakevicius for technical help and Marco White for assistance with the animal care. This work was sup- ported by National Eye Institute grants (EY-13163 to TWW and EY-06516 to HR), by the Association RETINA France (to RB) and by MBL fellowships (to TWW, MS, and RB). 84, 1, and 0.45, respectively. The slight asymmetry in Bo/l:mann values implies a small degree of dependence of GJa on Vm. 1C) In response to a +100 mV voltage step, the transjunctional current decayed toward a steady state level along a single exponential with a time constant (T) of 0.448 s. (D) The values of r remained relatively constant for voltage steps ranging from ±80 to ±120 mV and were not dependent on the polarity ofVf data are the means (±SDj of 8 experiments. (E) Hemichannel activitv of C.\35 recorded from single oocytes expressing Cx35. With the cells clamped at —40 mV, non-junctional currents were elicited by depolari-ing voltage steps (from —20 to +80 mV in 20 mV intervals! imposed for a duration of 5 s. When bathed in control solution, progressively larger currents were obtained as the depolarising voltage step exceeded +20 mV. With the addition of 100 fj.M quinine. activation of hemichannel activity occurred over the same voltage range, but (he magnitudes of the ounvard currents were greatlv enhanced. {F) Tail currents measured at the end of the voltage pulses were we/I fit bv single exponentials having ^ values of —0.125 s. Quinine had no significant effect on the gating kinetics of Cx35 hemichannels: data are the means (±SD) of 6 experiments. 168 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Literature Cited 1 . Bruzzone, R., T. VV. White, and I). L. Paul. 1996. Eur. J. BIOC/ICIH. 238: 1-27. 2. Vaney, D. I. 1994. Pn>K. Ret. Eye Res. 13: 301-353. 3. Becker, D. L., V. Bonness. and P. Mobbs. 1998. Cell. Biol. Int. 22: 781-792. 4. Mills, S. L., and S. C. Massey. 1995. Nature 377: 734-737. 5. Newman, E. A., and K. R. Zahs. 1997. Seience 275: 844-847. 6. O'Brien, J., M. R. Al-Ubaidi, and H. Ripps. 1996. Mol. Bio/. Cell 1: 233-243. 7. O'Brien, J., R. Bruzzone, T. \V. White, M. R. Al-Ubaidi, and H. Ripps. 1998. J. Neurosci. 18: 7625-7637. 8. Condorelli. D. F., R. Parent!, F. Spinella, A. T. Salinaro, N. Bel- luardo, V. Cardile, and F. Cicirata. 1998. Eur. J. Neurnsci. 10: 1202-1208. 9. Sohl, G., J. Degen, B. Teubner, and K. \\illecke. 1998. FEBS Lett. 428: 27-31. 10. Srinivas, M., R. Rozental. T. Kojima, R. Dermietzel. M. Mehler, D. F. Condorelli, J. A. Kessler, and D. C. Spray. 1999. J. Neuro- sci. 19: 9848-9855. 11 Al-Ubaidi, M. R., T. W. White, H. Ripps, I. Poras, P. Avner, D. Gomes, and R. Bruzzone. 2000. J. Neurosci. Res. 59: 813-826. 12 White, T. W., M. R. Deans, J. O'Brien. M. R. Al-Ubaidi, D. A. Goodenough, H. Ripps, and R. Bruzzone. 1999. Eur. J. Neurosci. 11: 18X3-1890. 1 3 Bruzzone, R., J.-A. Haefliger, R. L. Gimlich, and D. L. Paul. 1993. Mol. Bio/. Cell 4: 7-20. 14. Spray, D. C., A. L. Harris, and M. V. L. Bennett. 1981. J. Gen. Pliysiol. 77: 77-93. 15. Barrio, L. C., J. Capel, J. A. Jarillo, C. Castro, and A. Revilla. 1997. Bio/)/iv.v. J. 73: 757-769. 16. White, T. W., R. Bruzzone, D. A. Goodenough, and D. L. Paul. 1992. A/o/. Bio/. Cell 3: 711-720. 17 DeVries, S. H., and E. A. Schwartz. 1992. J. Pliysiol. (Lund. ) 445: 201-230. 18. Malchow, R. P., H. Qian, and H. Ripps. 1993. J. Neurosci. Res. 35: 237-245. Reference: Biol. Bull. 199: 168-170. (October 2000) Hydrogen Ion Fluxes from Isolated Retinal Horizontal Cells: Modulation by Glutamate Antlwnv J. A. Molina (Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607), Peter J. S. Smith1, and Robert Paul Malchow2 Retinal horizontal cells are second order neurons that receive direct input from photoreceptors. These cells are believed to play a crucial role in the formation of the surround aspect of the classic center-surround receptive fields of visual neurons. Debate still persists as to the molecular mechanisms used by horizontal cells to establish the surround portion of these receptive fields. One hy- pothesis, promulgated recently by Kamermans and colleagues ( 1 ), suggests that horizontal cells may exert their lateral inhibitory actions by modulating the calcium flux into the synaptic terminals of photoreceptors, thus altering the release of the photoreceptor neurotransmitter. Hydrogen ions are among several agents that have been proposed to act in this modulatory role (2). and in fact, the responses to light by second order retinal neurons are very sensitive to changes in extracellular pH (3, 4). In an elegant series of experiments. Barnes and coworkers (5) demonstrated that this pH-dcpendent modulation of synaptic transmission was due to the marked sensitivity of calcium channels in the photoreceptors to extracellular hydrogen ions. These investigators found that ele- vated concentrations of H f shifted the voltage-dependence of the calcium current activation curve of the photoreceptors to more depolarized levels and also reduced the calcium conductance. Moreover, small light-induced changes in extracellular pH within the intact retina have been reported by Oakley and Wen (6). Horizontal cells could thus exert their inhibitory influences by 1 BioCurrents Research Center, Marine Biological Laboratory, Woods Hole. MA. 2 Departments of Biological Sciences and Ophthalmology, University of Illinois at Chicago. Chicago. IL. modifying the concentration of hydrogen ions in the external milieu. In the present work, we have used pH-selective microelec- trodes to monitor the flux of hydrogen ions surrounding isolated retinal horizontal cells. In particular, we examined whether the amino acid glutamate could alter the flux of hydrogen ions re- corded from these cells. We reasoned that, if the release of hydro- gen ions from horizontal cells is indeed a key factor in the creation of the surround portion of retinal receptive fields, then such a flux should be modified by glutamate, the neurotransmitter believed to be released by vertebrate photoreceptors (7). The pH-selective electrodes were used in a self-referencing mode (8), which greatly enhances their signal sensitivity and stability, eliminating much of the electrical noise and drift inherent in such electrodes. In this format, the electrode is first placed just adjacent to the membrane of the cell, and a reading taken; the electrode is then moved a set distance away (typically 30 /am), and a second reading taken. The difference between the voltage read- ings at the two positions reflects differences in the free hydrogen activity at the two locations. This method allowed us to measure the small hydrogen ion fluxes that would otherwise have been lost in the noise of the recordings. pH selective electrodes were prepared by pulling thin-walled glass capillary tubing (o.d. 1.5 mm) to a tip diameter of 2-4 jum. The pipettes were silanized and back-filled with 100 mM potas- sium chloride, and the fluid was forced to the tip of the pipette by air pressure applied to the back of the pipette from a syringe. The pipette tip was then filled with a pH-selective resin (hydrogen ionophore 1 -Cocktail B, Fluka Chemical; the tip was placed in contact with a source pipette containing the resin, and about 50 /xm NEUROBIOLOGY 169 of the resin was then drawn up by suction on the back of the pipette. The resin employed here has a particularly high selectivity for hydrogen ions, and is reported to be more than 10'' times more sensitive to hydrogen ions than to either sodium or potassium ions (9). Isolated retinal horizontal cells were obtained by enzymatic dissociation of the retina of the skate (Rajti erinacea or R. ocellata ) as described in Malchow ft al. (10). Briefly, the animals were chilled in ice. cervically transected, and double pithed. The eyes were removed, and the retinas were isolated and placed for 45 min under gentle agitation into a skate-modified L-15 culture medium containing 2 mg/ml papain and 1 mg/ml cysteine. The retinas were then rinsed 8 times in media lacking papain and cysteine. and then mechanically agitated through a 5-ml graduated glass pipette. Single drops of this cellular suspension were placed in 35-mm plastic culture dishes that had previously been coated with 1% protamine sulfate and 0.1% concanavalin A. Cells were stored at 14°C for up to 4 days before use. Recordings were made in a skate Ringer's solution containing 2 mM of the pH buffer HEPES and no added bicarbonate. A 5 mM glutamate stock solution was prepared in skate Ringer and adjusted to pH 7.6 with 1 M NaOH. Glutamate was applied by adding 1 ml of the 5 mM glutamate solution to 4 ml of Ringer already present in the culture dish, resulting in a final concentration of 1 mM glutamate. Under these conditions, a steady differential signal was obtained from horizontal cells indicative of a higher concentration of hy- drogen ions near the membranes of the cells. The size of this signal decreased as the concentration of the pH buffer HEPES was increased, consistent with the hypothesis that the signal detected indeed reflected hydrogen ions. Moreover, as shown in Figure 1. the application of 1 mM glutamate resulted in a marked decrease in the size of the differential signal. A differential signal of approximately 100 juV was initially recorded from this cell. The 140 120 - 100 - 80 - 60 40 • 20 • AAV/1 0 -20 -40 200 400 600 800 1000 time(s) Figure 1. Effects of glutamate on the hydrogen ion flux recorded from a single isolated retinal horizontal cell. The differential voltage recorded from a pH-selective electrode is plotted as a function of time. Before the application of glutamate. a differential signal indicative of a higher con- centration of hydrogen ions near the membrane of the cell is observed. At the arrow, glutamate was added such that the final concentration in the dish was 1 mM. A marked decrease in the differential signal recorded hy the pH-selective electrode \vas obsen'ed. actual proton flux represented by this differential voltage can be calculated using an equation derived by D. M. Porterfield [in prep.; see aKo (12)] as follows: J= - D(A[FT] + [Buffer] * 0.25A[H+] * K;1) * Ar1 Where J is tlu- flux. D is the diffusion coefficient for hydrogen ions. A[H + ] the change in hydrogen ion activity between the two poles of measurement. [Buffer] is the buffer concentration expressed in moles per cm~\ Ka is the pK., of the buffer expressed in cm" \ and Ar is the distance in cm between the two measuring positions of the probe. Taking into account a small loss of the signal within the electronics of the amplification system (8). under our experimental conditions the 100 /J.V signal we observe is then estimated to be indicative of a proton flux of —75 pM cm"2 s"1. In 6 cells studied in this fashion. 1 mM glutamate reliably reduced the differential signal by an average of 60%. We thus conclude that glutamate. the presumed neurotransmitter from vertebrate photoreceptors. can indeed alter the flux of hydro- gen ions from horizontal cells. In this context, it is interesting to note that glutamate has previously been reported to promote an acidification of the internal milieu of catfish retinal horizontal cells as measured using the pH-indicator dye BCECF (11). We hypoth- esize that glutamate may shut down the transport of hydrogen ions from horizontal cells, thus trapping hydrogen ions in the interior of the cell. This would account for the increased intracellular acidity and the alkalinization of the extracellular milieu that we have observed. The alteration in extracellular pH induced by glutamate may be important in modifying signaling within the outer plexi- form layer of the retina. Indeed, extracellular alkalinizations in- duced by neuronal activity occur in several other regions of the nervous system (reviewed by Chester (13)), and excitatory amino acid receptors have been implicated in the generation of these phenomena. Thus, modulation of extracellular pH within the CNS by glutamate may be a common means by which synaptic activity is altered. Future experiments are planned in which specific phar- macological agents will be used to determine which transporter or transporters may be involved in the glutamate-induced changes in extracellular hydrogen ion concentrations. We are grateful to Kasia Hammar for her generous assistance with electrode preparation and cell culture, Naomi Rosenkranz for help preparing isolated cells, and Richard H. Sanger for electronic and computer assistance. This work was supported by grants EYO941 1 from the National Eye Institute. P41 RR01394 from the National Center for Research Resources, and a grant from the Campus Research Board of the University of Illinois at Chicago. Literature Cited 1. Venveij. J., M. Kamermans, and H. Spekreijse. 1996. Vision Res. 36: 3943-3953. 2. Kamermans, M., and H. Spekreijse. 1999. Vision Rex. 39: 2449- 2468. 3. Kleinschmidt, J. 1991. Ann. N.Y. Acad. Sci. 635: 468-470. 4. Harsanyi, K., and S. C. Mangel. 1993. Vis. Neurosci. 10: 81-91. ?. Barnes, S., V. Mere-hang, F. Mahmud. 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 10081-10085. 170 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 6. Oaklev, B. and R. Wen. 1989. ./. Physiol. (LonJ) 419: 353-378. 7. Copenhagen, D. R., and C. E. Jahr. 1989. Nature 341: 536-539. 8. Smith, P. J. S., K. Hammar, D. M. Porterfield, R. H. Sanger, and J. R. Trimarchi. 1999. Micmsc. Res. Tech. 46: 3W-417. 4. Fluka, 1991. Selectophore, lonophores for Ion-Selective Electrodes ami Opt rode s. Fluxa Chemie A. G. Ronkonkoma, New York. 10 Mali-how, R. P., H. Qian. H. Ripps, and J. E. Dowling. 1990. J. Gen. Physiol. 95: 177-188. 1 1. Dixon, D. B., K-I. Takahashi, and D. R. Copenhagen. 1993. Neu- nm 11: 267-277. 12. Smith, P. J. S. and J. Trimarchi. 2000. Am. .1. Ph\siol. (in press.) 13. Chester, M. 1990. Progr. Neurohwl. 34: 401-427. Reference: Biol. Bull. 199: 170-171. (October 2000) Odor-induced Oscillatory Activity in Drosophila CNS Jing W. Wang (Department of Biochemistry anil Molecular Biophysics, Howard Hughes Medical Institute, Columbia Universit\, New York, New York 10032) In mammals and the fruit fly, the vast array of odors in the environment is discriminated by a large number of receptor mol- ecules ( 1, 2, 3). Individual olfactory sensory neurons express only one of the many receptor genes (1,2, 3). Neurons expressing the same receptor gene project to the same glomerulus (4, 5, 6), providing the anatomical evidence for a spatial coding mechanism. Electrophysiological recordings from olfactory neurons suggest that the temporal pattern of their responses can also convey infor- mation about odor quality (7). Odor-induced oscillatory activity, an indication of synchrony, has been observed in phylogenetically different species, including molluscs, insects, and mammals (7. 8, 9. 10. 11. 12). The adult Drosophila antennal lobe, organized in spheroidal subcompartments termed glomeruli, receives about 1200 olfactory afferents from the antenna and 120 afferent fibers from the max- illary palp (13). Although the fly and mammals share the similarity that receptor neurons expressing the same receptor gene project to one or two glomeruli in a stereotypic manner (4, 5, 6), there are only 60 receptor genes and 43 glomeruli in Drosophila, in contrast to the 1000 receptor genes and 1800 glomeruli within the olfactory bulb of mammals ( 1, 2, 3). The lower complexity in anatomy and the rich behavioral repertoire in Drosophila makes it an attractive system with which to study olfaction. Moreover, sophisticated genetic tools and behavioral mutants can now also be used to study the olfactory system in Drosophila. Nevertheless, understanding mechanisms of odor discrimination in the CNS of the fly has been difficult due to a lack of physiological tools for functional studies. Odor-induced oscillations have been observed in several insect species, including the locust, cockroach, honeybee, bumblebee, and wasp (7). Local field potential (LFP) recordings show odor- induced oscillation at —10 Hz. which typically lasts for the dura- tion of odor stimulation. I have investigated this phenomenon in the Drosophila CNS. LFPs were recorded with glass electrodes (tip, 5 /urn) that were filled with Drosophila HL3 saline and Peppermint Local Field Potential Amyl Acetate Power Spectrum Density Peppermint Amyl Acetate Frequency Figure 1. Local field potential recordings of odor-induced oscillation in the CNS of Drosophila. The left panel shows five sequential responses to peppermint stimulation recorded from the same preparation. Responses to ainyl acetate from the same preparation are shown in the middle panel. Averaged power spectrum density from the five trials is shown in the right panel. The LFP response to peppermint appears to have a higher density at 2 Hz than the response to amyl acetate. NEUROBIOLOGY 171 positioned with a motorized manipulator (MP285. Sutler). A patch clamp amplifier (EPC 7. Heka) was used, and the signal was filtered (band pass at 0.1 to 20 H/.) with a signal conditioner (CyberAmp, Axon Instruments) and recorded with software (Axo- Scope. Axon Instruments) run on a PC. Adult flies (less than a week after eclosion) were lightly anesthetized with CO2 and de- capitated. The heads were immobilized with wax on a microscope slide with the antennae pointing upward. A small opening was made on the dorsal cuticle for the extracellular recording. Figure 1 shows LFP recordings from the CNS of the Canton-S wild-type fly that reveal an odor-induced oscillation. This phenom- enon was confirmed in 6 preparations. A power spectrum analysis indicates that the major frequency components are less than 4 Hz (Fig. 1 ). This LFP oscillation signal appears to be sensitive to the position of the electrode, and the coordinates taken from the manipulator suggest that the recordings may have originated in the antennal lobe. Future experiments with GFP-labeled antennal lobe may help in identifying the sources of the oscillatory activity. The patterns of oscillation in response to the same odor appear to be roughly similar in sequential recordings from the same animal. The LFP patterns generated in response to peppermint (from McCor- mick) and amyl acetate (from Sigma) were distinguishable by eye. Moreover, the power spectrum analysis indicates that peppermint generates slightly more high frequency components. This is the first LFP recording from the Drosophila CNS. The preliminary results presented here show that odor-induced oscil- lation occurs in Drosophila; this finding suggests that a temporal coding mechanism may be employed by the fly. and that the power of genetics may be applied in the future to decipher the physio- logical significance of the odor-induced oscillation. I would like to thank Alan Gelperin for his generous support. Leonardo Belluscio for critical comments on the manuscript, and Carl Zeiss. Inc., and Axon Instruments, Inc., for providing equip- ment. This research was carried out in the Grass Laboratory at the Marine Biological Laboratory, Woods Hole. Massachusetts, and was supporu d by the Grass Foundation. Literature Cited I Buck, L., and K. Axel. 1991. Cell 65: 175-187. 2. Clyne, P. J., C. (,. Warr, M. R. Freeman, D. Lessing, J. Kim, and J. R. Carlson. 1999. Neuron 22: 327-338. 3. Vosshall, L. B., H. Amrein, P. S. Morozov, A. Rzhetsky, and R. Axel. 1999. Cell 96: 725-736. 4. Gao, Q., B. Yuan, and A. Chess. 2(100. Nature Neuro.ici. 3: 780- 785. 5 Vosshall, L. B., A. M. Wong, and R. Axel. 2000. Cell 102: 147 159. 6. Ressler, K. J., S. L. Sullivan, and L. B. Buck. 1994. Cell 79: 1245-1255. 7. Stopfer, M., M. Wehr, K. MacLeod, and G. Laurent. 1999. Pp. 163-180 in Insect Olfaction. B. S. Hunsson, ed. Springer, Berlin. X. Adrian, E. D. 1942. J. Physio/. Lond, 100: 459-473. 9. Delaney, K. R., and J. B. Hall. 1995. J. Neurosci. Methods 68: 193-202. 10. Gray, C. M. 1994. Neurosci. 1: I 1-38. 1 1 Tank, D. W.. A. Gelperin, and D. Kleinfeld. 1994. Science 265: 1819-1820. 12 Lam, V.-W., L. B. Cohen, M. Waclumiak. and M. R. Zochowski. 2000. J. Neurosci. 20: 749-762. 13. Stacker, R. F., and V. Rodrigues. 1999. Pp. 283-314 in Insect Olfaction, B. S. Hansson, ed. Springer, Berlin. Reference: Biol. Bull. 199: 171-173. (October 2000) Circadian Rhythms in the Receptive Fields of the Liimilus Lateral Eye James M. Hilt (Marine Biological Laboratory, Woods Hole, Massachusetts 02543), Frederick A. Dodge, Ehud Kaplan, and Robert B. Barlow Hartline found that in the frog "a given optic nerve fiber re- sponds to light only if a particular region of the retina receives illumination." He called the region the receptive field of that fiber (1). Continuing Hartline's study of the frog retina. H. B. Barlow detected an inhibitory influence surrounding the excitatory region of the receptive field (2). In the lateral eye of the horseshoe crab Limiilitx polyphemus, the receptive fields of single ommatidia have both excitatory centers and inhibitory surrounds. The field of view of a single ommatidium defines the narrow excitatory center, whereas the neural network connecting neighboring ommatidia ( — 200) generates the wide inhibitory surround. A circadian clock in the animal's brain transmits signals to the lateral eye at night, changing its structure and function to increase the retinal sensitiv- ity (3) so that the animal can detect mates nearly as well at night as it can during the day (4). Several mechanisms underlying the remarkable nighttime sensitivity have been identified: they are increased photoreceptor gain, decreased photoreceptor noise, de- creased lateral inhibition, and increased photon catch as a conse- quence of an increased acceptance angle for each ommatidium (3). High retinal sensitivity at night is associated with highly vari- able ("noisy") optic nerve responses, which result from random photon events at low nighttime levels of illumination. Such noisy neural responses hinder our efforts to measure properties of the nighttime state of the eye and, thus, our development of an accu- rate cell-based model of retinal function. Our goal is to understand the neural code the eye sends to the brain at night as we have already done for the daytime state of the eye (5, 6). Here we report an analysis of retinal receptive fields and demonstrate how their properties change from day to night. Our method takes advantage of the remarkable linearity of the responses of the lateral eye to small modulations of the visual input. We employ time-varying sinusoidal stimuli and linear sys- tems analysis. Modulated square patterns are presented on a TV monitor having a grey background, with nighttime stimuli attenu- ated by a 4.25 log unit neutral density filter. After isolating the response of a single optic nerve fiber, we align the animal so that the optic axis of the recorded ommatidium views the center of the pattern. We use animals that had been entrained to the natural 172 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS lighting cycle, taking daytime measurements before 1730 h and nighttime measurements from 2200 to 0100. The square pattern is modulated ±10% relative to the background at 1 Hz. Using a strategy previously developed for studying retinal receptive fields in the frog (2), we measured the width of the excitatory center by illuminating the eye with square stimuli of increasing size. Be- cause the inhibitory surround extends into the excitatory center, responses are a mixture of excitatory and inhibitory inputs; but the responses to the smallest square stimuli are predominantly excita- tory. Figure 1A plots the amplitude of modulated optic nerve re- sponses as a function of the visual angle of the square stimuli during the day (gray points) and night (black points). For each data set, the ordinate scale ranges from no response (0%). to the theoretical maximum excitatory response (100%) that would be achieved in the absence of inhibition. The points plotted in Figure 1A show an increase in response for increasing stimulus size, up to 24° of visual angle, beyond which responses decrease because more of the inhibitory surround is illuminated by the square stimulus. The growth of the responses to expanding stimuli (smooth curves) are estimated from the responses to the smallest square stimuli, because the small stimuli minimally activate the inhibitory surround. Assuming that the excitatory center can be well represented by a two-dimensional gaussian function (3). the "pure" excitatory response is proportional to the volume of the excitatory center surface covered by a stimulus. Using this rela- tionship, we estimated the size of the excitatory center based on the recorded responses to the four smallest square stimuli and extrap- olated the theoretical maximum response of the excitatory center (100% on the ordinate in Figure 1A: Ep in Equation [1]). The smooth curves plot the theoretical responses, yielding excitatory centers with half-maximal width of 12° during the day (gray curve) and 16° at night (black curve). Because the size of the excitatory center increased at night, the recorded response reached only 85%' of its maximum theoretical value before decreasing as a result of surround inhibition. Although the inhibitory surround overlaps the excitatory center, the effects of surround inhibition are minimal for small squares. We therefore attribute the observed changes in Figure 1A to an expanded excitatory field width at night, arising from circadian changes in ommatidial structure, that is. a shift of photoreceptor position and migration of pigmented cells (3). The vectors in Figure IB plot the modulated optic nerve re- sponse in terms of its phase and amplitude relation to the stimu- lus— a sine wave with a direction of 0° (vertical) and a length of 1.0. To measure the strength of the inhibitory surround, we first determine the maximal excitatory response, as described above, and plot it as a vector in Figure IB (open circles). We next determine the response vector for full-field stimulation by modu- lating the entire TV monitor with a ± 10% contrast at 1 Hz (open squares); this vector represents the summed response of excitatory and inhibitory inputs. Finally, we measure the response vector for inhibition by modulating the surround while holding constant the stimulus to the center (crosses). Because the Limulus eye responds linearly to small amplitude stimulation, the effects of excitation and inhibition superimpose; i.e.. the sum of center and surround response vectors should equal the full-field response. The vector sum of excitatory and inhibitory responses during the day is plotted as a thin line that lies adjacent to the vector for the full-field o D. en A. 100% 80% 60% (D 1 40% 0% 5 10 15 20 25 Visual Angle (Degrees) Night 30 -1 O Center illumination x Surround illumination D Full-Field illumination 1 2 Cosine component Figure 1. A: Plot of the response of a single optic nen'e (ordinate) as a function of the size of the centrally located visual stimulus (abscissa). Davtime (gray points) and nighttime (black points) responses are normal- ized to their respective theoretical maximum excitatory response. As ex- pected, the response increases with the size of the stimulus. Solid lines show the growth of the response calculated on the basis of a gaussian- shaped excitatory center. B: Vector plots of responses to illumination of the center (unfilled circle), surround (crosses), and full-field (squares) stimu- lation. Vectors are plotted relative to the sine wave stimulus, which has an angle ofO° and a length of 1. As explained in the te.\t, responses to center and surround alone predict with reasonable accuracv the response to full-field stimulation, which drives both the excitatory center and the inhihitorv surround. Such vector addition confirms the linear properties of the Limulus lateral eye. The nighttime vectors show a reduction in the difference bet\\jll and 2 I/HI h. The top video frame was taken in the clear water of Great Harbor, Woods Hole. Massachusetts, where the grey/black sectors of the cylinder had a contrast of 69% /contrast = (LCrfy — i-Bi:,ct>Ai-cr^ + LB/Mt)]. The second video frame was taken near Stonev Beach. Woods Hole, where turbid water reduced the contrast of the grey/black sectors to 26%. Arrows indicate the times at which the undenvater scenes to the right were videotaped. At these times the black sector begins to enter the field of view of the recorded ommatidium, reducing its response rate. The "Day" records are responses to a single rotation of the cvlinder in a right to left direction (loop with arrow). The "Night" records \liow responses to nine consecutive rotations of the cylinder (thin black traces; period of rotation ~ 16 s) and their average (thick black trace). The peaks and valleys of the thin black traces reflect the hig/tly variable rate of discharge of the single optic nen'e fiber itniler /mr nighttime levels of illumination. are highly variable relative to those recorded during the day, and that the average response rate to the grey sector is about 3 impulses/s which is 6-fold lower than the mean daytime response rate of about 18 impulses/s (middle trace). We attribute the highly variable response rates to random photon events occurring at the very low nighttime levels of illumination. The nighttime sky during this experiment was heavily overcast and lacked moonlight. From radiometric measurements we estimate that ambient light decreased by about 106 to 107 relative to daytime levels. The circadian increase in lateral eye sensitivity cited above nearly compensates for such large reductions in ambient lighting. Exper- iments in the laboratory (R. Barlow and F. Dodge, unpub. obs.) indicate that the average response to the grey sector of -3 im- pulses/s is about 50<7f lower than expected for the low nighttime levels of illumination. The surgery performed to isolate the single optic nerve fiber may have partially damaged the fragile efferent fibers that carry the circadian clock's signal from the brain to the eye; as a consequence, the lateral eye may not have received the normal efferent input and thus the retina may not have shifted completely to its fully sensitive nighttime state. Nevertheless the eye's circadian increase in sensitivity was sufficient to detect the rotating black sector of the cylinder, which mimics a moving mate. Computational analyses of visual processing in the Limuhm brain indicate that retinal inputs may sum at the first synaptic level 178 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS (5). Spatial summation across a matrix of 5-10 ommatidia signif- icantly increases the signal-to-noise properties of responses re- corded at night. Indeed summing seven sequential optic nerve responses to the rotating cylinder yielded a relatively noise-free response. These experiments represent our first attempts to analyze lateral- eye responses of Limulus at night in the animal's natural habitat. The use of a periodic stimulus obviated the need for video docu- mentation of the visual stimulus, which is not feasible under nighttime lighting conditions. With this technique, we successfully recorded visual responses in the animal's habitat and found that the lateral eye transmits information to the brain about mate-like objects at night under dark overcast skies. Under such conditions Lintulus could see what we could not. Supported by the National Science Foundation. National Insti- tute of Mental Health, National Eye Institute, Research to Prevent Blindness, and the Central Lions of New York. Literature Cited 1 Passaglia, C., F. Dodge, E. Herzog, S. Jackson, and R. Barlow. 1997. Pn>c. Null. ACM/. Sci. 94: 12649-12654. 2. Barlow, R. B., L. C. Ireland, and L. Kass. 1982. Nature 296: 65-66. 3. Barlow, R. B. 1983. J. Neuruscience 3: 856-870. 4 Barlow, R. B., S. C. Chamberlain, and S. C. Levinson. 1980. Si-i- encc 210: 1037-1039. 5. Hitt, J.. C. Passaglia, F. Dodge, and R. Barlow. 2000. Ninth Annual Computational Neitroscience Meeting. Brugge. Belgium, p. 75. Reference: Bioi Bull. 199: 178-180. (October 2000) Do the Properties of Underwater Lighting Influence the Visually Guided Behavior of Limulus? Matthew A. Krutky1, J ilium L. Atherton2, Spence Smith, Frederick A. Dodge and Robert B. Barlow (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) In the spring, horseshoe crabs. Limit/us pol\phemus, migrate to the water's edge along the East coast of the United States to pair off and build nests ( 1 ). As they enter a nesting area, males use their lateral eyes to locate mates both day and night (2). They approach females and objects resembling them, such as rocks, patches of seaweed, or mate-like objects. What does a male see in a female? Her size and contrast are two important factors. Males are attracted to objects that approximate the size of females. They orient toward mate-like objects at distances up to 1.2 meters, detecting higher contrast objects better than lower contrast ones (3). How do the properties of underwater lighting in the animal's natural habitat influence whether a crab finds a mate during the day or at night? The approximate 1.000,000-fold reduction in ambient lighting after sundown has no appreciable effect. Their remarkable visual performance results in part from a circadian increase in lateral eye sensitivity of as much as 1.000,000 times at night (4). In this paper we consider another property of the animal's underwater habitat, termed "strobic lighting." In the shallow waters of nesting areas, overhead waves act like lenses, creating moving beams of sun- and moonlight that reflect off the sandy bottom and submerged objects. On average, the peak intensity of these beams is about three times that of ambient illumination. The fields of view of single ommatidia are wider than the moving beams of light. Because ommatidia sum the illumina- tion within their field of view, the amplitude of modulation of the light beams reaching the underlying photoreceptor cells decreases to about 70% contrast. The strobic illumination by the beams strongly modulates the firing rate of an ommatidium, with peak firing rates reaching three times the mean (5). Such strobic illu- mination might be expected to enhance the detectability of under- 1 Syracuse University, Syracuse, New York. 2 Allegheny College. Meadville. Pennsylvania. water objects, such as potential mates. Indeed, an earlier study suggested that strobic conditions enhance the visibility of low contrast mate-like objects, and that without strobing. Limulus is attracted to higher contrast objects (6). We have further explored the influence of strobic lighting by carrying out more field studies and combining the results with those collected over the past five years. We investigated the visual performance of Limulus during their springtime mating seasons at Mashnee Dike, Bourne, and North Monomoy Island. Chatham, both located in Massachusetts. Our study and those of previous years were carried out day and night under various weather conditions ranging from dense cloud cover to clear skies, yielding 105 to 107-fold diurnal changes in the intensity of ambient illumination, with an average change of about 106. In all our studies, a modified two-alternative forced choice technique adapted from human psychophysics was used (7). As shown in Figure 1, we placed on the sandy bottom a clear Plexiglas chute with a funnel at one end and a narrow chute at the other. Crabs entered the funnel and, upon exiting the chute, were pre- sented with the choice of a black or gray female-sized object; these were located 1 m from the exit of the chute and 1 m from each other, creating an equilateral triangle. The objects were either a hemisphere (diameter of 0.3 in) or a cylinder (height of 0.15 m; diameter of 0.3 m), both approximating the size of an adult female horseshoe crab. The objects were switched periodically during an observation period to avoid any effects of directional bias in behavior. Their black and gray tones represent the greatest range of contrast of the female carapace (8). The black object has a negative contrast of 37% against the background of sand and seawater. and the gray object has a positive contrast of 35%. Animals exiting the chute either approached and contacted one of the two targets or swam by them. In 1999 and 2000, about 60% of animals exiting the chute did not approach or contact either submerged object. The animals passing by both objects were not recorded in the years NEUROBIOLOGY 179 o \ r &i Figure 1. Left: Diagram of field experiment. Limulus approaches the Plexiglas chute and exits toward gra\ fG) ami black (B) mate-like objects located 1 mfrom the opening and I in from each other. Middle: Photograph taken under nonstrobic conditions showing an animal (outlined) exiting the chute and oriented toward the black object. Right: Photograph taken under strobic conditions showing an animal approaching the grn\ object which is highlighted by bright beams of light. Only the chute and part of the funnel are visible in the photos. Nonstrobic condition?, occurred when cither no sun or moon was visible, when the wind was calm, or when overhead waves were blocked by a clear-bottom Plexiglas box. preceding 1999. More than 99% of the animals studied were males, because animals in amplexus were prevented from entering the funnel, and <1% of single animals in nesting areas are females (1). We could not determine whether a specific male crab passed through the chute more than once in a single observation period, but since animals in nesting areas are abundant, such events are unlikely to have occurred. When an animal exited the chute, observers noted whether the lighting conditions were strobic or nonstrobic. In 1995 through 1998, nonstrobic conditions occurred naturally day and night, under cloudy skies or in calm water. In 1999 and 2000, we controlled strobing by placing a clear-bottomed box on the surface of the water above both targets. The Plexiglas bottom of the box prevented rippling wave action, thereby elimi- nating strobic lighting of the underwater scene (See Fig. I ). Table 1 summarizes the data collected in 2000 and during the five previous years. Taken together, the data for all six years ("Total" in Table 1) indicate that, under nonstrobic conditions. there is no significant difference between the number of animals attracted to the two objects day or night (P-values of 0.76 and 0.077 respectively, as determined by the x* test). Under strobic conditions, significantly more animals, 69 or 24% more, ap- proached the gray object during the day (P < 0.00005). The greater number of animals, 12, attracted to the gray target at night under strobic conditions, was not significant (P > 0.4). These field studies show that when the distribution of illu- mination in the animal's natural habitat is uniform (nonstrobic conditions), the animals detect black and gray mate-like objects about equally well day and night. This is understandable be- cause the black and gray objects have about the same absolute contrast, 37% and 35% respectively, against the underwater background. Under strobic conditions, significantly more ani- mals are attracted to the gray object during the day. but not at night. This is also understandable because, as described above, the moving, underwater light beams increase the contrast of the gray object, but not the black one. Indeed optic nerve recordings in the animals' natural habitat reveal bursts of activity in response to gray objects illuminated by strobic light (5). Why the gray objects are not more attractive under strobic conditions at night is not understood. The highly variable optic nerve discharge resulting from random photon events at low levels of nighttime illumination (9) might be masking the bursts of activity generated by strobic lighting. We conclude that the properties of ambient lighting can affect an animal's vision in its natural habitat, particularly during the day. Limulus is not unique. Strobic lighting appears to have a prominent role in the visual performance of other marine animals (10. 11). Supported by the National Science Foundation, National Insti- tute of Mental Health, National Eye Institute, Research to Prevent Table 1 Number of crabs that exited the chute and hit black or grey targets in the 1995-2000 mating seasons Strobic Nonstrobic Year Time Black Gray Black Gray 1995 Day 3 7 0 0 Night 13 in 13 1 1996 Day 16 25 44 41 Night 16 36 8 2 1997 Day 0 0 17 10 Night 33 35 10 12 1998 Day 11 13 21 18 Night 6 9 54 48 1999 Day 30 76 63 87 Night 28 TT 15 16 2000 Day 44 48 193 190 Night 4 2 5 11 Total Day 107 176 338 346 Night 100 112 139 11 1 Hits were divided into strobic and nonstrobic categories depending on the underwater lighting conditions when an animal left the chute. Strobic refers to moving beams of sun- and moonlight that reflect off the sandy bottom and submerged objects, whereas nonstrobic refers to the absence of this phenomenon. 180 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Blindness, and the Central Lions of New York. Special thanks to Lori Broderick, Hoyt and Deborah Ecker. Elizabeth Flynn. Jason Friedenfelds. Son Gibson, Carrie McGuinnes. Michelle Parshley. and Vanessa Ruta, and the Monomoy National Wildlife Refuge. U.S. Dept. of Interior. Chatham. MA. Literature Cited 1 Barlow, R. B., M. K. Powers, H. Howard, and L. Kass. 1986. Bwi Bull. 171: 310-329. 2 Barlow, R. B., L. C. Ireland, and L. Kass. 1982. Nature 296: 65-66. 3. Herzog, E. D., M. K. Powers, and R. B. Barlow. 1996. Visual Neurosci. 13: 31-41. 4 Barlow, R. B. 1983. J. Neuroscience. 3: 856-870. 5. Passaglia, C. L., F. A. Dodge, E. Herzog, and R. B. Barlow. 1997. Proc. Nail. Acad. Sci. USA. 94: 12,649-12,654. 6. Passaglia, C. L., M. E. McSweeny, K. M. Stewart, E. Kim, E. J. Mole, M. K. Powers, and R. B. Barlow. 1997. Biol. Bull. 193: 205-207. 7. Teller, D. Y. 1979. Infant Behav. and Dev. 2: 135-153. 8. Herzog, E. D., and R. B. Barlow. 1992. Visual Neurosci. 9: 571- 580. 9. Atheron, J. L., M. A. Krutky, J. Hitt, F. Dodge, and R. B. Barlow. 2000. Binl. Bull. 199: 176-178. 10. Loew E. R., and W. N. McFarland. 1990. Pp. 1-40 in Visual System of Fish. R. H. Douglas and M. B. A. Djagmoz. eds. Chapman and Hall. London. 11. Glantz, R. M., and A. Bartels. 1994. J. Neurophysioi 71: 2168- 2182. Reference: Biol. Bull. 199: 180-182. (October 2000) Startle Responses of Fish Without Mauthner Neurons: Escape Behavior of the Lumpfish (Cycloptenis liimpus) Melina E. Hale (Dept. of Neurobiology and Behavior, SUNY Stony Brook. Stony Brook, New York 1 1794-5230 and Grass Foundation Fellow, Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Fast start escape responses are the primary behaviors used by fishes to avoid an attacking predator. Of particular importance is the C-start type of fast start (reviewed by 1, also see 2. 3). During a C-start the fish rapidly turns away from a threatening stimulus into a "C" shaped body bend, called stage 1. Frequently, stage 1 is followed by u tail stroke to the opposite side of the body, stage 2. which propels the fish away from the stimulus. The C-sturt is initiated by the Mauthner cells, a pair of large reticulospinal interneurons (4. 5 1. Each M-cell has a large axon that crosses the body midline and extends the length of the spinal cord, exciting motoneurons that innervate the lateral muscle. In response to a stimulus from the right side of the body, the right M-cell fires an action potential that propagates rapidly down the axon to cause nearly simultaneous contraction of muscle on the opposite side of the body from the M-cell soma and the "C" bend away from the stimulus (6, 7). Although Mauthner cells have been identified in a large number of taxa broadly representing the phylogenetic diversity of acti- nopterygian fishes, a few species appear to lack these neurons (8). This study examines the startle behavior of one such species, the lumpfish (Cycloptenis liimpus). Two specific questions are ad- dressed. First, do lumpfish have a startle response that is distinct from routine swimming? If so. how does the behavioral pattern and performance compare with the M-cell initiated C-start of other fishes? The startle response was examined in larval lumpfish rather than in mature individuals. The larval lumpfish have a more generali/ed morphology than mature lumpfish, and so it was thought that the response ot the larvae to a startle stimulus may be more easily compared to other species. Additionally, it seemed that if the lumpfish were to have high performance behavioral responses to predation. it would be seen in the larvae because of greater vulnerability to predators due to less developed morphological defenses. It is possible that M-cells are present in larval lumpfish and are reduced or lost during development; however, morpholog- ical examination of the reticulospinal neurons of the larval lump- fish (n = 30) with retrograde labeling has not identified Mauthner neurons or homologous cells. For studies of behavior, lumpfish (n = 12: 6.2 ± 1.0 mm, total length) were hatched from eggs collected off the coast of Glouces- ter, Massachusetts, at approximately 6 m depth. Eggs and larvae were maintained in a 10-gallon aquarium with flow-through sea- water chilled to 1 1'C. Behavioral trials were conducted within a week of hatching. A tactile stimulus — touching the head with a fine gauge wire — was used to elicit startle behavior which was filmed in a small petri dish (3.5 cm diameter). The responses were captured on high-speed video (1000 Hz) taken with an EG&G Reticon digital camera imaging through a Zeiss Stemi SR micro- scope. Three trials from each fish (36 total trials) were analyzed with Microsoft Excel 98 and Scion Image 1 .6. Parameters exam- ined were the angles of head movement during stage 1 and stage 2, the latency between stimulus and response, and the durations of stages 1 and 2. The larval lumpfish respond to the stimulus with a C-start behavior pattern (Fig. 1A). Fish turned tightly away from the stimulus direction in stage 1 [Fig. I A. left column (0-24 ms)] with an average stage 1 angle of 146r:' ± 23°C degrees. Stage 1 was consistently followed by a stage 2 tail stroke [Fig. 1A, right column (24-56 ms)] and movement away from the stimulus. The stage 2 angle, generally in the opposite direction of the stage 1 NEUROBIOLOGY 181 Stage I Angle a Stage 2 Angle " Stage 1 Duration Stage 2 Duration a. — c = c '-= — c .o c. — E c Figure 1. A. /I typical startle response oflan-al lumpfish (Cyclopterus lumpus). Stage 1. the tight "C" bend away from the stimulus, lusts 24 ms (column I) and stage 2. the first propulsive tail stroke, follows from 24 10 56 ms (column 2). Data for the angle of movement and kinematic stage durations are shown in B, with comparative data from brown trout (Salmo truttaj. chinook salmon (Oncorhynchus tshawytscha), coho salmon fOncorhynchus kiisutch) (10. minimum mines in scaling relationships), zehmftsh (Danio rerio) (91 and herring (Clupea harengusj (12). turn, was consistently smaller than that of stage 1 (stage 2 angle = 50° ± 30°). The movement angles made by larval lumpfish during the C-start are comparable to those of other species (Fig. IB; e.g. 9, 10. 12. 13). Several important fast start performance variables are the latency of response to the stimulus and the duration of the kinematic stages. The latency between stimulus and initiation of movement of an M-cell initiated startle can take less than 4 ms (9) and the duration of the response is generally less than 100 ms ( 1 ). The latency of the lumpfish, recorded for a subset of the trials (one from each of 10 individuals) was 9 ± 2.1 ms. It was considerably longer than that of the larval zebratish (3.9 ± 0.2 ms) (9). The duration of stage 1 of the larval lumpfish was 22.8 ± 5.2 ms. and the duration of stage 2 was 26.3 ± 6.8 ms. Because the duration of the fast-start stages changes with size (11) and developmental stage (10), direct comparisons among species are difficult. Still, the durations of kinematic stages 1 and 2 of the larval lumpfish are in the same range of values as other immature fishes; all under 5 cm (Fig. IB; 9, 10, 12). The total duration of the fast start (stages 1 and 2) for the larval lumpfish is shorter than the fast start duration of most larger fishes (reviewed in 1). Although the lumpfish has a longer response latency to a startle stimulus than zebrafish larvae, the C-start of the larval lump- fish — in pattern and in the duration of response — has the charac- teristics of the M-cell initiated C-start. One explanation for the similarities in the startle response among taxa is that the Mauthner cell and its homologs are present in the larval lumpfish but have not yet been identified. Another is that alternative neural circuits can generate rapid C-start behavior and that the Mauthner cell and its homologs are most critical for rapid initiation of movement. If so. such mechanisms may be taxon specific since ablating the Mauthner cell and its homologs in the larval zebrafish results in a significant decrease in performance (9). The presence of a rapid C-start type escape behavior in the lumpfish, a species that appears to generate the fast start behavior without the Mauthner cell system, provides an exciting opportunity for comparative exami- nation of an evolutionarily conserved neural and behavioral system. I thank S. Van Sant and J. Fetcho for their contributions of fish and equipment, respectively. Thanks to S. Zottoli and M. Westneat 182 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS for helpful discussion and advice on this paper and to the reviewers of this paper for their comments. This project was supported by a Grass Foundation Fellowship and a NIH NRSA fellowship (MH 11861). Literature Cited 1. Domenici, P., and R. W. Blake. 1997. ./ E.\/>. Biol. 200: 1165- 1178. 2. Weihs, D. 1973. ./. Tlicor. Biol. 106: 1X9-206. 3. Webb. P. W. 1976. J. £.»/>. Biol. 65: 157-177. 4 Hackett, J. T.. and D. S. Faber. 1983. Ncnmseience 8: 317-331 5 Eaton, R. C., R. DiDomenico, and J. Nissanov. 1991. Brain Behav. Evol. 37: 272-285. 6. Foreman, M. B., and R. C. Eaton. 1993. J. Nenrosei. 13: 4101- 4133. 7 Jayne, B. C'., and G. V. Lauder. 1993. J. G™/> Pli\si<>l. A. 173: 495-508. 8. Zottoli. S. J. 1978. ./. Comp. Neiirosci. 178: 741-758. 9. Liu, K. S., and J. R. Fetcho. 1999. Neuron 23: 325-335. 10. Hale, M. E. 1999. J. £v/>. Biol. 202: 1465-1479. 11. Webb, P. W. 1978. J. £.v/i. Biol. 74: 21 1-226. 12. Blaxter, J. H. S., and R. S. Batty, 1985. J. Mar. Biol. Assoc. U.K. 65: 737-750. Reference: Biol. Bull 199: 182-183. (October 2000) Memory Consolidation in Hermissenda crassicornis David A. Epstein, Herman T. Epstein, Frank M. Child, and Alan M. Kuzirian (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Experiments with shell-less molluscs (Aplysin and Hermissenda) have revealed a number of processes that underlie learning by these organisms and also by some vertebrates. Hennissenda, for example, shows significant Pavlovian conditioning capabilities ( 1 ). Follow-up investigations on these molluscs dealt with the sensory stimuli needed for short-term memory (STM) and long-term memory (LTM) (2). The relationship of the two memories with in vitro changes in exci- tatory post-synaptie potentials (EPSPs) have also been investigated in the neural networks of these organisms (3). Many studies of the molecular aspects of these two different memory regimes have led to quite detailed descriptions of the events (4. 5. 6). Both Aplvsiu and Hermissenda have been tested for their recall of induced behavioral modifications after one, two, or many con- ditioning events (CEs). In Aplysia, the EPSP component of learn- ing produced by 1 CE was compared to that produced by 5 CEs (3). In Hermissenda, the comparison was made between 2 CEs and 9 CEs (2). Five to ten minutes after finishing one or two condi- tioning events, both animals exhibited significant behavioral recall (i.e. STM); but there was no recall after an hour or more (i.e. no LTM). The larger numbers of CEs, however, did induce LTM in both species. Since STM and LTM are clearly responding to a different set of conditions, we focused first on what might inhibit or block STM. This problem was partially anticipated in 1900, according to McGaugh (7) who cited Muller and Pilzecker as having found that "memory of newly learned information was disrupted by the learning of other information shortly after the original learning" (8). This concept led us to test, in Hermissenda. whether STM recall (at 5 min) might be blocked simply by the input of additional information (i.e. extraneous sensory stimuli) if the latter were applied within the first 5 nun after conditioning. The initial results of the blocking experiments, which showed that the simple sensory inputs blocking STM also blocked LTM. then led to the hypothesis that temporal consolidation of LTM could be detected by measur- ing when the blocking sensory input was no longer effective. Hermissenda (Sea Life Supply, Sand City, CA) were tested with 2 and 9 paired CEs for induction of STM and LTM. Conditioning events consisted of exposing the animals to 6 s of bright, white light (CS) explicitly paired with 4 s of strong orbital agitation (US) following a 2-s onset delay with an inter-trial interval of 1 min. Recall of the behavioral modification induced by associative con- ditioning was assessed by recording the animal's change in foot length when presented with 6 s of light alone. The conditioned response (CR) was foot contraction, the unconditioned response (UR) was foot elongation (9). Two paired conditioning events initiated behavioral recall after 5 min but not after 90 min; the LTM input of 9 pairings was recalled at both 5 and 90 min (Fig. 1A). The small and non-overlapping S.E.Ms for each point indi- cate statistical significance (P = <0.01, r = 3.18). After giving the animals the paired CS and US stimuli leading to STM. we tested two simple paradigms of blocking sensory stimuli. The first was a modification of the conditioning stimuli: dim orange light and very slow orbital rotation. The second block- ing stimulus tested consisted of rotating the tray containing the animals upside down and, after 5 s, rotating il upright again (rotational block). Both experimental paradigms blocked STM and LTM (Fig. IB). To determine the temporal specificity of LTM in Hermissenda. the following experiments were done. Animals were trained with 9 CEs, and the CR was measured at the usual 90 min. However, at selected time intervals (2. 25, 50, 55, 60, 65 min) post-condition- ing, the animals were rotationally blocked. Control animals re- ceived only the 9 paired CEs. When the animals' behavior was plotted, a clear and decisive LTM consolidation interval in Her- missenda appeared; consolidation occurred between 55 and 60 min (Fig. 1C). Presentations of rotational blocking prior to 55 min totally blocked memory consolidation. However, the stimulus given after 60 had no blocking effects, and the animals demon- strated the CR. The consistency of and surprisingly little variability in the response among the majority of the animals indicated the robustness of the paradigm. When the data were analyzed with Most and F-test statistics, they were found to be highly significant, whether compared between data points or to zero (P = ( training. Memory consolidation appeared to occur between 55 and 60 min. Statistical analyses using i-tests and F-tests were highly significant (P = * cxp( - A.vJ where F(.v) is the probability of having an interval greater than .v. and A is the mean mini frequency. Log-binned frequency distributions of mini-intervals indicated (4) that in most cases in --'- 14/24; P < 0.05, log-likelihood ratio test), multiple decaying exponentials are required for optimal fit of interval distributions. This indicates a clear divergence of spontaneous exocytosis from a random Poisson process (Fig. 1, A-B). The best fit of the interval distributions indicated that the area underneath the fast or bursting component (a,.lsl) could be as high as 66% or as low as 3% (mean a,as, = 12 ± 4%; n = 14) (Fig. 1, A-B). Since miniature events arise from a large popu- lation of independent synapses, could this divergence arise simply from the temporal averaging? The answer is "No," according to the following argument. If each synapse generates spontaneous events according to a Poisson process, then the probability of finding k events in the time interval At is: P(k) = exp(-ju,- Af) • JU.J1 k\ where /u, is the mean Poisson rate at the ith synapse. With a population of N independent synapses, the occurrence of minis at the soma will also be a Poisson process with a single parameter /j. which is just the sum of the individual parameters: Therefore, based on these simple mathematical considerations, if every synapse made onto an individual neuron is releasing in a random manner, whole-cell mini interval distributions should dis- play a single exponential component. Moreover, this conclusion would also be valid in the presence of a large variability in spontaneous quantal rates at different synapses, as previously reported in the same system (5). In agreement with these expec- tations we have used a technique that permits to us record minis from individual hippocampal synapses and have found that, even at the level of a single terminal, the generation of quanta diverges from a random memory-less Poisson process (Abenavoli et al., unpub.l. A transient up-modulation of quantal discharges, such as the NEUROBIOLOGY 185 A 10 B 70 - CL> O CO 10 J 10 0 Intervals (s) Figure 1. Distribution of intervals between minis at hippocampal synapses. A) Minis were acquired in voltage clamp using the Whole-cell recording configuration. Short trains of minis could be seen consistently under these conditions. In these experiments multiple exponentials were always required for best fit of mini-interval distributions indicating a divergence from Poisson's statistics. The histogram presented was best fitted b\ the sum {solid line) of two decaying exponentials {dotted lines). B) Summon' data for the area (af) of the short-inten'al component {range af = 3-66%, mean value = 12 ± 4%; range rf = 1.56 - 48.64 ms, mean value 20.37 ± 4.07 ins). one observed in the fast component of mini interval distribu- tions, might result from some sort of transient change in pre- synaptic Ca2+ levels (see ref. 6 for review). We have therefore tested the effects of cadmium (50 juM). a broad spectrum Ca2 + channel blocker. When cadmium was applied, no effects on mini frequency and mini amplitude were detected. In 9 cells, the average mini frequency in control conditions was 2.51 ± 0.75 Hz. and it was 2.56 ± 0.74 Hz after the application of cadmium. In these experiments, when log-binned distributions of mini- intervals were constructed, if multiple decaying exponentials were required for optimal fit in control conditions, they were also required in the presence of Cd2 + (;i = 4/4; P < 0.01 ). We also examined the effects of BAPTA, a high affinity, fast- binding Ca2+ chelator (7). BAPTA was introduced in all syn- aptic terminals impinging upon a postsynaptic neuron by per- fusing those neurons with the membrane permeable analog BAPTA-AM while recording synaptic events. Long-term appli- cation of BAPTA (>20 mint (in the presence of tetrodotoxin) produced no significant effect on mini frequency ( = 2.14 ± 1.30 Hz, = 1.74 ± 1.56, mean ± sd; n = 7). Importantly, interval-distributions of minis displayed no detect- able change after the BAPTA treatment (n = 3/3: P < 0.05; = 8 ± 3%; = 8 ± 3%). Taken together, these observations rule out a role in the divergence from a random Poisson process for a brief elevation in presynaptic Ca2 + , whether from an influx through the plasma membrane or release from internal stores. Our electrophysiological recordings re- vealed that the dynamics of spontaneous quanta is more com- plex than previously thought and cannot be simply predicted by applying the Poisson theorem (1-2). This is because short epochs of multiple quanta releases were consistently present in the recordings. The genesis of this phenomenon is independent of Ca2 + elevation in the presynaptic terminals. Our relative ignorance about the molecular organization of release sites precludes any deeper understanding of this synaptic behavior. Nonetheless, we can speculate that, since minis keep occurring in the absence of any incoming electrical activity, trophic support through minis would circumvent requirements for Heb- bian mechanisms to maintain some forms of synaptic plasticity (8). In particular, the rapid discharge within a burst would certainly lead to a temporal summation in the postsynaptic spine and dramatically increase the probability of calcium influx through postsynaptic NMDA channels. The input-output prop- erties of CNS synapses are an additional consideration. The results presented might also be relevant to the hypothesis that the release of multiple vesicles is happening under some conditions and in some neuronal systems during spontaneous and evoked exocytosis (9-12). Regardless of the frequency of mul- tivesicular exocytosis, this could certainly have an impact on the synaptic input-output characteristics of hippocampal synapses. since glutamate AMPA receptors are not saturated by the content of a single vesicle (3). A. Abenavoli was a recipient of an Armenise-Harvard fellow- ship. This research was supported by a Rand award. Telethon. Human Frontier and MURST grants to A.M. 186 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Literature Cited 7. Adler. E. M., G. J. Augustine, S. N. Duffy, and M. P. Charlton. 1991. J. Neiirosci. 11: 1446-1507. 1 Fan, P., and B. Kat,. 1952. ./. Physiol (U>nd.) 117: 109-128. 8 T y and Q L Co|li rid 1993. Nature 361: 31_39. 2 Rotshenker, S.. and R. Rahamimolt. 1970. Sr/«ire 170: 648-649. 9. korn, H., C. Sur, S. C harpier, P. Legendre, and D. S. Faber. 1994. 3. Forti, L., M. Bossi, A. Bergamaschi, A. Villa, and A. Malgaroh. . „„_ ,„_ 0_0 Pp. 301— 322 in Molecular and Cellular Mechanisms of Neurotrans- 1997. Nature 388: S74-S7a. . ,, ,. .. ,r,oi nii miner Release, L. Stiarne c; «/., eds. Raven Press, New York. 4. McManus, O. B.. A. L. Blatz. and L. Magleby. 1987. Pfliiwrs Arch 410-530-5^3 l(l Au8er- c-, S. Kondo, and A. Marty. 1998. J. Neiirosci. 18: 4532- 5. Malgaroli, A., A. E. Ting. B. \\endland, A. Bergamaschi, A. Villa, 4347 R. W. Tsien, and R. H. Scheller. 1995. Science 268: ]624-lh2K. ' ' Tong- G- and C. E. Jahr. 1994. Neim. Hemolysis was judged to be divalent cation-dependent because the divalent cation chelator. ethylenediaminetetraacetic acid, reduced hemolysis (Fig. 1A). The macromolecular osmolites dextran-8 (Mr 8-12 kDa) and, to a lesser extent, dextran-4 (Mr 4-6 kDa) reduced the extent of hemolysis (Fig. IB). This suggests that hemolysis is the result of hydrophilic membrane channels established in the plasma membrane of the target red blood cell by the hemolytic protein of the cuticular secretion. Protection by the dextrans is sug- gested to result from their ability to balance the osmotic pres- sure across the permeabilized cell membrane, which will reduce the flow of water through the hemolytic pore and into the cell and will prevent swelling and lysis (8). It is difficult to envision ways for macromolecular osmolites to protect the cell if the hemolytic process featured such other possible mechanisms as phospholipase action or detergent-mediated membrane reorga- nization. It is proposed that the cuticular secretion is one agent that helps maintain the cleanliness of the cuticle of Limulus. Its 80 -, 60 g <;V AND BlnCHI MISIKY 193 a) ® M-M M-M-M6 \1-Gn-Gn-Asn Glc-Glc-Glc-M-M-f/S A M-M v M-M-M6 M-M-M3 IB M-Gn-Gn-Asn M6 'M-Gn-Gn-Asn , Gn2-M3 M M-M6 (D GnT I M v2 M-M6 M-Gn-Gn-Asn . M-Gn-Gn-Asn , Gn2-M3 M3 I GnTII Gn2-M6 'M-Gn-Gn-Asn Gn2-M3 1 A = glucosidases B = mannosidases C = mannosidase II GnT = GlcN Ac-transferase 5 10 15 nmol I mg / h Figure 1. (a) Biosynthesis of N -linked glycans. Encircled numbers 1-6 designate structural formulae for glycopeptides resulting from sequential enzyme activities (A-C) and of GnTs (I, II). Briefly, compound I is acted upon by glycosidases (A), which remove glucose (Glc) to produce compound 2. This is modified by mannosidases (B) to yield the M5 structure (3), which in turn becomes a substrate for steps 4 -5 after the additional removal ofmannose residues by another mannosidase (C). Compound 6 results from GnT II activity upon the GnT I product. Succeeding structures are enriched, one GlcNAc at a time, b\ the actions of GnT III. IV. and V. This scheme is practically universal as inferred from many species studied, including lower invertebrates (7). (Fig. la adapted from ref. 2). (b) The results of trimannosyl substrate-product assays are presented for substrate concentrations ranging from 12.5 to 50 nanomoles. A straight line relationship was observed. The inset (lower right) represents the biosynthelic activity presumed to occur when GnT I converts trimannosvloctyl substrate into GlcNAc 2-trimannosyloctyl product. lb). This relationship established GlcNAc-trimannosyl octyl as the presumed product catalyzed by sponge lysate GnT I activity. The specificity of this synthetic acceptor substrate has been confirmed using a wide range of Gn-T I compounds, including cell lysate from vertebrate and non-vertebrate sources, as well as the cloned and expressed rabbit, human, and mouse Gn-T I gene product (9). Final confirmation of the GlcNAc-trimannosyl link requires addi- tional detailed NMR and spectroscopic analysis of scaled-up pu- rified product. The finding of a GnT I activity in the sponge raises the prospect of manipulating sponge N-glycan structures by the use of specific GlcNAc enzyme inhibitors. Gray cells, regarded as the immuno- cytes of the sponge (10), bear the highly N-glycosylated CD44 surface antigen (T. Simpson and W. Kuhns, unpub. datal that may be an appropriate substrate for such chemical interventions. CD44 is likely to be involved in allograft rejection events (11), and if so, alterations in its structure by de-N-glycosylation may play a role as biological response modifiers. In summary. GnT I enzyme activity appears to function in a manner very much like its counterpart in higher species. Since the sponges are the most ancient eukaryotes with a multicellular lineage, the occurrence of cellular GnT I speaks to its importance in cell functions and to its remarkable conservation over time. Literature Cited Curr. Opin. Struct. Biol. 2: Glycoproteins and Hu- 1. Drickamer, K., and .). Carver. 1992. 653. 2. Brockhausen, I., and W. J. Kuhns. 1997. man Disease. R. G. Landes, Austin, TX. 3. Schachter, H., and I. Brockhausen. 1993. Pp. 263-332 in Glyco- conjugates, Composition, Structure and Function. H. Allen and E. Kisailus. eds. Marcel Dekker, New York. 4. Jarchow, J., J. Fritz, D. Anselmetti, A. Calabro, V. Hascall. D. Gerosa, M. Burger, and X. Fernandez-Busquets. 2000. ./ Struct. Biol. (in press). 5. Misevic, G., J. Finne, and M. Burger. 1987. J. Biol. Chem. 262: 5870-5877. 194 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS d. Kaltenbach, J., K. Hudock, M. Burger, G. Misevic, and W. J. Kuhns. 1996. Bio/. Bull. 191: 296-297. 7. Kornfeld, S., and R. Kornfeld. 1985. Anim. Rev. Biochem. 54: 631-664. 8. Kaushal, G., and A. Elbein. 1994. Methods En-ymol. 230: 316- 320. 9 Reck, F., M. Springer, H. Paulson, I. Brockhau.sen, M. Sarkar, and H. Schachter. 1994. Carhohyjr. Res. 259: 93-101 10. Humphreys, T., and E. Reinherz. 1994. Imiminol. Today 15: 316- 320. 11 Fernandez-Busquets, X., and M. Burger. 1999. Microsc. Res. Tech. 44: 204-218. Reference: Bio/. Bull. 199: 194-195. (October 2000) A Ca+2-Independent Cytolytic System from the Blood of the Marine Snail, Busycon caiialiculum Peter B. Armstrong* and Rengasamy Asokan (Molecular and Cellular Biology, University of California, Davis. California 95616) The immune system is the ensemble of cell-based and humoral agents that protect the organism against parasites that have gained access to the internal milieu. One of the important immune activ- ities for a variety of animals is the outright lysis of foreign cells that contact the blood ( 1 ). Cytolytic systems are frequently ex- plored as if they were hemolytic systems, where the foreign cell that is destroyed is the mammalian erythrocyte. The red cell is convenient for the assay of cytolysis because its hemolysis is easily quantified by the release of hemoglobin into the bathing medium. Here we report the presence of a hemolytic system in the plasma of the marine snail. Busycon. Blood was obtained as follows. The foot of the adult whelk was wounded, and the blood flowing from the wound was collected and then immediately centrifuged to remove the cells. Hemocyanin was removed from the plasma by centrifugation (40.000 RPM. 4 h). The hemocyanin-free plasma was dialyzed into Tris-buffered saline (0.15 M NaCl, 50 m/WTris, pH 7.3). Hemolysis was assayed with rabbit red cells as described previously (2). The hemolytic activity of hemocyanin-depleted Busycon plasma is shown in Figure 1 . Hemolysis is progressive, requiring 4 h for completion. The hemolytic activity is unaffected by the inclusion of EDTA in the hemolysis assay, and thus is independent of divalent cations. The lack of a Ca + 2 dependence distinguishes this hemolytic system from one reported previously in Busycon (3). Hemolysis is reduced at low ionic strength, showing a broad activity maximum in buffers containing in excess of 0.2 M NaCl. The hemolytic activity is inactivated by trypsin treatment of the plasma, indicating a proteinaceous character to the hemolytic system. The hemolytic activity of plasma is thermolabile, showing complete inactivation by treatment of plasma at 40°C for 0.5 h. The hemolytic protein(s) are retained by a PM30 Amicon filter and pass through a YM100 filter, indicating a molecular mass between 30 and 100 kDa. The hemocyanin-free plasma shows 13 distinct protein bands in this interval by SDS-PAGE. with prominent bands at 78. 55, 43. 35. and 34 kDa. At least three mechanisms can be envisioned for hemolysis: the insertion of the hemolytic protein into the lipid phase of the plasma membrane to create a hydrophilic trans-membrane pore, the enzy- matic modification of the lipid head groups by a phospholipase, and the initiation of membrane-lipid phase-transitions by a deter- 1 Molecular and Cellular Biology. University of California, One Shields Avenue. Davis, CA 95616. (pbarnistrongfe'ucdavis.edu). gent-like, or a surface-active protein (4). To test the first possibil- ity, we determined the effects of macromolecular osmolites. The macromolecular osmolite dextran-4 (Mr 4-6 kDa) reduced hemo- lysis significantly (from 57.0 ± 2.0% to 8.0 ± 2.3% in one trial). This suggests that hemolysis in the present system is produced by insertion of the hemolytic protein into the plasma membrane, generating hydrophilic channels that allow water to flow into the cell in response to the high internal concentration of macromolec- ular osmolites, principally the protein hemoglobin. The presence of osmolites in the external milieu larger than the channel pore size (e.g., dextran-4), at concentrations sufficient to balance the os- motic pressure of hemoglobin in the cell, would protect the cell from osmotic rupture (5). The molecular size of dextran-4 is approximately 1.7 nm (6). indicating an effective pore size for the membrane-associated hemolytic protein as no larger than this value. The hemolytic system of Busycon is sensitive to the presence of lipopolysaccharide (LPS) from the cell wall of gram-negative bacteria (Fig. IB). The reduction in the hemolytic action at higher concentrations of LPS may derive from the binding of the hemolytic agent to this important signature molecule of the gram-negative bacterium, reflective of an anti-bacterial action 80 n _ 60 - _ o a) X B 40 - 20 - iO 1 5 dilution of Busycon plasma in all samples 03 040 Busycon plasma (fraction [Lipopolysaccharide] (mg/ml) of volume of assay) Figure 1. Hemolytic activity from the plasma of Busycon canalicula- tum. Hemocyanin was removed h\ ultracentrifugation, and the supernatant wax dial\:ed into Tris-hiiffered saline l<>. 15 M NaCl, 51) inM Trix, pH 7.31 Figure I A, dependence of hemolysis on the presence of hemocyanin-free Busycon plasma. Figure IB, sensitivity of hemolysis to lipopolysaccharide. PHYSIOLOGY AND BIOCHEMISTRY 195 of the hemolytic agent. Although the concentrations of LPS used for Figure IB are high, the actual concentration of LPS at the surface of the bacterium is far higher than the solution concentrations used in this trial. The possibility that the hemo- lytic agent does bind to gram-negative bacteria deserves further investigation. The ability to destroy foreign cells that come in contact with the blood is an important defense strategy for a variety of animals. In mammals, the cytolysis of foreign cells is conducted by the com- plement system, a multi-component ensemble of plasma proteins whose membrane attack elements are activated by a proteolytic cascade that, itself, is initiated by a variety of stimuli indicative of parasitic invasion (7). The complement system is found only in the deurostomate animals (i.e., the echinoderms and the chordates) and is absent from protostomate animals (8, 9). In the latter, the relatively few cytolytic systems that have been characterized are less complex than the vertebrate complement system, with some the province of a single protein that both recognizes and binds to the foreign cell and mediates its cytolytic destruction (5, 10). Only a few cytolytic systems have been reported in the plasma of molluscs (11, 12), and we have not found any reports for gastro- pods. The systems reported from bivalves (11, 12) are Ca+2 dependent, suggesting that they are based on different effector molecules than the system described here from Bnsycon. Supported by Grant No. MCB-97-26771 from the National Science Foundation. Literature Cited 1. Canicatti, C. 199(1. Experientia (Basel} 46: 239-244. 2. Swarnakar. S., R. Asokan, J. P. Quigley, and P. B. Armstrong. 200(1. Biticlwm. J. 347: 674-685. 3 Biswas, C.. and P. B. Armstrong. 1999. Biol. Bull. 197: 276-277. 4. Oliver, A. E., F. Tahlin, N. J. Walker, and J. H. Crowe. 1999. Biochim. fi/Vy./n v Actu 1416: 349-360. 5. Hatakeyama, T., H. Nagatomo, and N. Yamasaki. 1995. ./ Biol. Client. 270: 3560-3564. 6. Scherrer, R.. and P. (ii-rhardt. 1971. ./. Bucteriol. 107: 718-735. 7. Law, S. K., and K. B. M. Reid. 1988. Complement. IRL Press, Oxford. 8. Smith, L. C., L. Chang, R. .). Britten, and E. H. Davidson. 1996. J. IniiiiitiHil. 156: 593-602. 9 Al-Sharif, W. Z., J. O. Sunyer, J. D. Lambris, and L. C. Smith. 1998. J. Immimol. 160: 2983-2997. 10. Armstrong, P. B., S. Swarnakar, S. Srimal, S. Misquith, E. A. Hahn, R. T. Aimes, and J. P. Quigley. 1996. J. Bioi Chem. 271: 14,717-14.721. 11. Anderson, R. S. 1981. /Vr. Comp. Imiiiunot. 5: 575-585. 12. Yoshino, T., and T.-L. Tuan. 1985. Dev. Camp, liimnniol. 9: 515- 522. Reference: Biol. Bull. 199: 195-197. (October 2000) Modulation of the Development of Plutei by Nitric Oxide in the Sea Urchin Arbacia Punctiilata Diane E. Heck, Lydia Louis. Michael A. Gallo and Jeffrey D. Luskin {Departments of Pharmacology and Toxicology, Rutgers University, and Environmental and Community Medicine, UMDNJ Robert Wood Johnson Medical School, Piscatawa\, New Jersev 08854) Nitric oxide, a reactive free radical, has recently been identified as a key mediator of intercellular signaling in numerous species ( 1 ). It is produced enzymatically from 1-arginine by the nitric oxide synthase family of oxidoreductases (2). Nitric oxide regulates a variety of physiological functions including relaxation of vascular smooth muscle, long-term potentiation, tumor cell apoptosis, and cytostasis (3). In addition, inappropriate or excessive production of nitric oxide has been implicated in tissue injury (4). Nitric oxide is known to initiate biochemical effects through binding to iron and iron-sulfur-containing proteins and modulating their activity (5). Nitric oxide can also modify DNA and is known to alter growth factor-mediated transcription processes (5). At the present time, no clear role for this free radical species in regulating development has been defined. We have previously reported that nitric oxide synthase inhibi- tors alter fertilization and differentiation of sea urchin eggs (6). In the present studies, we examined the direct effects of nitric oxide on the development of the sea urchin Arbacia punctulata. For these studies, fertilized eggs were prepared from sea urchins, as de- scribed by Hinegardner (7), and maintained at 24°C. Embryos were exposed to S-nitroso-N-acetylpenicillamine (SNAP. Molec- ular Probes. Eugene, OR I, an agent that spontaneously releases nitric oxide, for 1 h at various developmental stages. These stages included: immediately after fertilization, following the first and fifth divisions, the morula and prism stages, and at two points in pluteal development, i.e., 24 and 48 h after fertilization. Treated embryos and untreated controls were further evaluated at regular intervals for 72 h. Both morphological abnormalities and transient delays in development were observed, but particular effects were dependent on the developmental stage at the time of treatment. Morphological abnormalities were quantified at the morula. prism, and pluteus stages. In initial experiments, we determined the effects of a range of concentrations of SNAP (2 nM-2 /J.M) applied to sea urchin eggs immediately after fertilization. Embryo mortality with little cell division was observed when embryos were treated with concen- trations of SNAP in excess of 200 nM. However, both transient and permanent developmental changes were found after treatment with lower concentrations of the nitric oxide releasing agent (see further below). For our studies, we used 20 nM SNAP; at this 196 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS concentration, prominent morphological alterations occurred with- out significant toxicity. Developmental lesions are often induced by temporally re- stricted biochemical changes that eliminate critical downstream events or cause them to occur inappropriately. The resulting ab- errant development reflects a stage-specific sensitivity to a phar- macological agent or event. To determine the developmental stages at which sea urchin embryos are sensitive to nitric oxide, we next exposed fertilized eggs to SNAP during various stages of development. Following fertilization, sea urchin eggs divide syn- chronously until the fifth division. At this stage, the embryo is designated a morula, and the uniformly distributed pigment gran- ules become unevenly distributed to specific cells, with most going to the 8 macromere cells. When SNAP was applied after fertili- zation or first division, we observed no morphological alterations in the morula. Both drug-treated embryos and controls contained 2-4 darkly pigmented cells. Sea urchin embryos hatch and become free-swimming shortly after the initiation of motility. Later, about 18 h after fertilization, the free-swimming sea urchin embryos become pyramidal, and enter the prism stage. When such embryos were exposed to SNAP, no anatomical aberrations were observed. However, embryos treated with SNAP at any stage prior to hatching exhibited delays in development (Table 1 ). Thus. 18 h after fertilization, embryos treated with SNAP prior to the morula stage had not progressed beyond the blastula stage. In addition, embryos exposed to SNAP before hatching contained fewer than the 6-8 darkly pigmented cells observed in controls. Developing sea urchins generally progress to the pluteus by about 24 h after fertilization. At this stage, plutei that had been treated with SNAP immediately after fertilization, or the first division, exhibited gross morphological alterations. Additionally, the pigment cells aggregated and were fewer in number (Table 1; Fig. 1. compare panels A and B; other data not shown). This effect was associated with fewer animals progressing through develop- Tahlo 1 Changes in \CLI urchin embryo development in response to treatment with SNAP Developmental stage 24 hours after fertilization (embryos/50 fj.1) Developmental stage at treatment Morula Prism Pluteus Untreated 1 ± 2.3 0 ± 0.6 1 3 ± 3.9 Following fertilization1 24 ± o.l 7 ± 3.2 0 ± 0.3 First Division 27 ± 0.6 14 ± 2.3 1 ± 3.3 Division 5 19 ± 4.0 10 ± 1.4 0 ± 3.4 Morula 1 ± 4.3 4 ± OS 9 ± 2.3 Prism 0 ± 3.4 0 ± 0.9 2 1 ± 4.5 Pluteus 2 ± 3.3 1 ± 0.3 13 ± 2.6 ' Approximately I05 embryos were suspended in 10 ml of sea water supplemented with 20 nA/ SNAP at the indicated developmental stages. Alter 1 h. embryos were transferred into 100 ml of SNAP-free sea water. Embryos were then evaluated for progress through development at 24 h after fertilization. Each point represents the average number of embryos in cadi stage ot development tor 5 experiments ± SEM. Figure 1. Effect of nitric o\idc mi the development of sen urchin eggs. Immediately following fertilization, sea urchin eggx were treated with 20 nM SNAP. After 1 h llie embryos were washed and a/lowed to develop. Punch A and B are sea urchin embryos 24 h after fertilisation; panels C and D show embryos 48 h after fertilization. Panels A and C are control 1'inhrvos: panels B and D are embryos that have been treated with SNAP. merit. Those that did develop further exhibited diminished arm extension when evaluated 48 h after fertilization (Fig. 1. compare panels C and D and not shown). As these plutei continued to develop, the poorly extended arms were often oriented 90° to the longitudinal axis rather than parallel to it. Aberrant morphology was less pronounced in embryos exposed to nitric oxide after division 5. In addition, when evaluated 24 h after fertilization, the number of pigment cells remained unchanged in embryos treated with SNAP before division 5, while about 4 times as many darkly pigmented cells were identified in untreated embryos. When observed 24 h after fertilization, delayed development was again evident in embryos treated with SNAP at all stages prior to hatching. However, following exposure to SNAP at the morula stage, embryos appeared normal in development by 48-72 h after fertilization. No effects on morphology or development were ob- served in embryos exposed to nitric oxide at the prism stage or as plutei either 24 or 48 h after fertilization (not shown). Anatomical changes resulting from exposure to nitric oxide were preceded by an apparent inhibition of pigment cell division. These results indicate that sea urchin embryos exposed to nitric oxide before their development into morula show permanent morphological changes, whereas exposure at later stages has no apparent perma- nent effects. Taken together, our results demonstrate that brief exposure of sea urchin embryos to nitric oxide during early development causes irreversible abnormalities in plutei. including skeletal PHYSIOLOGY AND BIOCHEMISTRY 197 aberrations and changes in the proliferation and migration of pigment cells. The effects of nitric oxide applied at later stages are reversible. We speculate that nitric oxide may act as a negative regulator of pigment cell division and skeletal extension in vivo. Literature Cited I Hohbs, A. J., A. Higgs, and S. Moncada. 1999. Anini. Rev. Phur- imicol. To.vicol. 39: 191-220. 2. Hemmens. B., and B. Mayer. 1998. Methods Mol. Bint. 100: 1-32. 3. Moncada, S. 1999. ./. R. Soc. Meil. 92: 164-169. 4. Laskin, J. D., D. E. Heck, and D. L. Laskin. 1994. Tn-mh Emln- crinol. Melah. 5: 377-3S2. 5. Lane, P., and S. Gross. 1999. Semni. Nephrol. 19: 215-229. 6. Heck, D. E., J. I). Laskin, S. Zigman, and W. Troll. 1994. Biol. Bull. 187: 24S-249. 7. Hindgardner. R. 1975. Pp. 10-25 in The Sea Urchin Emhr\<>. Biochenn\ir\ unJ .\l is a western hlot probed with ap-205 showing « single hand at 21)5 kD. Panel B: Lane I is cooinassie blue stained SDS-PAGE gel showing a myosin H-enriched fraction prepared from clam oocytes. Lane 2 is the corresponding western hint of the fraction probed with ap-205 and showing a single hand at 205 kD. Panel C: Lane I is a gel of protein-A purified ap-205 showing til least four hands: the antibody at 55 kl~> and ihiee sennn proteins that co-pnrif\ with the antibods. Lane 2 shows an immunoprecipitation with ap-205. Six proteins are seen: the /mo- serum proteins, msosin II at 205 kD. and actin at 45 kD. Lane 3 is the corresponding western hlot probed with ap-205: the antibod\ ret t>gni~cs the four serum proteins unil ms'osiu 11 CELL BIOLOGY 203 myosin II (gift of Donald Kaiser). Therefore, the ap-205 antibody exhibited high affinity and specificity for clam myosin II. In immuno-precipitation (IP) experiments with ap-205, proteins of 205 and 45 kD. the respective molecular weights of myosin II and actin, were present in the precipitate (Fig. 1C, lane 2). A blot of the ap-205 immune-precipitate probed with ap-205 (Fig. 1C. lane 3) identified the 205 kD band as myosin II, and a blot with an antibody to actin identified the 45 kD band as actin (data not shown). The 4 additional bands seen on both the gel and blot were present in the protein A-purified antibody (Fig. 1C. lane 1 ); there- fore, they represent serum proteins rather than proteins in the oocyte extracts. The IP data provided evidence that the antibody binds to native myosin II and may serve as a function-blocking antibody. We therefore examined the effects of the ap-205 anti- body on actin-dependent vesicle transport. The protein-A purified ap-205 antibody was concentrated to 3 mg/ml and buffer-exchanged into vesicle motility buffer (T buffer. pH 7.2) for antibody-inhibition experiments. An antibody (aQLLQ) made to squid myosin V (5) that does not detect oocyte proteins on western blots was used as the control. Extracts were prepared for motility assays with either 0.38 or 1.0 mg/ml of ap-205, and a control sample was prepared at the same time with aQLLQ. The motile activity was determined in the control and the treated samples at regular intervals for a period of 1.5 hours. Motile activity in the control remained at 412 ± 96 vesicles/ minute/field (v/m/f) for the observation period, while the extracts treated with 0.38 mg/ml ap-205 decreased to 232 ± 5 1 v/m/f (43% inhibition). At 1.0 mg/ml ap-205, motile activity decreased to 199 ± 58 v/m/f. while the control remained at 363 ± 71 v/m/f (45% inhibition). These data showed that ap-205 inhibited 40% to 45% of the motile activity in clam oocyte extracts. We plan to use affinity-purified ap-205 to determine whether motile activity is inhibited completely. In summary, the inhibition of vesicle transport by a myosin II-specific antibody provides evidence in support of the conclusion that myosin II in clam oocytes functions as a vesicle motor. The lack of 100% inhibition by ap-205 may suggest the involvement of other myosin motors in actin-based vesicle transport in oocytes. The vesicles in these extracts are probably ER-derived. and myosin II may therefore be involved in the transport of ER vesicles during the early events of fertilization and embryonic development. Supported by NSF grants DCB 9974709 and DBI-9977613 and an MBL fellowship sponsored by the Josiah Macy. Jr. Foundation. Literature Cited 1 . Kuznetsov, S. A., G. M. Langford, and D. G. Weiss. 1992. MI/HIV 356: 725-727. 2. Langford, G. M. 1995. CHIT. 0/im. Cell Biol. 7: 82-88. 3 Langford, G. M., and B. J. Molyneaux. 1998. Brain Res. Rev. 28: 1-8. 4 DePina, A. S.. and G. M. Langford. 1999. Microsc. Res. Tech. 47: 93-106. 5. Tabb, J. S., B. J. Molyneaux, D. L. Cohen, S. A. Kuznetsov, and G. M. Langford. 1998. ./. Cell Sei. Ill: 3221-3234. 6. Dekker-Ohno, K.. S. Hayasaka, V. Takagishi, S. Oda, and N. Wakasugi. 19%. Bruin Re\. 714: 226-230. 7. Takagishi, V.. S. Oda. S. Hayasaka, K. Dekker-Ohno, T. Shikata, M. Inouye, and H. Yamamura. 1996. Nenruxci. Lett. 215: 169- 172. 8. Prekeris, R.. and 1). M. Terrian. 1997. ./. Cell Biol. 137: 1589- 1601. 9 Wu, X., B. Bowers, Q. Wei, B. Kocher, and J. A. Hammer 3rd. 1997. ./. Cell Sci. 110: 847-859. 10 Hill, K. L., N. L. Catlett. and L. S. Weisman. 1996. J. Cell Biol. 135: 1535-1549. 1 1. Govindan, B., R. Bowser, and P. Novick. 1995. J. Cell Biol. 128: 1055-1068. 12. Rogers, S. L., and V. I. Gelfand. 1998. CHIT. Biol. 8: 161-164 13. Woellert, T., A. S. DePina. and G. M. Langford. 1999. Biol. Bull. 197: 41-42. 14. Stow, J. L., K. R. Fath, and D. R. Burgess. 1998. Trends Cell Biol. 8: 138-141. 15. Ikonen, E., J. B. de Almeid, K. F. Falh, I). R. Burgess, K. Ashman. K. Simons, and J. L. Stow. 1997. J. Cell Sci. 110: 2155 2164 16. Musch, A., D. Cohen, and E. Rodriguez-Boulan. 1997. J. Cell Biol. 138: 29l-3()h, 17. Buss, F., J. Kendrick-Jones, C. Lionne, A. E. Knight, G. P. Cote, and J. P. Luzio. 1998. J. Cell Biol. 143: 1535-1545. 18. Ruderman, J. V., V. Sudakin, and A. Hershko. 1997. Methods En-\mol. 283: 614-622. Reference: Biol. Bull. 199: 203-205. (October 2000) Interaction of Actin- and Microtubule-Based Motors in Squid Axoplasm Probed with Antibodies to Myosin V and Kinesin Phillip Stafford, Jeremiah Brown and George M. Langford {Dartmouth College, Hanover, New Hampshire 03755) Myosin V, an actin-dependent molecular motor highly ex- pressed in neurons, transports ER vesicles on actin filaments in the giant axon of the longfin squid. Loligo pealei ( I ). The amino acid sequence of squid brain myosin V is similar to those of mouse and human myosin Va (2). In a recent study. Huang et al. (3) showed that the rod-tail domain of ubiquitous kinesin (aa680 to aallOO) and the AF6/cno globular tail domain of MyoV (aa!643 to aa 1800) bind to each other. This led to the hypothesis that the two motors form a complex on vesicles through tail-tail interaction. The direct interaction of these motors could provide a mechanism by which vesicles move efficiently from microtubules to actin filaments, as postulated in the dual filament model of vesicle transport (4-7). In this study, we used antibodies raised to squid brain myosin V (aQLLQ) and squid brain kinesin (H2 antibody provided by 204 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Dr. S. Brady) to determine whether kinesin binds to myosin V in squid neurons. In addition, we used recombinanl tail frag- ments of mouse MyoV and mouse kinesin (constructs provided by Dr. Huang) to test whether these fragments form heterolo- gous complexes with squid brain kinesin and myosin V, respec- tively. The plasmids containing the glutathione S-transferase (GST)-labeled mouse AF6/cno tail-globular-domain (GST- MyoV-tail aa!569 to aa!768 without the coiled medial tail domain) and the histidine (His)-labeled, kinesin-tail fragment of mouse ubiquitous kinesin (aa763-856) were expressed in E. cnli. The bacterially expressed mouse GST- or His-tagged frag- ments were purified on affinity columns and used in binding assays of squid brain extracts. The specificity of aQLLQ and H2 was determined by probing blots of squid brain extracts with the two antibodies. aQLLQ recognized a single protein of 196 kD in blots of squid brain extracts (Fig. 1A, lane 2), and H2 recognized a single protein of 120 kD (Fig. 1A. lane 3). These antibodies were then used in immunoprecipitation experiments to determine whether myosin V and kinesin interact directly. Squid brain extracts were incubated with the aQLLQ and then with protein-A beads. The precipitate was collected by centrifugation and run on a SDS-PAGE gel (Fig. IB, lane 1). Proteins in the 50-55 kD range representing the antibody were the primary components in the precipitate. A blot of the immuno-precipitate probed with aQLLQ showed a myosin-V band at 196 kD, protein bands at 50-55 kD representing the antibody, and one unknown protein at about 100 kD. The unknown protein may represent a serum protein that binds to the antibody Antibodies 2 3 IP: QLLQ Ab: QLLQ H2 H2 H2 rf* B C Figure 1. Protein si:e murker is listed on the left, dashes indicate the corresponding vi;c\ on the gels In the right. Pane/ A: Lane I is a coomassie-stained SDS-PAGE gel showing total st/nid optic lobe homog- eiuite. lune 2 i\ an immunoblot using an antibod\ to the M\oV tail, QLLQ, lane 3 is the -.ante him probed with H2, an antibody against the kinesin heavy chain (provided b\ Dr. Scott Brady). Panel B: Lane I shows a gel />/ an immunoprecipitation using QLLQ to /mil down MyoV from si/iiid opth lobes. Lane 2 is a western probed with QLLQ showing MvoV at 196 kit anil the QLLQ antibody nt —511 kl) and a possible M\o\'-tail break- down product at — 100 kD. Lane 3 is a western probed with H2. Panel C: Lane I shows a gel from an immunoprecipitation using HI to pull down ki/u'\in. Lane 2 is a wc\lcrn prohcd with H2. during protein-A purification or a breakdown product of myosin V. A blot of the immunoprecipitate was probed with H2, and a protein of 120 kD representing kinesin was revealed (Fig. IB. lane 3). Therefore, these data support the hypothesis that kinesin is a binding partner of myosin V and the interaction may function to regulate the motor activity of myosin V. In corollary experiments, squid brain extracts were incubated with the kinesin antibody H2 followed by incubation with pro- tein-A beads. The precipitate was collected by centrifugation and analyzed by SDS-PAGE. Multiple bands were observed, including a protein of 120 kD and one of 200 kD (Fig. 1C. lane 1). The antibody (50 kD) was the other major band on the gel. The 120 kD protein was shown to be kinesin by probing a blot with H2 (Fig. 1C, lane 2). The H2 antibody recognized several other unknown proteins. These results complement those obtained with aQLLQ and support the conclusion that kinesin and myosin V bind to each other in neurons. The purified mouse GST-MyoV-tail fragment and the His- labeled kinesin-tail fragment were analyzed by SDS-PAGE, trans- ferred to nitrocellulose, and probed with aQLLQ and H2. aQLLQ and H2, respectively (data not shown), did not detect the GST- MyoV-tail fragment and the His-kinesin-tail fragment. The aQLLQ antibody was made to a 14-amino acid synthetic peptide in the AF6/cno domain of squid myosin V (8). so its failure to recognize the mouse protein fragment was not surprising. Affinity isolation experiments were performed to determine whether the GST-MyoV-tail fragment binds to squid brain kinesin. Squid brain extracts were incubated with the GST-MyoV-tail fragment and then with glutathione beads. The beads were washed 5x with PBS and eluted by the addition of 3 bead-volumes of 1 X glutathione. then analyzed by SDS-PAGE. A blot of the precipitate probed with an antibody to squid brain kinesin (H2) did not show kinesin as one of the proteins in the complex (data not shown). In a similar experiment, purified mouse His-kinesin-tail fragment was ana- lyzed for its ability to interact with squid brain myosin V. Squid brain extracts were incubated with the His-labeled kinesin frag- ment followed by incubation with the His-antibody. The antibody kinesin-fragment was precipitated with protein A-Sepharose beads and analyzed on SDS-gels. Blots of the immuno-precipitate probed with an antibody to squid brain myosin V (aQLLQ) did not show myosin V as one of the proteins in the complex. Therefore, these data suggested that heterologous complexes do not form between the mouse MyoV-tail and native squid kinesin and between mouse kinesin-tail and native squid myosin V. In summary, these experiments demonstrate that squid brain my- osin V and squid brain kinesin are binding partners in neurons. The mouse recombinant fragments and squid native proteins did not form complexes that could be detected on blots. Therefore we plan to produce squid recombinant proteins to determine whether vesicle transport is inhibited in axoplasm. The inhibition of vesicle transport by tail fragments of myosin V and kinesin has been shown in cells grown in culture (9. 10) but has not been demonstrated in vitro. Supported by NSF grunts DCB 9974709 and DBI-9977613 and an MBL fellowship sponsored by the Josiah Macy. Jr. Foundation. Literature Cited 1 Tabb, J. S., B. J. Molyneaux, D. L. Cohen, S. A. Kuznetsov, and G. M. Langford. 1998. J. Cell Sci. Ill: 3221-3234. CELL BIOLOGY 20? 2. Mnlyneaux, B. .1., M. K. Mulcahey, P. Stafford, and G. M. Lang- ford. 20(10. Cell Motil, Cytoskeleton 46: 108-1 IS. 3. Huang, J. D., S. T. Brady, B. VV. Richards, D. Stenoien, J. H. Rcsau, N. G. Copeland, and N. A. Jenkins. 1999. Nunirc 397: 267-270. 4. Langford, G. M. 1995. Curr. Opm. Cell Biol. 7: S2-88. 5. Langford, G. M., and B. J. Molyneaux. 1998. Bruin Rex. Rev. 28: 1-8. 6. Langford, G. M. 1999. FASEB J. 13 Suppl 2:5248-250. 7. DePina, A. S., and G. M. Landlord. 1999. Mieroxc. Rex. Tech. 47: 93-106. 8. Molyneaux, B. J., and G. M. Langford. 1997. Biol. Hull 193: 222-223. 9 Wu, X., B. Bowers, K. Rao, Q. Wei, and J. A. Hammer, 3rd. 1998. J. Cell Biol. 143: 849-418. 10. Wubbolts, K.. M. Fernande/.-Borja, I. Jordens, E. Reits. S. Dus- seljee, C. Eche\erri, R. B. Vallee, and J. Neefjes. 1999. J. Cell Sci. 112: 785-795. Reference: Biol. Bull. 199: 205-206. (October 2000) Microtubule-Dependent Nuclear Positioning and Nuclear-Dependent Septum Positioning in the Fission Yeast Saccharomyces pombe P. T. Tran[ , V. Doye , F. Chang , ami S. Inoue (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) The correct placement of the cell division plane is important for cell shape, size, and orientation, and for the proper partition of cellular determinants for development. The position of the division plane has been shown to be dependent on mierombules in many organisms ( 1 ). What molecular mechanism of the fission yeast ensures that the division plane and septum will be properly posi- tioned? Fission yeast is a rod-shaped cell that divides by medial cleav- age. The nucleus is located at the geometric center of the cell, attached to multiple bundles of dynamic, anti-parallel microtubules that push on the nucleus (2-4; our unpublished results). The position of the division plane and septum coincides with the position of the interphase nucleus (5). By examining the effect of depolymerizing microtubules within the cell, we tested the hypoth- esis that microtubules dictate the central position of the nucleus; and the position of the nucleus, in turn, dictates the future position of the septum. A wild-type fission yeast strain (h~ leul-32 nup!07-GFP nmt- GFP-atb2), expressing ( 1 ) fusion of a nuclear pore protein to the green fluorescent protein and (2) fusion of the green fluorescent protein to tubulin, was used to visualize both the nucleus and the microtubules. For imaging, cells grown to mid-log phase in liquid yeast media (EMM + 5 /ng/ml thiamine) at room temperature (21°-23°C) were mounted between a coverslip- and slide-sealed chamber containing a thin pad of 2% agarose and yeast media (YE5S). Methyl-2-benzimidazole-carbamate (MBC), a potent in- hibitor of microtubule polymerization, was used from fresh stock (100X in DMSO) at the final concentration of 25 /xg/ml to depo- lymerize the microtubules. Time-lapsed images (1-h interval. 1-s exposure time) were digitally acquired at room temperature (21°- 23°C) with Metamorph Software (Universal Imaging Corp.) con- trolling a CCD digital camera (Orca-1. Hamamatsu Corp.). at- tached to a Leica DMRX microscope stand equipped with DIC optics, as well as with a PL Fluotar 100X/1.3NA oil-immersion objective (Leica Corp.) and a mercury arc lamp for wide-field epi-fluorescent microscopy. 1 Columbia University. New York. NY 10032. 2 Institute Curie, Pdris, France. To determine whether microtubules play a role in the placement of the nucleus at the cell center, we used time-lapse microscopy to examine the position of the nucleus in intetphase cells treated with the microtubule-depolymerizing drug MBC. Nuclear position in a cell can be expressed as the ratio of two lengths: the length from the center of the nucleus to the shorter cell tip (Lshorl), and the length from the center of the nucleus to the opposite longer cell tip (Llong). The ratio Lsh,,rl/Llc,nt, = 1 when the nucleus is exactly at the cell geometric center, and Lvh,,rl/L1(>nt, < 1 when the nucleus is off-center. MBC-treated cells, which have no microtubules and ultimately die, continued to lengthen for several hours at a rate similar to that of control cells. ~ 1 .5 /j,m/h. During a 2-h period, the control cells grew from an average length of 9.16 ± 1.04 /urn to 12.16 ± 1.47 jixm (N = 23 cells). Almost all nuclei were posi- tioned in the middle of the cell, with an average Lshort/Llong ratio of 0.96 ± 0.03; and -967r of cells had better than 0.90 ratio. In contrast, MBC-treated cells grew from an average length of 8.73 ± 1.55 fxm to 11.45 ± 2.01 /xm (N = 40 cells), with many offset nuclei, an average Lshl,n/Llonp ratio of 0.80 ± 0.15: and only -28% of cells had better than 0.90 ratio (Fig. 1 A). Clearly, while MBC did not affect the cell growth rate, the proper central posi- tioning of the nucleus was dependent on microtubules. To test whether the position of the nucleus dictates the position of the division plane and septum, we examined the position of the septum in MBC-treated cells, which have off-center nuclei. Lack- ing microtubules. MBC-treated cells showed a delay in the cell cycle, and a curvilinear or "bent" growth pattern (6. 7). Whereas control cells exhibited a cell cycle time of ~4 h, the cell cycle in MBC-treated cells was significantly delayed to —6-8 h. However. MBC-treated cells attempted to divide at the end of the cell cycle delay, and eventually each cell formed a septum at the site of the offset nucleus that "cut" the nucleus. The new daughter cells subsequently died. Figure KB, C) illustrates the formation and position of the septum in control and MBC-treated cells, respec- tively. Thus, the septum formed at the position of the nucleus, even when the nucleus was not at the center of the cell. Our preliminary results are consistent with our hypothesis that the central position of the nucleus is dependent on microtubules. and that the position of the nucleus, in turn, may dictate the 206 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS A 100 — 80 § 40 O" 0) U_ 20 • C ontrol cells D ME.'! -treated': -His n. n. 0 4 U 5 0 6 07 08 09 Ratio L short Figure 1. Cells treated with MBC exhihil a misplaced nucleus and septum. Fission yeast cells expressing nucleoporin-GFP ami GFP-lnhidin were imaged at room temperature (21"-23"C) with time-lapse fluorescence microscopy. lAI Pint ot distribution f'ret/iieney of nuclear positions ex- pressed as the ratio £.1/l,,r(/L;,,,li;. where /,,,„„, is the length Irom the center of the nucleus to the short cell tip and /.,„„,. is tin- length from the center of the nucleus to t/ie opposite long cell lip. (B) Wild-type cells through one cell cvclc- Lett panels arc I>IC images s/un\ ing septum formation at the middle of the icll I at 0 h and later ul 4 hi. Right panels arc fluorescent images of nuclear membrane and microtubules. Microtubules span the length of the cell during interphase. As the cell grew, nuclei were posi- tioned h\ Jsnnnn, mi, roinhnles at the center of the cells where siihsci/ucnt cell division and scptanon o, i iirred. creating two daughter cells of ap- pro\inui!cl\' cifiuil length. \i>t< ihe disappearance <>/ interphase nncrotu- hulcs in the cell cytoplasm iind the appearance «/ the nntotic spindle inside the cell nucleus during nnlosis i at 4 hi. tCl MBC-treated cells through one cell c\cle. No microtubules weic present in MBC-treated cells. \Vithoui position of the plane of cell division and the septum. Cells which have lost their microtubule cytoskeleton can continue to grow, and can undergo septation and cytokinesis after a cell cycle delay. Our results are consistent with phenotypes seen in studies of tuhulin mutants (7). However, our experimental conditions ensure an almost complete loss of the microtubule cytoskeleton; and our time-lapse microscopy allows long-term viewing of the develop- ment of phenotypes. It has been proposed that cytokinesis factors may be localized to the nuclear region by association or movements on microtubules (8). However, our studies suggest that microtubules may not be strictly required for the assembly or localization of the ring at the nucleus. P.T.T. thanks Dr. Rudolf Oldenbourg for generously providing lab space and equipment during this study. This work was funded in part by NIH and March of Dimes grants to P.T.T. and F.C. Literature Cited 1. Field, C., R. Li, and K. Oegema. 1999. Curr. Opin. Cell Biol. 11: 68-80. 2. Drummond, D. R., and R. A. Cross. 2000. Curr. Biol. 10: 766-775 3. Hagan. I. 1998. J. Cell Si i. Ill: 1603-1612. 4. Tran, P. T., P. Maddox, F. Chang, and S. Inoue. 1999. Biol. Bull. 197: 262-263. 5. Chang, F., and P. Nurse. 1996. Cell 84: 141-194. 6. Sawin, K. E.. and P. Nurse. 1998. J. Cell Biol. 142: 457-471. 7 Toda, T., K. Umesomo, A. Hirata, and M. Vanagida. 1983. J. Mol. Biol. 168: 251-270. 8. Chang, F. 1999. Curr. Biol. 9: 849-852. microtubules, the nuclei nvrc offset, the cell c\cle was delaved. no spindles were formed, and subsequent division planes ami septum were also offset, creating "cut" nuclei and daughter cells of unei/iial length. The DIC panels from fl-4 h show a cell will] a "hirlh \car. " not to he confused with the septum. Bar = III p.in. CELL BIOLOGY 207 Reference: Kin!. Bull. 199: 207-208. (October 2000) The Role of Microtubules During Blastodisc Formation of the Squid, Loligo pealei Karen Crawford (Department of Biology, St. Mary's College of Maryland, St. Mary's City. Maryland 20686) After fertilization, cytoplasm streams from the vegetal region of the squid egg towards the animal cap to form a blastodisc where meroblastic cleavage will occur (1. 2). This process begins at fertilization, accelerates after second polar body formation (90 min, at 20°C), and continues through third cleavage (6.5-7.0 h). A blastodisc cap is formed, although at a slower rate, in eggs that have been artificially activated with 10 /u.g/ml A23187 (Molecular Probes) (3). To explore the role of the cytoskeleton in this process, /';; vitro fertilized (4) or activated embryos were placed in small petri dishes lined with 0.2% agarose (Sigma, Type II) and filled with 20°C Millipore-filtered seawater (MFSW). The dishes were placed on ice and cooled to 4°C. Exposure to cold was chosen to perturb cytoplasmic movements targeting microtubules (5). so that the effect on the embryos could be easily reversed. Cold treatment periods were selected to include the first and second polar body meiotic divisions (20 min and 1.5 h respectively), and the first (3.5 h), second (4.0 h) and third (6.5 h) cleavage events. Treatment periods were 20 min to 3 h. 3 to 4 h. 4 to 5 h. 5 to 6 h and 6 to 7 h of development. After treatment, dishes of embryos were removed from the ice and allowed to return to room temperature (20°C). Embryos were compared to control embryos for blastodisc forma- tion, the presence of polar bodies, and cleavage pattern. Cleavage in squid is bilateral (Fig. la). First cleavage occurs along the line between the polar bodies and the apex of the embryo where the male pronucleus enters the egg. Second cleavage occurs perpen- dicular to this, and third cleavage is unequal and distinguishes the future right and left sides of the developing embryo. Exposure to cold inhibited blastodisc cap formation in all em- bryos treated prior to cytoplasmic streaming: it also arrested streaming in embryos treated after second polar body formation. Twenty minutes after removal from cold exposure, precleavage stage embryos develop a blister-like swelling of clear cytoplasm surrounding the male pronucleus. Activated eggs do not form blisters of cytoplasm when removed from cold treatment, although a small crescent of cytoplasm may form over the female pronu- cleus after 50 minutes. Over this same period of time, the polar bodies that are present swell to more than 4 times their normal diameter of 10 /u.m and then slowly return to normal size. Over the next 20 min the blister of cytoplasm around the male pronucleus relaxes into a small but growing blastodisc cap that resembles a normal cap in most (95%) cases. Abnormal cap formation was observed in about 5% of the embryos examined and included displacement of the cytoplasm to one side of the animal pole or splitting of the cap at the apex into two regions. Normal cleavage did not occur in these cases. In contrast to control squid embryos, which form two polar bodies, in vitro fertilized embryos treated during polar body formation possessed one (59/73. 37%) or two (3/73. 4%) and more frequently no (43/73 or 59%) polar bodies. Similar results were observed in activated eggs treated with cold during polar body formation. Fertilized embryos that failed to complete their meiotic divisions often possessed three nuclei at the apex of the blastodisc cap prior to cleavage, indicating that cold shock at this early stage induces polyploidy. These embryos sel- dom underwent normal cleavage. Interestingly, in contrast to the 2%-10% of control-activated eggs that underwent a cleavage event, 60% (79/132) of activated eggs treated with cold during their meiotic divisions possessed cleavage furrows. Embryos treated with cold from 3 to 4 h, the time when control embryos undergo first cleavage, possessed two polar bodies (as did all other embryos treated at later times), formed normal blastodisc caps, and cleaved normally. In contrast, even though first cleavage begins at 3.5 h, embryos treated from 4 to 5 h of development and returned Figure 1. Cold exposure inhibits specific cleavage furrows in the squid enibnn. Individual furrows are numbered in order of their appearance, (at Control third cleavage stage embryo, animal pole view. Note that third cleavage is iniei/iial mid that the polar bodies (pb) lie adjacent to the first cleavage furrow, which marks the midline body axis, (b) Embryo treated with cold exposure from 4 to 5 h after fertilization. The first cleavage farrow is missing, anil third cleavage is equalized, (c) Embryo treated with cold exposure from 5 to 6 h after fertilization. The second cleavage furrow is missing in this embryo, while first and third cleavage furrows are present and normal. Images magnified 650 X. 208 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS to room temperature failed to retain their first cleavage furrow (Fig. Ib) in 90% of the cases examined (36/40). In these embryos, because the polar bodies mark the region through which first cleavage will form, it is possible to determine that second cleavage occurred normally, while third cleavage — which is normally un- equal— was equalized to mirror the cells in the future dorsal region of the embryo. Most embryos treated between 5 and 6 h of development retained a reduced first cleavage furrow at the center of the blastodisc and formed normal second and third cleavage furrows (76/89 or 85%), while other embryos from this group developed without a second cleavage furrow (Fig. Ic). This pattern was also observed in embryos treated between 6 and 7 h. Surpris- ingly, most of the embryos from all treatment groups continue to develop and at 48 h appeared fairly normal, although they often possessed clumps of large cells, uneven blastoderm yolk boarders, and regions where cell layers appeared thicker than controls. These results suggest that the ordered movement of cytoplasm, which forms the blastodisc in the squid, is disturbed by cold treatment. Cold exposure also induced polyploidy and perturbed cleavage furrows. The failure to retain or create specific cleavage furrows may be due to the direct action of cold on the microfila- ments responsible for furrow formation, cell membranes, or spe- cific factors that regulate mitosis. However, the formation of the blister-like swellings of cytoplasm around the male pronucleus. likely initiated by the sperm centriole to form microtubule arrays, suggests that cytoplasmic movements are rapidly resuming and may disturb the previously formed or forming microfilaments responsible for cleavage. That the polar bodies, which are little more than unwanted chromosomes and microtubules, swell rapidly during this same period further suggests that microtubules may be partially responsible for these events, although this does not rule out the possibility that cold exposure results in destabilization of membranes in these cells. Microtubules originating from the sperm pronucleus are crucial for the reorganization of cytoplasm after fertilization in frog eggs (6). The result that cold exposure can equalize third cleavage in squid embryos is nearly identical to what was reported when squid embryos were treated with the micro- filament inhibitor cytochalasin B. although first cleavage furrows were still present in some of those embryos (7). To address the importance of microtubules and microfilaments alone and in con- cert to blastodisc formation and cleavage in the squid, it will be necessary to selectively challenge each element with specific in- hibitors and characterize their appearance over time with immu- nohistochemistry. This work was supported by a Research Opportunity Award from the National Science Foundation to Karen Crawford. Literature Cited 1. Brooks, W. K. 1881). Anniv. Mem. Roslon Sue. N.H. 1-22. 2. Arnold, J. M. 1968. Dev. Biol. 18: 180-197. 3. Crawford, K. 1985. Biol. Bull. 169: 540. 4. Klein, K. C., and L. A. Jaffe. 1984. Biul. Bull. 167: 518. 5. Yahara, I., and F. Kakimoto-Sameshima. 1978. Cell 15: 251-259. 6 Elinson, R. P., and B. Rowning. 1988. Oct. Biol. 128: 185-197. 7 Arnold, J. M., and L. D. Williams-Arnold. 1974. J. Emhryol. Exp. Morphol. 31: 1-25. Reference: Biol. Bull. 199: 208-209. (October 2000) Cytoplasmic Proteins on the Surface of Discharged Microsporidian Sporoplasms Earl Weulner (Bioloi>\, Louisiana State University, Baton Rouge, Louisiana) The spore cell of an intracellular microsporidian parasite is a missile which, when activated, explosively discharges an invasion tube. The spore contents (sporoplasm) pass through the invasion tube and are introduced into a target cell. Evidence reported earlier indicates that the membrane surrounding the newly discharged sporoplasm cell is derived in part from the polaroplast organelle of the spore ( 1 ). An early accepted model for microsporidian sporo- plasm discharge held that the membrane everts with the cytoplas- mic face shifting outward during extrusion (2). If this is what happens, it would seem that cytoplasmic proteins might remain attached to this membrane and end up on the surface of the discharged sporoplasm. Evidence presented here indicates that cytoplasmic tubulin and dynactin proteins are on the surface of discharged microsporidian Sporoplasms, thus supporting the idea of membrane eversion during spore extrusion. In this study, proteins were identified from the sporoplasms discharged from spores of the microsporidian, Spmxiteu lophii. The protocol for isolating the sporoplasms was reported earlier (3). Sporoplasms examined immunocytochemically for surface tubulin (using IgG monoclonal or polyclonul primary antibody, with fluo- rescein-coupled secondary antibody) revealed an even, but some- times patchy labeling (Fig. 1A). Similar results were found when sporoplasms were tested with fluorescein-labeled colchicine. In a follow-up experiment, sporoplasms were incubated in tubulin as- sembly medium with fluorescein-coupled tubulin. The results showed a preferential bordering of the labeled tubulin around the sporoplasms (Fig. IB). The S. lophii sporoplasms were also tested for surface dynactin proteins by using antibodies for pl50gl"cd. dynein intermediate chains, and dynein heavy chains. The site of binding was visual- i/ed with colloidal gold or fluorescein-coupled secondary anti- body. The results showed some uneven labeling for p!50glued (Fig. 1C) and dynein light chains, but no dynein heavy-chain labeling was apparent. Western blot analyses revealed substantial levels of pl50glued and dynein light-chain proteins. The positioning of these proteins onto the sporoplasm surface suggests that the sporoplasm membrane at first faces the cytoplasm within the spore, but shifts to the outside during spore discharge. Surface dynactin is an important component in the movement of membranous structures within cells. Recall, moreover, that 5. CELL BIOLOGY 209 lupliii parasitizes the central nervous system of different species of angler fish of the genus Luphiits, the infections being particularly evident in the cranial ganglia, dorsal root ganglia, and the su- pramedullary neurons. Surface dynactin. therefore, is prob- ably involved in positioning the microsporidian parasites in neu- ronal cell bodies within the central nervous system of their piscine hosts (4). Literature Cited 1 . Weidner, E., VV. Byrd, A. Scarborough, J. Pleshinger, and D. Sibley. 1984. ./. Pi;>t,>:<>«l. 31: 145-198. 2. Lorn, J., and J. Vavra. 1963. Ada Protocol. 1: 279-283. 3. Weidner, E., and A. Findley. 1999. Bio/. Bull. 197: 270-271 4. Weidner, E., and T. King. 1998. Hi,,/. Bull. 195: 229-228. Figure 1. Spraguea lophii sporoplusnis with tubulin labeling. (A) Anti- tubulin fluorescence confined to sporoplasm surface. (B) Time-interval recordings of fluorescein-coupled tubulin bordering spherical sporo- plasms. (C) Anti-dynactin plSO*1""' label with patchy positioning on sporo- plasms. All scale bars represent 5 IJLIII. Reference: Bio/. Bull. 199: 209-211. (October 2000) Adhesion of a Viral Envelope Protein to a Non-Self-Binding Domain of the Aggregation Factor in the Marine Sponge Microciona prolifera Roger MacKenzie1, David Newman2, Max M. Burger*, Rene Roy4, ami William J. Kuhns5 (Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Studies of the Microciona aggregation factor (MAP) have thus far been focused upon its self-binding characteristics. This study demon- strates for the first time an MAP non-self-binding domain. The pur- pose of this study has been two-fold: (a) to purify the binding motif as 1 Institute for Biological Sciences. National Research Council of Can- ada, Ottawa, Ontario. 2 Natural Products Branch. National Cancer Institute, Frederick, MD. 3 Friedrich Miescher Institute, Basel, Switzerland. 4 Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada. 5 Hospital for Sick Children, Toronto, Ontario. Canada. a potential pharmacoactive mierobicide; and (b) to characterize non- self adhesins as participants in the cross-species binding of microbes to sponge cells. Cell-cell aggregation in Microciona prolifera is mediated by its aggregation factor, a species-specific compound ex- hibitins a unique sunburst structure at high magnifications, with a molecular weight of 2 x 107 Da (1, 2). There is a bracelet-like protein core composed of multiple beads, each attached to a protein-carbo- hydrate arm. Multiple anionic glycans on the arms polymerize to form a viscous gel in the presence of calcium. A sulfate disaccharide and a pyruvate trisaccharide mediate self-binding to adjacent arms and to cell membranes (3, 4). A hyaluronic acid (HA)-like compound stabi- lizes the core-arm connections (5). Binding inhibition studies of MAP 210 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 120 100 § 80 | 60 "o c 40 0) o Qj a. 20 0 -20 400 300 200 100 CC 3100 I 125 0 100 75 50 25 0 -25 0 • Infected Treated Culture Q Uninfected Treated Culture — 100%, 50% Ref. Lines Viral Cylopathic Effect 0 1 2 Q of Sample Concentration (pg/ml) 200 400 600 200 400 600 110 100 90 80 70 60 50 40 30 20 'ercent of Uninfected Untreated Control Cultur D Dose (pg/ml) Percent of Protection 1.19X 1CT1 56.99 3.76 x 10~1 55.68 1.19X 10° 88.99 3.76X10° 105.80 1.18x10+1 96.46 3.75x10+1 95.03 1.18x10+2 95.21 3.75X 10+2 86.80 200 150 100 50 0 -50 400 I I I I 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 Time (sec) Figure 1. Summary of the antiviral propertv of MAF in CEM lymphoblastoid cells infected in//? human immunodeficiency virus (H1V-1} (a, b). Sensorgrarns indicate MAF inhibition of rgp!20-rCD4 binding bv MAF ft', d), whereas synthetic sitlfated and pyruvylated constructs show no inhibition (e, f). (a, b) Results of forma-an XTT assays for viral inhibitory properties of MAF derivatives. Round black dots and diamonds depict infected treated culture and Uninfected treated culture respectively. MAF gave >50% protection at a dose as low as 0. 1 p.g/ml (EC50), while demonstrating little or no toxic effects. Lower dotted line demonstrates viral pathogenic effect in infected untreated culture. Straight dashes are 50% ami 100% reference lines, (c-f) Sensorgrarns which schematize the relative binding affinities of self and non-self MAF binding epitopes. The small letters that accompany each set ofcun'es represent the following: a = rpg!20 — rCD4 binding, b = rpg/20 + putative inhibitor — rCD4 interaction, c = putative inhibitor - rCD4 reaction. The constants for these reactions are given b\ the terms k:l and kj. where k:l refers to the association constant and ktl describes the dissociation constant. The values for rpg 1 20 at the stated concentration were 1.2 X Iff1 and 3.3 x 10 4 respectively and indicated a strong binding affinity between rpg/20 and rCD4 (a curves). The reactions did not differ appreciably when gp!20 was in mixture with the sit/fate or pyruvylated compounds (b cun'es). However, both MAF I and MAF ds were reactive as inhibitory compounds. MAFI a/so possessed a strong affinity for rCD4. as indicated by the c cun'e shown on the left middle drawing. fractions were carried out using as a binding model recombinant (r) gp!20 human immunodeficiency vims (HIV-1) envelope protein and lymphocyte rCD4 protein receptor. MAF was prepared from cuttings of sponge branches and pro- cessed as described elsewhere (6). The clear gel derived from cesium chloride gradient ultracentrifugation was dialyzed repeat- edly against 200 X volume de-ionized water and then lyophilized (MAF-I). The result was a truncated molecule, as shown by elec- tron microscopy — one having morphologically intact bracelets, but lacking arms (7). Low molecular weight anionic glycans were prepared from MAF-1 by ethanol precipitation (8) followed by recovery and lyophili/ation of the ethanolic supernatant (MAF-ds). CELL BIOLOGY 21 Compositional analyses of MAFds demonstrated a high sulfate and carbohydrate content; spectroscopic analysis showed a major peak that had a mass/charge ratio of 3 kDa and represented about 60% of the solids in the dried sample. Neither fraction was active in aggregation assays at levels above 20 /ng/ml, although freshly derived MAP was active at a level of 0.5 /j,g/ml. Binding of the HIV-1 envelope protein gp!20 to target lympho- cytes via CD4 peptide is necessary for syncytium formation and viral entry and multiplication in target cells (9). Inhibition of gp!20-CD4 binding by MAP fractions was evaluated using surface plasmon resonance as the detection principle for molecular inter- action analysis (10). Instrumentation provided by BIACORE Com- pany (Piscataway, New Jersey) permits ligands to be immobilized on a gold sensor chip upon which a light beam is directed. A continuous flow system permits injection of binding compounds alone or in mixture with inhibitors over the ligands. Binding causes a change in the angle of the light beam, with an association phase beginning at analyte injection and a dissociation phase at the end of injection. The changes are recorded as a sensorgram. The inhibition of HIV-1 by MAP compounds was assayed in infected lymphoblastoid cells by using a colorimetric method in which a colorless compound (formazan XTT) is metabolically converted by healthy cells, but not dead cells, to an orange -colored deriva- tive (II). The results of MAP titrations for two MAP fractions in formazan assays indicated that amounts as low as 0. 1 ;ug conferred protection on more than 50% of the cells (EC50) while simulta- neously showing little or no toxic effects toward non-infected cells (Fig. la, h). Data for the binding of recombinant rgp!20 to rCD4 gave a dissociation constant of 17 nM, which agrees well with the value of 19 nM previously reported by Wu el til. (12). At concen- trations of 0.75 mg/ml, both MAP derivatives inhibited binding (Fig. Ic, d). Tests for binding inhibition using synthetic constructs and polymers of the MAP self-binding epitopes sulfuted disaccha- ride and pyruvylated trisaccharide were completely negative (Fig. le. f). Of the four compounds, only MAF-1 showed any binding to rCD4 at the end of the injection cycle, while MAF-ds was bound to rgp!20, but not to rCD4. In summary, a partially purified MAP derivative (MAFds) in- hibited the replication of the human immunodeficiency virus (HIV) by specific gp!20 binding and interfering with syncytium formation between the viral gp!20 envelope protein and the lym- phocyte CD4 antigen. Thus, in pure form, it will qualify at a clinical level as a microbicide or as a microbistatic agent, similar in its chemical properties to HIV-inhibitory products derived from some other marine invertebrates (13). Although it seems unlikely that MicriH-imiti would encounter the HIV virus in its natural surrounding, we propose that this model typifies cross-species binding (as demonstrated by a non-self adhesin unrelated to the known self-binding epitopes), and it may provide the sponge with a means of immobilizing symbionts or other forms that are re- quired for nutrition or for disposal by macrophages. Literature Cited 1. Humphreys, S., T. Humphreys, and J. Sano. 1977. ./. Siiprumol. Struct. 1: 339-351. 2. Fernandez-Busquets, X., and M. M. Burger. 1999. Microsc. Res. Tech. 44: 204 -2 IS. 3 Spillmann, D., J. Thomas-Gales, J. van Kuik, J. Vliegenthart, G. Misevic, M. Burger, and J. Finne. 1995. J. Bid. Cliem. 270: 5089-5097. 4 Spillmann, D., K. Hard, J. Thomas-Gates, J. Vliegenthart, G. Misevic, M. M. Burger, and J. Finne. 1993. J. Bio/. Cliem. 268: 13378-13387. 5. Jarchow, J., J. Fritz, D. Anselmetti, A. Calabro, V. Hascall, D. Gerosa, M. M. Burger, and X. Fernandez-Busquets. 2000. ./ Struct. Biol. (In press). 6. Misevic, G., J. Finne, and M. M. Burger. 1987. J. Biol. Cliem. 262: 5870-5877. 7. McLaurin, J., T. Franklin, W. Kuhns, and P. Fraser. 1999. Ainy- loul. Int. J. Ev/>. din. Invest. 6: 233-243. 8 Cardellina, J., R. Munro. K. Fuller, T. Manfredi, M. McKee, H. Tischler, K. Bokesch, J. Gustafson, J. Beutler, and M. Boyd. 1993. J. Nat. Prod. 56: I 1 23- 1 124. 9. Chimule, N., and S. Phawa. 1996. Microbiol. Rev. 60: 386. 10. Inagawa, J., J. Okazaki, K. Morimoto, and S. Hashimoto. 20(10. Pp. 37-57 in Real Time Analysis of Bionmleciilar Interactions. Appli- cations of BIOCORE. K. Nagata and H. Handa, eds. Springer Verlag, Tokyo. I 1. Weislow, O.. R. Kiser, D. Fine, J. Bader, R. Shoemaker, and M. Boyd. 1989. J. Nail. Cancer Inst. 81: 577-586. 12. Wu, H., D. Myszka, S. Tendian, C. Brouillette, R. Sweet, I. Chaiken, and W. Hendrickson. 1996. Proc. Natl. Acad. Sci. USA 93: 15030-15035. 13. Beutler, J., T. McKee, R. Fuller, M. Tischler, J. Cardellina, K. Snader, T. McCloud, and M. Boyd. 1996. Antivir. Chem. Cliento- ther. 4: 83-88. 212 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Biol. Bull- 199: 212-213. (October 2000) Fertilization-induced Changes in the Fine Structure of Stratified Arbacia Eggs. I. Observations on Live Cells with the Centrifuge Polarizing Microscope Mukoto Coda1, Mario H. Burgos2, and Sliinya I none {Marine Biological Luboratory. Woods Hole. Massachusetts) Eggs of the sea urchin (Arbacia punctulata), when suspended in isopienic seawater and subjected, by centrifugation for several minutes, to a gravitational field a few thousand times greater than the earth's, stratify into several layers. As shown earlier by Har- vey, who used supra vital dyes ( 1 ). the layers are, from top (centripetal pole) to bottom: oil cap, a large clear zone immediately below the oil cap containing the nucleus, the mitochondria! layer, yolk granules, and the pigment granule layer. Observing this phenomenon with a recently developed cen- trifuge polarizing microscope (CPM) (2. 3), we find that cen- trifugation introduces to the upper part of the clear zone a negatively birefringent curtain of material that drapes down 1 Kyoto University. Japan 2 IHEM. UNC-CONICET, Argentina from the oil cap and surrounds the isotropic nucleus (Fig. 1A, B). The negative birefringence increases with both time and speed of centrifugation. The negative sign of birefringence (larger refractive index perpendicular to the texture), fluores- cence staining with brefeldin A, and electron microscopy of fixed cells (5) suggest that this negatively birefringent material is a pleated series of endoplasmic reticulur membranes, strati- fied and oriented by the centrifugation. When a stratified egg is fertilized, the birefringence, viewed with the CPM, disappears in a few seconds, a surprising obser- vation (Fig. 1C-E). Furthermore, the egg concurrently starts floating up in the Percoll-seawater density gradient (Fig. 1F-H). As the egg floats up, the fertilization envelope rises and its positive birefringence increases over the next 3 to 4 min. In the next 10 or so min. the negative birefringence below the oil cap 16:25:06 A 16:25:52 16:26:06 16:26:10 D 16:26:12 16:26:16 16:26:38 H ^^^^Hill^^^^F 16:27:10 Figure 1. Fertilization of vr« urchin egg observed with the CPM. After the unfertilized Arbacia eggs were centrifugally stratified at 3000 X gfor iihmil I? nun, the CPM was Mopped, and u drop of sperm suspension was plated mi the wall of the specimen chamber above the Pereo/l seinvitler containing the egg.s. Upon re-starting the CPM, the sperm suspension crept down and fused with the egg suspension. Sperm then swum and reached the eggs (at about I'' 2d nut, which were Mill stratified, hut hail roniuletl up fliiring the few minutes that the CPM was stopped. (A. B): Negatively birefringent curtain uf material sho\\ \ in the upper region of the clear rone .surrounding the nucleus of unfertilized eggs. (C-E): During about the first 10 s after fertilization, the neganvi birefringence disappears. \c *copc, the only C-\ccption heing the annulate liimellae. which lie inu'i i>:> I<:J lo be "coarse fibrous components. " CELL BIOLOGY 213 gradually returns again, but with a much more complex align- ment of material. To test whether these changes reflect a rise in cytosolic Ca2 + (released from the endoplasmic reticulum [ER], an intracellular Ca2 + storage organelle). we observed the responses of unfertilized stratified eggs to the calcium ionophore A-23187 (6). Whether in normal or Ca2+-free seawater, the eggs indeed responded exactly as when fertilized. We surmise that elevation of cytosolic Ca2 + in the seconds immediately following fertilization (7) is correlated with the transient breakup of the ER (8, 9), and that this breakup is manifested as the rapid loss of birefringence. As the Ca~+ level drops again, the ER must re-assemble into large layered sheets since the negative birefringence reappears. In control experiments, inactivated eggs, that had received the identical history of centri- fugation, retain their negative birefringence for more than 30 min. Following fertilization, the egg may become less dense due to exocytosis and swelling of the cortical granules, or by uptake of water by the egg or the egg jelly. While not observed when stratified eggs were fertilized in normal, Ca2 + -containing seawa- ter, eggs activated with the Ca2 + ionophore suddenly fall in the density gradient after steadily rising for several minutes. Since this fall is accompanied by a sudden release of diffuse material sur- rounding the fertilization envelope, swelling of the egg jelly may be primarily responsible for the increased buoyancy of the egg following its activation. Support of research by Hamamatsu Photonics KK, Olympus Optical Company, Kyoto University, and the Marine Biological Laboratory is gratefully acknowledged. We thank Drs. B. Kaminer and M. Terasaki for extensive discussions of their work relating to changes in cytosolic Ca2 f and ER organization following fertili- zation. Literature Cited 1. Harvey, E. B. 1941. Bu>l Hull. 81: I 14-118. 2. Inline. S., R. A. Knudson. K. Suzuki, N. Okada, H. Takahashi. M. lida, and K. Yamanaka. 1997. Microxc. Microanal. 4: 36-37. 3. Inoue, S. 1999. h\\SHH .1, (.Vn/v/J 13: sl85-s!90. 4. McCulloch, D. 1952. ./. /:v/> 7n,'l. 119: 47-65. 5. Burgos, M. H., M. Coda, and S. Inoue. 2000. Biol. Hull. 199: 213-214. 6. Steinhardt, R. A., and 1). Epel. 1974. Prac. Null. Aciul. Sci. USA 71: 1915-1919. 7 Gilkey, J. C., I,. F. Jaffe, E. B. Ridgeway, and G. T. Reynolds. 1978. J. Cell Bwl. 76: 448-466. 8 Henson, J. H., D. A. Begg, S. M. Bealieu, D. J. Fishkind, E. M. Bonder, M. Terasaki, I). Leheche, and B. Kaminer. 1989. ./. Cell Biol. 109: 144-161 4 Terasaki, M., and L. A. Jaffe. 1991. ./. Cell Biol 114: 929-940. Reference: Biul. Bull- 199: 213-214. (October 2(100) Fertilization-induced Changes in the Fine Structure of Stratified Arbacia Eggs. II. Observations with Electron Microscopy Mario H. Burgos[, Makoto Goda2, and Shin\a Inoue (Marine Biological Lahoratory, Woods Hole, Massachusetts) Unfertilized Arbacia eggs are stratified by centrifugation: the centripetal pole is occupied by an oil cap, which crowns a large clear zone containing the nucleus ( 1 ). When such eggs are ob- served with the centrifuge polarizing microscope (CPM). a curtain of negatively birefringent material, draping down from the oil cap, is introduced to the upper part of the clear zone (2). When stratified eggs are fertilized or activated by the Ca2 + ionophore A23 1 87, this birefringence disappears within a few seconds — even before the fertilization envelope starts to elevate. Its sign, and the fluorescent staining by brefeldin A, suggest that the negative birefringence is due to a stack of membranes, stratified and aligned by centrifuga- tion, and oriented more or less parallel to the direction of the centrifugal force. To evaluate this proposal further, we investigated the birefrin- gent region of the egg by electron microscopy. We used 2% glutaraldehyde in phosphate-buffered saline made up into 700-mA/ sucrose to prevent swelling of the Arbacia egg. Eggs placed in fixative without sucrose swelled up to about eight times the vol- 1 IHEM. UNC-CONICET, Argentina 2 Kyoto University, Japan ume of the unfixed egg. lost their microvilli. and (reversibly) lost their negative birefringence. Thin sections of stratified non-activated eggs, fixed with su- crose-glutaraldehyde, retained their negative birefringence and revealed that the birefringent region is occupied by stacks of smooth and rough endoplasmic reticulum (ER; Fig. 1A). The ER surrounded the nucleus and was aligned more or less parallel to the axis of centrifugation. A small number of Golgi membrane stacks were found amidst the ER. but with random orientation. At the lower region of the ER. we found stacks of annulate lamellae (3.4). These are most likely the refractile rod- and plate-like structures that are seen in centrifuged eggs by light microscopy, especially clearly in DIC. They tended, at first, to lie parallel to the axis of centrifugation. but changed their orientation as time elapsed after the centrifuge was stopped. In centrifugally stratified eggs fixed about 5 min after fertil- ization— well after the negative birefringence had disappeared, but before it re-appeared — the distribution of the Golgi and annulate-lamellar material was basically unchanged. However, the ER was no longer in large sheets oriented along the cen- trifugal axis; rather, the sheets had fragmented into smaller vesicles (Fig. IB), as was anticipated from their loss of bire- 214 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS ER ne B f-ER Figure 1. Ultrastrncnire of upper eleur ;one in centrifugally stratified Arhacia egg. A. Unfertilized: B. fixed uhoitt 5 win after fertilization. After centrifiigalion on a Percoll-seawater density gradient for about 30 min {A), and after an additional 5 inin centrifugation following fertilization (B}. the eggs were fixed in 2% glularaldehyde phosphate Imfter made up in 700-inM sucrose solution. After confirming, with 1-p.m sections of the unfertilized eggs, that the negative birefringence in the upper part of the clear zone remained intact, the cells were post fixed with osmium, dehydrated, embedded in Epon. and sectioned for electron micro.scnp\. In A. the upper region of the clear zone contains a dense stack of ER, oriented more or less in the direction of the centrifugal force (long arrow t. In B. onl\ small pieces of ER remain, consistent with the (transient) loss of the negative birefringence, at: annulate lamellae. ER: endoplasmic reticiilum. f-ER: fragmented ER. ne: nuclear envelope. Scale bars 0.5 urn. fringence. Because the birefringence of activated live eggs does return in the upper half of the clear zone after about 10 min, albeit with less ordered alignment of the birefringence axes, the EM of cells fixed at that stage would be expected to again show stacks of large ER membrane sheets, but with the stacks ori- ented along less uniform axes. These observations suggest that the birefringence observed in live eggs with the CPM is a good indicator of membrane aniso- tropy, distribution, and especially their dynamic changes. In addi- tion, centrifugally fragmented mini-cells could well prove to be a useful source for several isolated membrane components of the cells. We thank Hamamatsu Photonics KK, Olympus Optical Com- pany, Kyoto University, and the Marine Biological Laboratory for support of this project. We also thank Louis Kerr and Christina Stamper of the MBL Central Microscope Facility for their coop- erative help with electron microscopy. M.B. was supported by an MBL Chairman of the Board Fellowship. Literature Cited 1. Harvey, E. B. 1941. Biol. Bull. 81: 114-118. 2. Goda, M., M. H. Burgos, and S. Inoue. 2000. Biol. Bull. 199: 212-213. 3. Afzelius, B. 1955. E\p. Cell Re.s. 8: 147-15S. 4. Swift, H. 1956. J. Biophys. Biochem. Cytol. iSnppl.t 2: 4 1 5-4 IS and 4 plates. CELL BIOLOGY 215 Reference: Bio/. Hull. 1"W: 215-217. (October 2(100) Optimization of Homogenization Conditions Used to Isolate mRNAs in Processes of Myelinating Oligodendrocytes Robert M. Gould1, Concetto M. Freund1, John Engler2, and Milan- G. Morrison3 Many of us visualize the myelinated nervous system from light microscope (LM) images. Almost all white matter space is occupied by myelinated fibers, each sheath having a thick- ness that relates to the caliber of its axon. Myelin sheaths are made by oligodendrocytes during nervous system development. The enormous space occupied by myelinated fibers leaves oli- godendrocytes so far apart that it is impossible to see connec- tions between cell bodies and myelin sheaths in adult tissue sections. A picture of how myelin sheaths form comes from reconstruc- tions of morphological images. Oligodendrocyte precursors mi- grate to each of the regions where axons are developing. When they arrive, they replicate and send out processes, which select, ensheath, and myelinate axons that have reached a certain size. Each process must successfully compete for the axon segment that it myelinates and then produce and maintain a sheath of identical dimensions to its neighbors. This morphological picture represents a multitude of complex process that requires coordinated expres- sion of many gene products. At present only a very small number of these genes are known. The immediate goal of our research program is to identify new genes involved in myelination and determine their contributions. As a first step we developed a method to identify a population of mRNAs that are important for myelination in rat brain. This mRNA population is selectively translated near sites where myelin sheaths assemble. Myelin basic protein, a dominant myelin protein, is selectively synthesized in these sites, for unlike other myelin proteins, MBP enters myelin within min- utes of its synthesis. For MBP to enter myelin so rapidly, not only MBP mRNA, but also all other components needed for its translation, must be transported from the oligodendrocyte soma to each myelin sheath assembly site. We reasoned that the capacity to synthesize proteins at sites distant from the oligo- dendrocyte soma would not be limited to a single protein. Furthermore, as we identified other proteins synthesized near to where myelin basic protein was incorporated into myelin. we would broaden our understanding of how myelin sheaths are assembled. David Colman and his collaborators (1,2) provided a starting point for our studies. They showed that MBP mRNA behaves differently from mRNAs for other myelin proteins when brain samples are subjected to subcellular fractionation. MBP mRNA purifies in myelin vesicles whereas mRNAs for other known myelin-related proteins do not. We used rat brain myelin 1 N. Y. S. Institute for Basic Research in Developmental Disabilities, Staten Island, NY. : Dept. of Biology, North Carolina State University, Raleigh, NC. ' Bay Paul Center for Molecular Biology and Evolution. Marine Bio- logical Laboratory, Woods Hole, MA. mRNA as starting material and suppression subtractive hybrid- ization to isolate cDNAs that represent mRNAs which co- localize with MBP mRNA in myelin sheath assembly sites. In the initial study (3), we compared two different homogenization media, one isoosmotic (0.32 M sucrose) and one hypertonic (0.85 M sucrose). Although we obtained more myelin RNA from samples homogenized in hypertonic sucrose, we worried that this RNA had higher levels of contaminating RNA than samples obtained from tissue homogenized in isoosmotic su- crose. We thus conducted most (2 of 3) suppression subtractive hybridization studies with myelin RNA prepared from isoos- motic homogenates (4). However, when we counted the novel mRNAs obtained in screens with samples prepared under each condition, we found a far more diverse population was obtained when samples homogenized in 0.85 M sucrose were used (4). Some of the mRNAs with particular relevance to myelin sheath biogenesis, SH3pl3 or endophilin 3 (5) and dynein light inter- mediate chain (6) were obtained from the screen with the hypertonic sample (4). During the summer of 2000, we analyzed 90 (25 were se- quenced) subtraction products prepared from samples homog- enized in 0.32 M sucrose and 90 subtraction products (46 were sequenced) prepared from samples homogenized in 0.85 M sucrose. We used colony hybridization and hybridization of inserts prepared from mini-prep samples to identify cDNAs derived from MBP and MOBP (myelin-associated oligodendro- cytic basic protein) mRNAs. These RNAs are known to be located in oligodendrocyte processes (7), since these were highly enriched in myelin (3; Fig. 1). The remaining cDNAs were sequenced in the Bay Paul Center sequencing facility at the Marine Biological Laboratory in Woods Hole, Massachu- setts. Confirming results from our recent study (4), we found that far more (30 versus 13) novel cDNAs were obtained from myelin prepared in 0.85 M sucrose. Most of these cDNAs relate to known mRNAs (Table I). A significant portion of the mRNAs generate proteins involved in regulating protein syn- thesis, namely eukaryotic translation elongation factors alpha and delta and ribosornal proteins L7a and L21. A few, kinesin light chain, rab7 and evectin. increase the number of proteins with recognized functions in membrane trafficking and biogen- esis. We have analyzed four cDNAs from the 0.32 M sucrose subtraction product and six cDNAs from the 0.85 M sucrose subtraction product by northern blot comparisons (starting ma- terial RNA versus myelin RNA). All of them have mRNAs that are highly enriched in myelin. Among the known mRNAs analyzed so far were ferritin light chain, eukaryotic elongation factor alpha- 1 and kinesin light chain. Future studies aim to locate the mRNAs and proteins in myeli- nating tissue. In addition, we will use this approach to identify mRNAs located in myelin sheath assembly sites in spiny dogfish. 216 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS MBP MtO Know n EST Unknow n Distribution of cDNA sequences Figure 1. Number and distribution ofcDNAs obtained from subtractive hybridization studies with rat brain samples homogenized in 0.85 M sucrose (black bars) or 0.32 M sucrose (gra\ bars). Clearly larger numbers of novel (30 vs. 13) cDNAs, many of which are related to known mRNAs (see Table I}, are obtained in the sample homogenized in 0.85 M sucrose. This difference is mainly due to the higher numbers (58 vs. 33) of cDNAs derived from niYc/in basic protein (MBP) or myelin-associated oligodendrocytic basic protein (MOBP) mRNAs in the 0.32 M sucrose sample. Other abbreviations: Mito, sequences related to the mitochondria! genome: Known, sequences in the non-redundant GenBank database: KR, sequences related to known GenBank sequences but with less than 50% of the set/uence matching the known: EST, sequences in the GenBank EST database; Unknown, sequences unrelated to sequences in either the non-redundant or EST databases. The results of this study will, in particular, help us to select appropriate conditions to homogenize dogfish brain, which exists in an environment of far higher tonicity (approximately I M) than mammalian brain. This work was funded by the National Multiple Sclerosis Soci- ety grant RG2944 (RMG) and the G. Linger Vetlesen Foundation (HGM). Funds for John Engler were from the Marine Models in Biological Research Proaram (NSF grant, DBI-99 12287). We Identities of "known" mRNAs obtained in this study Table 1 Name Accession* Size (region )t Homogenization Astrocytic phosphoprotein AJ243949 1565-2073(2341) 0.85 M Phosphodiesterase I D28560 2282-2898(3216) 0.85 M Ferritin light chain NM_008064 605-886(886)** 0.85 M Glial maturation factor NMJI04124 6-291 (4131) 0.85 M Eukaryotic translation elongation factor « 1 X63561 584-1449(1714) 0.85 M Eukaryotic translation elongation factor 81 NM_001960 37-281 (991) 0.85 M Ran? NM_009005 1179-1361 (2089) 0.85 M Ribosomul protein L7a XI 50 13 417-838(851)** 0.85 M Ribosomal protein L21 X15212 268-543 (554)** 0.85 M RANP- 1 D50559 1237-1540(1712) 0.85 M Zinc finger homeodomain enhancer protein U51583 1172-2094(3403) 0.85 M Kinesin lighl chain A, B, C M75146 1322-1705(2308) 0.32 M KPL-i. evectin AF081582 933-1411 (1903) 0.32 M Lens epithelial protein U20525 660-705(2308) 0.32 M * GenBank accession number. t Size refers to the nucleotide sequence in the known that matches the cDNA sequence we obtained. The size of the known is in parentheses. ** Highly related to known mRNAs — there are differences evident in comparison of aligned sequences. CELL BIOLOGY 2 I 7 would like to thank Dr. Mitchell Sogin for the generous use of the 3. Gould, R. M., C. M. Freund, and E. Barbarese. 1999. J. Neitro- Bay Paul sequencing facility. chem. 73: 1913-1924. 4 Gould, R. M., C. M. Freund, F. Palmer, and D. L. Feinstein. 20(10. Literature Cited J' Neurochem- 5. Ringstad, N., Y. Nemoto, and P. De Camilli. 1997. Proc. Null. 1. Colman, D. R., G. Kreibich, A. B. Frey. and D. D. Sabatini. 1982. Acad. Sci. USA 94: 8569-8574. J. Cell Biol. 95: 598-608. 6. Hughes, S. M., K. T. Vaughan, J. S. Herskovits, and R. B. Vallee. 2 Gillespie, C. S., L. Bernier, P. S. Brophy, and D. R. Colman. 199(1. 1995. / Cell Sci. 108: 17-24. J. Neurochem. 54: 656-661. 7. Holz, A., et al. 1996. J. Neurosci. 16: 467-477. 218 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Reference: Bint. Bull. 199: 218-219. (October 2000) Modeling the Effects of Land-Use Change on Nitrogen Biogeochemistry in the Ipswich Watershed, Massachusetts R\an Kirkbv, Luc Claessens, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino (The Ecosystems Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543) The Ipswich River Basin, which is located in northern Massa- chusetts and drains into the Plum Island Sound Estuary, covers a 400-km2 area composed of forest, wetlands, open and agricultural land, and a gradient of low- to high-density residential and com- mercial land ( 1 ). Over the last century, population growth and land-use changes in the basin have altered the land cover of the watershed. The United States Geological Survey (USGS) has recently modeled the hydrology of the Ipswich River Basin using a precipitation-runoff model called Hydrological Simulation Pro- gram-Fortran (HSPF) (2). Their intent was to develop a better understanding of the effects of water withdrawal on the water budget of the river basin (1 ). In addition to the hydrological data that has been collected by the USGS, we have monitored nutrient loading to build a better picture of the effects of land-use change on nutrient biogeochemistry in the Ipswich river basin. To further investigate nutrient processing in the watershed, the USGS HSPF model ( 1 ) was modified to include simulation of nutrient process- ing on land and in the Ipswich River and tributaries. Considering the projections that have been made for urban development in the Ipswich watershed, the ability to accurately model the resulting changes in nutrient processing may be an important tool in under- standing the health of the Ipswich ecosystem. It could also become an important aid in planning future development that minimizes harmful effects to the watershed. HSPF, in addition to simulating hydrology, is capable of simu- lating nutrient processing, sediment transport, pH and gasses, phytoplankton, and algae dynamics in a watershed. Nitrate-pro- cessing components for stream reaches and land areas were added to the HSPF model. Initial values required by the model were obtained from a database (3) which contains parameter values used in similar HSPF projects in the northeastern United States. Nitrate processing and output from different land types was further cali- brated using an empirical relationship between fractional cover of agricultural and forested land in small catchments \v/™\ nitrate concentration in the streams into which they drain (Fig. 1A, B). In this calibration, the only nitrate input was atmospheric deposition; so the differences in nitrate output between the two land types (Fig. 1 B ) represent the different values chosen for constants in equations governing the simulation of nutrients in the two land-use types. A more rigorous calibration of the model, which is in progress, will include comparing simulated data on nitrate concentration in the Ipswich River with data we are collecting. All simulations run on the model were driven by meteorological input for the years 19H9 to 1993, but future work on the model will include adding more recent meteorological data. The base simulation was run using 1991 land-use data for the watershed; other land-use change sce- narios were run by modifying the areas of different land types in certain parts of the river basin. Calibration of the model resulted in simulated nitrate output from forest and from open plus agricultural land (Fig. IB) that coincided with the empirical relationship for fractional cover ver- sus nitrate concentration (Fig. 1A). The base concentration of nitrate in first-order streams draining only forested lands was approximately 10 /u,M, whereas the value for streams fed by agricultural and open pasture land was closer to 70 p,M (Fig. IB). Modeled nitrate transects along the main stem of the Ipswich River show a strong trend of decreasing concentration near the head of the river, followed by a slowly decreasing concentration toward the Ipswich dam (Fig. 1C). This same general trend is seen in data collected for the same month, although in a different year (Fig. 1C). Stream-flow data along the Ipswich River reveal the opposite trend: a quick increase in flow near the head of the river, followed by a slower increase moving towards the mouth of the Ipswich River (Fig. 1C). Seasonally, nitrate concentrations at the mouth of the Ipswich River reach a peak during winter and spring (Fig. ID). Similarly, river flow at the mouth has its highest peak in the spring and another, smaller peak in the winter (Fig. ID). Data we have collected show similar correlation between peak discharge and peak nitrate concentrations (4). The opposite trends in stream flow and nitrate concentration along transects from the head to the mouth of the Ipswich River suggest that the decreasing nitrate concentration may be due, at least partially, to a dilution effect. The other factors contributing to diminishing nitrate concentration in the river are in-stream pro- cesses, such as denitrification and uptake by plants and algae, that can be examined using the model. One purpose of continuing to examine nutrient processing with this model is to help determine what processes are the most important contributors to the trends that have been observed and modeled. The HSPF model can be used to examine different scenarios for land use by modifying the areas of different land types in the basin. A 12-knr residential development was modeled at different loca- tions in the watershed. The results from those scenarios indicated that nitrate concentration would increase at the mouth of the river, and that the increase would be greater the closer the development is to the mouth of the river. The model predicts that urbanization in the watershed will have a smaller effect if it occurs farther upstream and on tributaries, as opposed to farther downstream and on the main stem of the river. One simulation of two different scenarios showed that a 12-knr residential area built in the lower watershed on the main stem would increase nitrate concentrations at the mouth of the river by approximately 5 (U/W, but an identical development on a tributary feeding into the main stem at the same location would produce roughly baseline conditions at the mouth. The model can be used to look more closely at the sources and sinks of nitrate in the river basin to better characterize the pro- cessing of nitrogen and other nutrients in the watershed. ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY 219 B 0-0 0.2 04 06 0.8 1 .0 Land-Use Fractional Cover (% urban + agriculture) 40 _. 30 , 20 River km 10 200 150 100 z 140 120 _ 100 3 80 2 60 * 40 20 0 M A - Forest Open/ Agriculture I JlJ FMAMJ JASOND Month w £ 15 J u. 10 a 5 0 FMAMJJASOND Month Figure 1. (A) An empirical relationship ben\-een the forest cover in a catchment and the nitrate concentration in streams draining the catchment. (B) Modeled nitrate concentration timeseries in first-order streams with all forest and all open plus agriculture contributing land area. (C) Modeled nitrate concentration and stream flow transect along the main stem of the Ipswich River in July 1993. and observed nitrate data from July 1998. (D) Modeled nitrate concentration and river flow at the mouth of the Ipswich River over the course of 1993. This research was funded through the NSF-EPA Water and Watersheds program DEB-9726862. Literature Cited 1. Zarriello. P., and K. Rics. 2000. A Precipitation-Runoff Model for Analysis of the Effects of Water Withdrawals on Streamflow, Ipswich River Basin, Massachusetts. United States Geological Survey, Denver, CO. 2. Bicknell, B. R., J. C. Imhof'f, J. L. Kittle, A. S. Donigian, and R. C. Johanson. 1993. HyJmloxic Simulation Program— FORTRAN (HSPF): User's manual for release 10.0. EPA 600/3-84-066. U.S. Environmental Protection Agency, Environmental Research Labora- tory, Athens. GA. 3. EPA HSPFParm site. http://www.epa.gov/docs/ostwater/BASINS/ support.htm [21 Aug. 2000], 4. The Ecosystems Center Plum Island Estuary data, hup:// ecosystems.mbl.edu/pie |2I Aug. 2000]. Reference: Bio/. Bull. 199: 219-221. (October 2000) Solute Dynamics in Storm Flow of the Ipswich River Basin: Effects of Land Use Anne Perring, Michael Williams, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino (The Ecosystems Center, Marine Biological Laboratory; Woods Hole, Massachusetts 02543) The Ipswich River in northeastern Massachusetts has supplied surrounding suburban communities with water since the 1800s. With current projections of increased urbanization in the water- shed ( 1 ), solute fluxes from developed areas may have an impact on the ecology of the Ipswich River. Solute fluxes from storm flow are particularly important since storms commonly flush solutes from storage reservoirs, thereby increasing the mass transfer of solutes to the aquatic system (2). The objectives of this study were to observe solute dynamics in storm flow in three first-order catchments of the Ipswich River basin to infer how increased development will affect the aquatic system. The three catchments were selected to represent the end-mem- bers of different land-use areas commonly found in the Ipswich River basin. The catchments represent predominately urban (URB), agricultural (AG) and forested (FOR) areas. The baseline discharges were 100, 0.4 and 1 0 1/s at the URB, AG and FOR sites, respectively. Rain volume at each site was measured using manual rain gauges, and samples for chemical analyses were collected. 220 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Baseline samples of stream water were collected before and after a storm that occurred 15-16 June, 2000. Each hour during the storm, filtered and unrtltered samples of stream water were col- lected and stage measurements were taken. Discharge was esti- mated from stage measurements. Stream water and rain samples were filtered immediately with glass-fiber filters, stored on ice in the field, and refrigerated at the laboratory until analysis. All filtered samples were analyzed for NH4 colorimetrically, for Cl, NO,, and SO4 using ion chromatography, and for Na, K, Ca, and Mg by atomic absorption. Unfiltered samples were analyzed for acid neutralizing capacity (ANC) and pH. Total rainfall at the sites ranged from 19 to 46 mm. Maximum stage observed was 25, 12, and 3 cm above base flow at the URB, AG and FOR sites, respectively; maximum discharges were 400, 140, and 70 L/s. Solute concentrations in rain were similar among all sites and much lower than those found in stream water. Base flow values for all solutes were lower at the FOR site than at the other, more developed sites. Sodium and Cl concentrations were high at the URB site, probably because of salting roadways in winter months. Calcium concentrations were high in the AG site, perhaps due to the addition of lime to agricultural fields. During the storm, solute concentrations in stream water of the FOR site were relatively invariant compared to the URB and AG sites (Fig. la-c). Concentrations of NH4 and NO, increased at the beginning of the storm, and trends were similar at the AG and URB sites (Fig. la). Concentrations of other solutes at the URB and AG sites decreased with the onset of the storm (Figs. Ib, c). After the storm, solute concentrations at the AG site increased rapidly toward base flow values (Figs. Ib, c). Stream water discharge from the URB site was the highest of the three catchments, as was the net flux of solutes measured (Fig. Id). The variations in solute concentrations observed are primarily due to site-specific differences in the relative proportions of groundwater and overland flow inputs to the stream. The propor- tion of these inputs is commonly regulated by the type and amount of ground cover in a particular catchment. The lack of forest cover in agricultural areas and impermeable surfaces in urban settings increase overland flow inputs to streams during storms (3). In forested catchments, runoff is typically smaller than in more de- veloped catchments because soil and vegetation allow much of the precipitation to percolate slowly to the groundwater table. In contrast, soils in predominately agricultural catchments can be- come quickly saturated during storms, causing larger inputs of water to enter a stream in the form of overland flow and diluting solute concentrations. As a storm subsides, solute concentrations in stream water will typically return to base flow levels as the ratio of groundwater inputs to overland flow increases. Urban settings characteristically have large amounts of impervious ground cover preventing rain from percolating to groundwater reservoirs, thereby increasing the proportion of overland flow (urban runoff) to the stream. Hence, the large decreases in solute concentrations observed at the AG and URB sites during a storm are probably due to a larger overland flow component in these catchment streams. In contrast, the increases of NH4 and NO, concentrations at these sites must be due to strong sources of nitrogen in overland flow and groundwater at the beginning of a storm that may be linked to the application of fertilizers in developed settings. Our results show that there are marked differences in the solute 1a ~ 6000 5 Time(h) 10 15 & ~ 4000 - Q) T3 '§ 2000 c. 0 n • • • • --* i^ V *~^r On 0 5 10 Time (h) 15 I o 1C 5 Time(h) 10 15 | 3000 -, H 2000 - 1 1000 U. •K n 4 D Net Flux URB D Net Flux AG • Net Flux FOR [NH4] 1d [CIJ/10 Solute Figure 1. Concentrations of ammonium (A), chloride (Bl, and calcium (C) plotted against time. Relative fluxes of ammonium, chloride (divided by 10) and calcium tit the three stiu1\ sites (D). dynamics of storm flow among streams in areas characterized by different land uses. Because anthropogenic inputs of nitrogen are associated with the eutrophication of receiving waters, further study is required to determine the impact of increased NH4 and NO, export from urban and agricultural catchments on the aquatic ecology of the Ipswich River. Literature Cited I Pontius, R. G. Jr., L. Claessens, C. S. Hopkinson, Jr., A. Mar- y.ouk, K. B. Rastetter, L. C. Schneider, and J. Vallino. 2(1(10. In ECOLOGY. BIOGEOCHEMISTRY. AND POPULATION BIOLOGY Proceedings of the 4'1' International Conference on Integrating CIS and Environmental Mode/ing (GIS/EM4): Problems, Prospects anil Research Needs. Banff, Alberta. Canada. 2-8 September 2000. (In press). l. Wood, E. F., M. Sivapalan, and K. Beven. 1990. physics. 28: 1-18. 3. Jordan, T. E., D. L. Cornell, and D. E. Weller. 1997. Qual. 26: 836-.S4S. 221 Reviews of Gt-o- J. Environ. Reference: «;>>/. Bull. 199: 221-223. (October 2000) Fate of Anthropogenic Nitrogen in a Nearshore Cape Cod Aquifer Elizabeth J. Westgate1, Kevin D. Kroeger, Wendy J. Pabich, am/ Iran Valiela (Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Nitrogen loading from land is a principal cause of eutrophica- tion of shallow estuaries ( 1, 2. 3). In regions such as Cape Cod, Massachusetts, which are underlain by unconsolidated sands, the major mechanism that transports nitrogen to estuaries is ground- water flow, and the major nitrogen source (primarily in the form of nitrate. NO,) is often wastewater from septic systems (1, 2. 3). Wastewater nitrate concentrations decrease during travel in groundwater due to dilution with clean groundwater and to loss by denitrification (4). The loss of nitrogen during flow between a septic tank and receiving estuary can be calculated by determining the reduction in concentration of dissolved inorganic nitrogen relative to the change in concentration of a passive tracer that accounts for dilution. We investigated losses of nitrate for a domestic septic system in the watershed of Quashnet River, Cape Cod. Effluent from septic systems moves downgradient within plumes containing high con- centrations of nitrate. In addition, the study area has plumes derived from fertilized turf or fields. To sort out the different plumes, we measured boron (B, a passive tracer derived from laundry detergents and associated with wastewater sources [5, 6. 7]) and potassium (K, associated with both wastewater and fertil- izer sources [8, 9]) in the samples of groundwater. To calculate loss of nitrate along the plumes, we collected samples from nine wells downgradient from the septic system. Each well was furnished with 14 ports that allowed us to sample groundwater at intervals of 1-2 m. We collected 300 ml of water from 129 ports during June and July 2000 and measured concentrations of nitrate (NO, + NO2) and ammonium (NH4) using colorimetric and fluoro- metric techniques, respectively. We selected samples with nitrate concentrations above 8 \iM and conductivities less than 4.000 /j,S/cm for measurements of B and K. These samples were analyzed by Ward Laboratories (Kearney, NE). Examination of vertical and horizontal profiles of nitrate and am- monium suggested that there were three distinct plumes within our well field (Fig. 1 ). The upper plume moved along near the surface of the water table and contained the highest nitrate concentration of the three plumes; at nearly 3000 \j.M, it was similar to literature values (8) for septic effluent that has just left the leaching field. The nitrate, B. and K concentrations in this plume differed considerably from those of the other plumes (Fig. 2, A and B). In contrast, the lower plume showed no increase in nitrate relative to increase in B (Fig. 2, A). It did, however, show a positive relationship to K, and at a given K concentration had a 1 Lafayette College, Easton, PA 18042. much higher nitrate concentration than did the upper plume (Fig. 2, B ). This evidence suggests that the lower plume might be due to fertilizer use upgradient of our septic system. The middle plume had no significant relationships between nitrate and B or K, perhaps because of the small number of samples and the low concentrations. The concentrations of nitrate. B, and K from the middle plume do, however, fit on the lower portions of the curves for the upper plume (Fig. 2, A and B). These circumstances lead us to think that the middle plume was probably the leading edge of a plume from a septic system located farther upgradient from our septic system. We therefore used data for the upper and middle plumes in our examination of the fate of septic- system nitrogen in this watershed. Concentrations of nitrate and B diminished as water parcels aged (age. Fig. 2, C and D, calculated from Vogel equations [10] that predict time since recharge as a function of depth in aquifer). To allow for dilution, we normalized the data by expressing concentrations as NO3/B (Fig. 2. E). We estimated the NO,/B in the effluent that had just left the septic system (age 0) by using a literature value (8) (Fig. 2, E, upper dashed line). The NO,/B values we used came from a Cape Cod site near our study area, and the data dated from 1992. only a 7-8 year difference from our date of collection. We presume that differences in B were therefore a reasonable proxy for those in our study system. We calculated losses of NO, as the difference between the age 0 nitrate concen- tration, allowing for dilution, and the measured nitrate concentra- tion. Losses of nitrate in excess of dilution were quite rapid, with rates reaching 50% loss at 0.2 years (Fig. 2, F). The loss rates diminished with time, which suggests that, if these data are rep- resentative of losses elsewhere, N losses by denitrification and retention take place primarily near the septic system source. Ex- trapolating the curve of Figure 2 (F), we find that near-complete losses may be reached at 4.8 years, which is equivalent to 480-730 m from the septic system, assuming a travel rate of 100-150 m per year (11). As a minimum estimate of loss, we also calculated loss relative to our highest measured NO,/B ratio (Fig. 2, E, lower dashed line). If our initial NO,/B ratio were closer to this measured value, our estimate of time to 50% NO, loss would increase to 0.6 years; but the estimate of time to 100% loss was not affected. The extrapo- lation to 100% loss assumes that the relationship between percent loss NO, and age continues to hold beyond our oldest sample. This would not be the case if the availability of labile organic carbon were to limit NO, loss before 100% loss is achieved. 222 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 4- 0 £ > LU -8- Soil surfac 0 20 40 Distance from shore (m) . water table, and aauifer throin'h our field ofmiiltml, 60 Figure 1. Vertical cross section from the soil surface, water table, and aquifer through our field of multiple sampling wells (elevation relative to mean tow water [MLW]). The numbers are concentrations of NO, (fxMJ/oc water samples collected from each of the 14 ports in each of the 9 wells. Although the wells were not all in one plane, for simplicity they are shown as if they were. Contour lines are drawn to indicate NO, concentrations of 32, 128, 512, and 2048 ;uM. Position of salty water determined from salinity of water samples. — 3000-, 12000 - g 1000- 0.2 B (ppm) — 3000 - a 2000 - g 1000- Z 0 §-3000- ., B 52000- /9 o 1000 • Z 0- ^f • ddlt -9-^^"^^ 0 10 20 K (ppm) ~ 0.4-i I 0.3- 3 0.2- D • . CQ 0.1 - n . • • . • • • ••• CD 12000 - 8000 4000- 0 1 2 Age (y) E 1 Age (y) 120- -2 80- co i 40 H 0 1 Age (y) 1 Age (y) Figure 2. A: NO, concentration versus B concentration for samples collected from upper (•), middle • and lower (A) plumes. B: NO, concentration versus K concentration for all three plumes. C: NO, concentration versus age for upper and middle plumes. D: B concentration versus age for upper and middle plumes. E: NO, to B ratio versus age for upper and middle plumes. F: Percent loss of NO, versus age for upper and middle plumes. ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY 223 If coastal zone managers wish to regulate septic nitrogen loads, they could concentrate on management of septic systems that lie within 480-730 m of the shore, since these appeared to be the major contributors of nitrate to receiving estuaries. Septic sources farther upgradient probably contribute less significantly. This research was supported by an internship from the Woods Hole Marine Science Consortium to Elizabeth Westgate, an MIT Sea Grant (#65591) awarded to Ivan Valiela and Harold Hemond, and National Estuarine Research Reserve Fellowships to Kevin Kroeger and Wendy Pabich. Literature Cited 1 Valiela, I., et al. 1992. 2. Valiela, I., et al. 1997. Estuaries 15: 443-457. Eco/. Appl. 7: 358-380. 3. Valiela, I., el al. 200(1. Biogeochemistry 49: 277-293. 4. Wilhelm, S. R., S. L. Schiff, and J. A. Cherry. 1994. Ground Watt, 32: 905-91(1. 5. Barf:. S. 1998. Water Res. 32: 685-690. 6. LeBlanc, D. R. 1984. U.S. Geological Survey Water-Supply Paper 22IS 7. Barber, L. B. II, E. M. Thurman, M. P. Schroeder, and D. R. LeBlanc. 19X8. Environ. Sci. Technol. 22: 205-211. 8. DeSimone, L. A.. P. M. Barlow, and B. L. Howes. Open-File Rep.-U.S. Geol. Surv. 1995, No. 95-290. 9 Bjerg, P. L., and T. H. Christensen. 1992. ./. Hydrol. 131: 133- 149. 10. Vogel, J. C. 1967. Pp. 355-369 in Isotopes in Hydrology. Proceed- ings of IAEA-SM-83/24, Vienna. 11. LeBlanc, D. R., et al. 1991. Wat. Res. 27: 895-910. Reference: Biol. Bull., 199: 223-225. (October 2000) Effects of Nitrogen Load and Irradiance on Photosynthetic Pigment Concentrations in Cladophora vagabitnda and Gracilaria tikvahiae in Estuaries of Waquoit Bay Michelle Denault , Erica Stieve, and Ivan Valiela (Boston University Marine Program. Marine Biological Laboratory, Woods Hole, Massachusetts 02543) Two major controls of activity and standing crop in macroalgae are nitrogen supply and irradiance ( 1 ). Increased nitrogen loads increase production (2) and biomass of macroalgae such as Cladophora vagabitnda and Gracilaria tikvahiae (E. Stieve. un- pub. data). Lower light availability lowers growth rates of mac- roalgae, although this effect varies among species (3). Because of exponential attenuation and self shading within algal mats, the irradiance available for benthic algae depends on water depth ( 1 ). Supply as well as storage of nitrogen and photons affect the concentration of photosynthetic components in macroalgae (1,4). Photosynthetic pigments such as phycoerythrin also act as nitrogen pools, and macroalgae acclimate to different irradiance regimes by changing pigment concentrations (5). To examine the effects of different nutrient supplies on photosynthetic pigment concentrations in a green and a red alga, we collected samples of Cladophora vagabunda (L.) van den Hoek and Gracilaria tikvahiae McLachlan from five estu- aries within Waquoit Bay (Childs River. Eel River, Quashnet River, Sage Lot Pond, and Timms Pond) that are exposed to different nitrogen loads (6). To study the effect of irradiance on pigment concentration we collected samples at a range of depths (80 to 210 cm). Irradiance at each sampling depth was measured using a spherical underwater sensor attached to a Li-Cor DataLogger LI-1000. Samples were collected during one day in early June. At each site, six samples of benthic macroalgal material were collected and sorted to isolate fronds of C. vagabunda and G. tikvahiae. The samples were sorted by species. Chlorophyll a, b, and carotenoids were extracted as described by Figueroa et al. 1 Brown University, Providence. RI 02912. (7); phycobiliproteins were extracted as described by Beer and Eshel (8). Pigments were extracted within 36 h of collecting and were kept at 5°C until extraction to avoid pigment degradation. Concentrations of pigments were determined by use of a Perkin Elmer UV/VIS spectrophotometer (8. 9. 10). Chlorophyll a and carotenoid concentrations were measured in both C. vagabitnda and G. tik\'ahiae. Chlorophyll b concentrations were measured in C. vagabunda. Phycoerythrin concentrations were measured in G. tikvahiae. To further ascertain the internal storage of nitrogen and carbon under different nitrogen and irradiance regimes, we dried macroalgal samples and measured percent nitrogen and per- cent carbon in a Perkin Elmer elemental analyzer according to the manufacturer's instructions. Concentrations of chlorophyll a (Fig. 1A) and carotenoids (Fig 1 B ) in both species of macroalgae increased as nitrogen load to the estuaries increased. There was no consistent difference between upstream and downstream sites within the estuaries, and there was no apparent effect of different salinities at the sites of collection (range of 10%, and K. Lajtha. 1992. Extiiaricx 15: 443-457. Figueroa, F., S. Soluna, .1. Aguilera, C. Jimenez, J. Mercado, B. Vinegla, A. Flores-Mova, and M. Altamirano. 1997. Mm: Ecol. Prog. Ser. 151: 81-40. Beer, S., and A. Eshel. 1985. Aust. J. Mm: Fre.ilm: Rex. 36: 785-792. Jeffrey, S., and G. Humphrey. 1975. Biochcm. Phyxiol. Pftan:.. 167: 191-144. Dawes, C. 1998. Pp. 380-382 in Marine Botany . John Wiley. New York. Lapointe, B. 1985. ./. Exp. Mai: Bit,!. Ecol. 93: 21 1-222. Reference: Biol. Bull. 199: 225-226. (October 2000) Differences in Properties of Salt Marsh Sediment Between Hayed and Reference Sites Adena Greenhaum (Wellesley College) and Anne Giblin: The practice of haying salt marsh grasses began in colonial times. Early settlers began harvesting marsh grasses for fodder, and the practice has continued to the present ( I ). Current haying techniques remove more than 90% of aboveground plant biomass, and could have a number of effects on processes within the marsh. Salt marsh food webs are based on detritus, so it can be hypoth- esized that removal of plant biomass could alter food webs. Nu- trient cycles, benthic algal biomass. microbial processes, and spe- cies composition could also be affected (2, 3). This study examined the effect of detritus removal on several sediment prop- erties to assess the long-term effects of haying. Sediment cores were taken from Plum Island Sound intertidal marsh, a long-term ecological research site located in northeastern Massachusetts. To study the effects of detritus removal, we mea- sured several characteristics of sediment in areas where the marsh grass is hayed by commercial farmers. We compared the results to those of reference areas, which are not hayed. We sampled two areas that are hayed every other year, a practice the commercial farmers recognized produced the highest hay yield. Duplicate cores were taken from each of two hayed and refer- ence sites. All cores were taken from high marsh areas that were heavily dominated by Spartina patens. Measurements of the fol- lowing sediment characteristics were taken at 2 cm intervals above 10 cm and 5 cm intervals below 10 cm, to a depth of at least 25 cm. Bulk density, a measure of soil density, was expressed as ratio 1 Ecosystems Center, Marine Biological Laboratory, Woods Hole. MA. between the weight and volume of sediment; percent organic- matter was measured by loss of ignition; total sulfur was measured using a LECO sulfur analyzer; sedimentation rates were calculated using Pb profiles (4); and total phosphorus was measured using the technique of Krom and Berner (5). These properties were mea- sured to examine sediment composition. There was no significant difference in bulk density between surface sediments in the hayed and reference sites (Fig. 1). Bulk densities for both the hayed and reference areas decreased from a range of 0.37 to 0.34 g cm ' at the surface to 0.22 g cm""3 at 12.5 cm. Below this depth, there was a slight difference in bulk density values. The hayed areas increased to a maximum of 0.34 g cm ' at 23 cm. However, the bulk density of the reference sites re- mained around 0.23 g cm 3. Judging from surface values for bulk density, current haying practices apparently do not compact the sediments. Percent organic matter was similar between the managed and natural areas to a depth of 12.5 cm. Values ranged from 31.7% to 44.5% organic matter. Deeper sediment samples of reference plots had a slightly higher percent organic matter than the hayed sites, but the difference between them was not significant. This indicated that the removal of biomass from the hayed sites does not affect organic matter content. Haying did not appear to affect the total sulfur content in the sediment. In both hayed and reference areas, total sulfur in- creased from about 0.65% at the surface to a maximum of 2.2% between 17 and 22 cm, and then decreased to about 1.55% at a depth of 30 cm. 226 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS Bulk Density (g/cm ) 00 0.2 04 0.6 Organic Matter (%) 20 40 60 Total Sulfur (%) 1 2 5 - _ 10 • ~ 15 20 • 25 • 30 a 1 5 i _ 10 § c. 15 20 • 25 30 15 • 20 • 25 30 Pb (ug/g) 0 20 40 60 80 100 Total P (urrVg) 0 10 20 30 40 50 5- ~ 10 o S 15 D 20 25- 30 - - -A - - Reference Sites — | Hayed Sites Figure 1. Measures of bulk density, percent organic matter, total sulfur, sedimentation rate, and total phosphorus in ha\ed and reference sites of salt marsh sediment in Plum Island Estuary. Values between hayed and reference sites for percent total sulfur, along with bulk density and percent organic matter deviated slightly below 12.5 cm. This sediment was deposited more than 25 years ago, and since land practices before this time are unknown, it is difficult to hypothesize reasons for the deviation. Sedimentation rates can be estimated by using stable lead pro- files to approximate dates of deposition. The lead profiles for hayed and reference areas were not significantly different. Al- though the cores were not deep enough to reach pre-industrial background values, we used a previous study to establish back- ground levels (Schmitt. unpub. data). Using both sets of data, we calculated a sedimentation rate of 0.54 cm y~' for both the reference and the hayed areas. The lack of differentiation in sedimentation between the hayed and natural marsh is surprising given that the haying process removes a large portion of the aboveground biomass. One possible explanation is that most of the organic matter making up the peat comes from other sources. Belowground biomass could contribute significant amounts of organic matter to detritus, especially since a large percent of biomass in a salt marsh could be below ground. Organic matter washed in with the tide could settle as detritus as well. The profile for the hayed sites was more variable than that of the reference site. Perhaps the tractor and trailer used for haying disturbs the surface sediment as it travels over the marsh. There was a significant difference between total phosphorus measured in hayed and reference sites. The hayed areas had less total phosphorus than the reference sites from the surface until 22.5 cm deep in the sediment, where the values for the two areas converged. One explanation is that the input of phosphorus to the marshes of the area is very low. and the periodic removal of biomass from the system eventually leads to a measurable loss in phosphorus. Of the sediment properties we examined — bulk density, percent organic matter, total sulfur, sedimentation rate, and total phospho- rus— only the last was affected by harvesting the aboveground biomass every other year. Current haying practices on the marsh did not significantly alter most of the properties we measured. However, other processes in the marsh could be affected by the decrease in phosphorus, and other systems could respond differ- ently to comparable practices. This research was supported by the Plum Island Sound LTER and a Research Experience for Undergraduates NSF fellowship. Thanks to Linda Deegan, Hap Garritt, and Nat Weston for advice and assistance with sampling. Literature Cited 1 . Teal, J., and M. Teal. 1969. Lift- ami Death of the Suit Marsh. Pp. 2 1 , 183. 2. Knapp, A. K.. and T. R. Seastedt. 1986. Bioscience 36: 662-668. 3 Bouchard, V., and J. Lefeuvre. 2000. Aquat. Bot. 67: 23-42. 4. Ng, A., and C. Patterson. 1982. Geochem. Cosmochim. Ada 48: 2307-2321. 5 Kroni, M. D., and R. A. Berner. 1981. Geochem. Cosmochim. Acta 45: 207-216. ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY Reference: Bio/. Bull. 199: 227-228. (October 2000) 227 Population Genetic Structure of the Goosefish, Lophius ainericanus Hemant M. Chikarmane, Alan M. Ku-irian {Marine Biological Laboratory, Woods Hole, Massachusetts), Robbin Kozlowski1, Mark Kuzirian,2 and Tony Lee3 Lophius ainericanus Cuvier & Valenciennes 1837 (1). the goosefish, anglerfish, or monkfish, is common in coastal waters of the northeastern United States. Its geographic range extends from the northern Gulf of St. Lawrence south to Cape Hatteras, North Carolina (2, 3). The highest fish concentrations are found along the shallower depths of the shelf from 70 to 100 m, but there is also a significant deep-water population below 190 m. Adult fish migrate seasonally in response to spawning, food availability, and optimal temperatures (3°-9°C) (2). The species is also dispersed through the drifting of egg rafts. Total dispersal time from embryonic development through larval and juvenile stages can extend to several months until benthic recruitment occurs. Sexual maturity is reached between 3 and 4 years of age (3). Goosefish is the fourth largest commercial species in the U.S. fishery, and number one in demersal species landings. Goosefish landings have risen steeply through the 1980s, reaching approxi- mately 28,800 mt ($35 million) for 1997 (4). Since the 1980s, the Canadian contribution to the fishery has declined precipitously, and now the major landings occur in the southern regions of the species range. In their autumn survey data, the Northeast Fisheries Science Center, Woods Hole, Massachusetts, has documented recent sharp declines in goosefish abundance, from 2.24 kg/tow in 1986 to 0.74 kg/tow in 1996. The New England and Mid-Atlantic Fishery Management Councils (NEFMC and MAFMC) conse- quently designated goosefish as overexploited and at low abun- dance (5). The 23rd Stock Assessment Workshop at the Northeast Fisheries Science Center concluded that it was not possible to delineate the stock structure for goosefish because of the lack of genetic, tagging, or migration studies. Nevertheless, the Councils divided the coastal population into northern and southern stocks (41°N latitude) for stock management purposes. This formula led to fishing restrictions being placed geographically, and made cer- tain areas uneconomical to fish. Because of the lack of definitive stock data for goosefish (5), we undertook a population genetic- study of goosefish in eastern waters from the Canadian border to North Carolina. We used random amplification of polymorphic DNA and PCR (RAPD-PCR) (6) to analyze the genetic structure of the sampled populations. Eight representative sampling sites were chosen, extending from Maine (42°40' N, 68°20' W) to North Carolina (35°40', 75°00'), from depths to about 300 m. Fish were collected from September 1999 to June 2000. Up to 45 fish were sampled at each location. Tissue samples were collected in tissue preservation buffer (7). Genomic DNA was purified by standard phenol-chloroform pro- cedures, and was finally dissolved in Tris-EDTA (TE) buffer (8). DNA fingerprinting was performed by RAPD-PCR (6), using 10 1 Cape Cod Community College, W. Barnstable, MA. 2 University of Rhode Island, Kingston, RI. 3 Duke University. Durham, NC. /nl per reaction. Amplification products were separated by electro- phoresis on 1.2% agarose gels in 0.5x TBE (8). Gels were stained with ethidium bromide and photographed under UV light. The presence or absence of amplification products was scored manu- ally. Cluster analysis was performed with the RAPDistance pack- age (9). Six fish, three each from Georges Bank and New York/New Jersey sites were first screened with the seven primers shown in Table I. As expected, the number of amplification products per primer varied, ranging from 2 to 9. and very few bands were polymorphic (Table 1 1. On the basis of the initial screening, a subset of 6-8 DNA samples from each site was analyzed, using primers 101 and 103; Figure 1 shows data for primer 103. There appeared to be no significant differences between indi- viduals or between populations, with either primer. Polymorphic bands were present in a minority of individuals, usually one or two. A set of eight fish collected off Martha's Vineyard, Massa- chusetts, by the Marine Biological Laboratory, was examined with an additional set of primers (115, 119. 130. and 143). Again, the band distribution was very homogeneous (data not shown). Of the 22 identifiable bands produced by these primers, 21 were present at a frequency of 100%. Band 22 was present at a frequency of 58%. All the MBL samples were clustered as one group by the RAPDistance package. These results taken together imply that the fish populations are relatively homogeneous genetically across all geographic sampling sites, the level of polymorphism within pop- ulations being as low as that between populations. Fish caught at shallower (<200 m) depths could not be differentiated from those at lower (>200 m) depths, neither could those collected north or south of the 41°N line. For the primers tested, there was no amplification product (or the absence of one) that uniquely char- acterized a particular population. The trend in the data is clear even though only a subset of samples was analyzed with two primers. We are currently examining the entire sample set with more primers to reinforce the validity of our results. Table I RAPD primer sequences and polymorphic bands for Lophius americanus Primer Sequence Number of bands Polymorphic bands 101 GCGGCTGGAG 9 1 103 GTGACGCCGC 7 2 104 GGGCAATGAT 3 0 105 CTCGGGTGGG 8 1 106 CGTCTGCCCG 8 1 107 CTGTCCCTTT 2 2 108 GTATTGCCCT 7 1 The primer numbering system and sequence is from the University of British Columbia RAPD primer kits ( 10). 22X REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS GB NY/NJ NC ME Figure 1. Representative RAPD-PCR profile* with Primer 103 fur individual goosefish samples from indicated locations. GB-Georges Bank, NY/NJ-New York/New Jersey, NC-North Carolina. ME-Maine. The homogeneity of the goosefish populations off the eastern coastline of the United States suggests that there is unrestricted gene flow across the region. This is very plausible considering the preferred temperature profile and migratory patterns of the adults, and the long dispersal times of the embryos, larvae, and postlarval juveniles (2. 3). These data will have serious implications for management of the goosefish fishery. The study results run counter to the current NEFMC/MAFMC policy of dividing the fishery into northern and southern stocks. Any management plan will be dif- ficult to implement because the spawning stock biomass is un- known. More data is also needed to determine the location of the standing reproductive population, and to assemble specific tempo- ral data on when spawning occurs over the fish's geographic range. Integration of the published data on seasonal abundances (NMFS Spring/Autumn Bottom Trawl Surveys) with yearly temperature profiles along the coastlines might suggest some possible avenues to pursue these answers. Such data will assist in defining the natural and fishing mortality rates (F) and what the Flhreshold should realistically be for this commercially important species. This work was supported in part by the Monkfish Defense Fund. H.M.C. and A.M.K. are indebted to Kathy Downey of the MDF for acquainting them with the problems of the goosefish fishery, and for organizing the fishermen for sample collection. We thank the Aquatic Resources Division, MBL, for collecting some goosefish used in this study. Literature Cited Histoire Naturelle des 1. Cuvier, G., and A. Valenciennes. 1837. Poissons. 12. Bertrand. Paris. 2. Grosslein, M. D., and T. R. Azarovitz. 1982. MESA New York Bight Atlas Monograph. N.Y. Sea Grant Institute. Albany, NY. 3 Bigelow, H. B., and W. C. Schroeder. 1953. Fish. Bull. 74: 53. 4. NOAA technical memo. 1999. Our Living Oceans. Report on the Status of U.S. Living Marine Resources. U.S. Department of Com- merce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service. Washington, DC. 5. Idoine, J. 1998. Pp. 88-89 in Status of Fisher,' Resources off the Northeastern United Stales for /W,V. S. H. Clark, ed. U.S. Department of Commerce, National Marine Fisheries Service, Woods Hole, MA. f.. Williams. J. G. K., A. R. Kubelik. K. J. IJvak, J. A. Rafalski, and S. V. Tingey. 1990. Nucl. Acids Res. 18: 6531-6535. 7. Asahida. T., T. Kobayashi, K. Saitoh, and I. Nakayama. 1996. Fish. Sci. 62: 727-730. 8. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press. Cold Spring Harbor, NY. 9 Armstrong J. S., A. J. Gibbs, R. Peakall, and G. Weiller. 1994. The RAPDistance Package. ftp://life.anu.edu.au/pub/RAPDistance [21 Aug. 2000). K). Primer Kits. NAPS Unit, University of British Columbia Biotechnol- ogy Laboratory, http://www.biotech.ubc.ca/services/naps/primers.html [21 Aug. 2000]. PUBLISHED BY TITLE ONLY 229 Published by Title Only Bewley, Arnaud Variations in white and black oaks as a function of their distance from the coast. Chiao, Chuan-Chin, and Roger Hanlon Visual mechanism of body patterning in young cuttlefish, Sepia pharaonis. Dayel, Mark Analysis of keratocyte motility and fine structure. Haines, Jamie, Matthew Cieri, and Linda Deegan Food choice convergence of benthic and pelagic fishes along an estuarine gradient. Hinckley, Eve-Lyn From forest to shore: the path of total dissolved nitrogen in a pristine coastal system, Martha's Vineyard, Massa- chusetts. Horowitz, Julie, Linda Deegan, and Robert Garritt Stable isotope analysis of detrital food webs in hayed and imhayed salt marsh. Janowitz, Tobias, P. Koulen, and B. E. Ehrlich Characterization of ryanodine receptors from zebrafish skeletal muscle. Kreitzer, Matthew, Naomi Rosenkranz, and Robert Paul Malchow Effects of extracellular ATP on skate retinal horizontal cells. Schwartz, Jesse, and Les Kaufman Mechanistic mass-balance models and community ecol- ogy. Sergeant, Kara, Matthew Cieri, and Linda Deegan Bivalve grazing pressure on primary producers in a New England estuary. Silver, Robert B., and Steven A. Farber Calcium-independent perinuclear phospholipase A2 and leukotriene B4 regulate the calcium signal required for nuclear envelope breakdown in sand dollar (Echinarac- nius parma) cells. Wolf, Amelia, Craig Tobias, and Bruce Peterson Dissolved inorganic nitrogen uptake kinetics and relative preference indices for estuarine primary producers. 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Microscopy & Imaging Systems One Zeiss Drive Thornwood, NY 10594 800.233.2343 Fax 914.681. 7446 micro@zeiss.com www.zeiss.com ZEISS Volumi THE Number -> BIOLOGICAL BULLETIN DECEMBER 2000 Published I >\ I IK \l.u iuc I'm .1. ,-i« .il I .ibi >i .11. .1 \ Galileo, Hooke and Leeuwenhoek for the incentive. We walk in the footsteps of visionaries. And see with the eyes of disciples. J^tfm^ Galileo up close: astronomer, physicist, gig mathematician, dethroner of Aristotle, defender j of Copernicus and, yes, credited with making Jouble convex lenses and the first biological observations with a microscope. A compound microscope. In 1624. Hooke: 43 years later, the compound microscope reveals living things are composed of cells. Leeuwenhoek: invents the first practical microscope and discovers bacteria. Armed with the knowledge and inspiration of these and other Olympians, secure in the history of our own accomplishments and aware that the mind's eye should always focus on challenging the enigmatic as well as the undeniable, Olympus has created the BX2 series of clinical and research microscopes, noteworthy for their enhancements in optics, imaging and ergonomics. And each so individual, it deserves the nomenclature My Microscope. From possibility to actuality, it is wonderful to behold. A posse ad esse est mirabile visu. The BX41 The new standard fnn^ in laboratory f microscopy vis-a-vis ~ *£ „- " ' i performance, ver- satility, operation. Uncanny digitized imaging: enhanced with the DP11 digital camera, delivering high-resolution image recording beyond the norm. I nusually insightfu / optics: observe / magnifications from J / 1.25x to lOOx / without changing 1 / the condenser. / Choice of condenser / includes Abbe, swing / out, phase-contrast / and darkfiel Unerringly precise- motion stage: selectable for right/ left hand. D speed, ease, comfort, am accuracy. Uncommonly easy-to-operate controls: extended fine-focus knob; forwarded-positioned illumination intensity dial; tactile X-Y control. Welcome to the ne Series The new standard in ergonomic microscopy. A dramatic metamor- phosis: innova- <&3Si^ - live, intelligent, insightful design improvements specifically addressing the needs of those spending hours looking through a microscope. The lowest low-position stage: three inches lower than standard microscopes, offering the promise of minimum effort and hand movement when changing specimens. The tilting, telescoping observation tube: a marvel of adjustability, it can be changed to accommodate any 80mm higher and 70mm "oser to the operator than a convention- al tilting tube. And the eye- irward by 45mm 1 0-25 degrees. rfhe BX51 The new stan- \ • ./ dard in research ^j^^^ microscopy. ^ . The words "new" and "standard" really don't do it justice. How about "ingenious" or "original," "paragon" or "prototypical." Prepare yourself for a higher power. The 6-position filter cube turret: Not 4, not 5, but 6, so single and multi- band imaging of new fluorochromes and fluorescent proteins is faster, simpler and exclusively tailored. The fluorescence excitation bal- ancers: another Olympus exclusive. A continuously variable excitation bandwidth. Visualization is enhanced, differentiation of multi-labels is immeasurably improved, capturing of images is at an unheard-of level. with prevalent FITC and Cy3 with prevalent DAPI and Cy3 with balanced fluorescence emission wider mirror aspherical lens The recta ours alone. t5y cleverly matcning the camera's field of view, only the area requiring fluorescence excita- tion is exposed. Which means that the surrounding areas are protected /.) E. LInusual words in journal titles should be spelled out in full, rather than employing new abbreviations invented by the author. For example, use Rit Visi/ulcifje/ags Islendinga without abbreviation. F. All single word journal titles in full (e.g., Veliger, Ecol- ogy, Brain). G. The order of abbreviated components should be the same 6. Reprints, page proofs, and charges. Authors may pur- as the word order of the complete title (i.e.. Proc. and Trans. chase reprints in lots of 100. Forms for placing reprint orders are placed where they appear, not transposed as in some BIOLOGICAL sent with page proofs. Reprints normally will be delivered about 2 . ,. to 3 months after the issue date. Authors (or delegates for foreign ABSTRAC. rs iistiniis). authors | will receive page proofs of articles shortly before pubh- H. A few well-known international journals in their preferred cation. They will be charged the current cost of printers' time for forms rather than WORLD LIST or USAS1 usage (e.g.. Nature. corrections to these (other than corrections of printers' or editors' Science, Evolution NOT Nature, Land., Science, N.Y.; Evolution, errors). Other than these charges for authors' alterations. The Lancaster, Pa. ) Biological Bulletin does not have page charges. CONTENTS for Volume 199 No. 1 . AUGUST 2000 RESEARCH NOTE Bolton, Toby F., Florence I. M. Thomas, and Celere N. Leonard Maternal energy investment in eggs and jelly coats sur- rounding eggs of the echinoid Arbaria punctulata .... 1 PHYSIOLOGY Johnson, M. L., P. M. J. Shelton, E. Gaten, and P. J. Herring Relationship of dorsoventral eyeshine distributions id habitat depth and animal size in mesopelagic de- capods I' Dietz, T. H., A. S. Udoetok, J. S. Cherry, H. Silvernian, and R. A. Byrne Kidney function and sulfate uptake and loss in ihc freshwater bivalve Toxolasma texasensis 14 Vollmer, Steven V., and Peter J. Edmunds Allometric scaling in small colonies of the scleraiiin- ian coral Sitlrmslmi siderm (Ellis and Solander) .... 21 DEVELOPMENT AND REPRODUCTION Torrado, Mario, and Alexander T. Mikhailov Frog Lim-1-like protein is expressed predominantly in the nervous tissue, gonads, and early embryos of the bivalve mollusc M\tilit\ galloprovincialii 29 Gibson, Glenys D., and Jennifer M. L. Harvey Morphogenesis during asexual reproduction in I\- gosf>i ECOLOGY AND EVOLUTION Barnes, David K. A., and Mathew H. Dick Overgrowth competition between clades: implica- tions for interpretation of the fossil record and over- growth indices 85 41 Annual Report of the Marine Biological Laboratory. . Rl No. 2, OCTOBER 2000 RESEARCH NOTE PHYSIOLOGY Hourdez, Stephane, Jason Lamontagne, Pat Peterson, Roy E. Weber, and Charles R. Fisher Hemoglobin from a deep-sea hydrolhei mal-vent copepod 95 Marsh, Adam G., Patrick K.K. Leong, and Donal T. Manahan Gene expression and enzyme activities of the sodium pump during sea urchin development: implications for indices of physiological state 10(1 Hill, Richard W., John W.H. Dacey, and Ahser Edward Dimethylsulfoniopropionate in giant clams (Tridac- nidae) 108 CONTENTS: VOLUME 199 ECOLOGY AND EVOLUTION Baker, Shirley M., Jeffrey S. Levinton, and J. Evan Ward Particle transport in the zebra mussel, Dreissena puly- morpha (Pallas) 116 Lajeunesse, T.C., and R.K. Trench Biogeography of two species of Symhindinium (Freudenthal) inhabiting the intertidal sea anemone Aiithopltura elrgniittwmii (Branch) 12h NEUROBIOLOGY AND BEHAVIOR Rosendial, Joshua J.C., and Francisco Bezanilla Seasonal variation in conduction velocity of action potentials in squid giant axon 135 DEVELOPMENT AND REPRODUCTION Misamore, M.J., and J.W. Lynn Role of the cvtoskeleton in sperm entry during fertilization in the freshwater bivalve Drmsena polymorpha ... 144 SHORT REPORTS FROM THE 2000 GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY Hitt, James M., Frederick A. Dodge, Ehud Kaplan, and Robert B. Barlow C.ircadian rhythms in the receptive fields of the Litnu- ln\ lateral eye 171 Fay, Richard R., and Peggy L. Edds-Walton Frequency response of auditory brainstem units in toadfish (O(u>anuf ttiu) 173 Yamagnchi, Ayako, Leonard K. Kaczmarek, and Darcy B. Kelley Intrinsic membrane properties of laryngeal 1110- toneurons that control sexually differentiated vocal behavior in African clawed frogs, Xenopiu laevis .... 175 Atherton, Jillian L., Matthew A. Krutky, James M. Hitt, Frederick A. Dodge, and Robert B. Barlow Optic nerve responses of Limului in its natural habi- tat at night 176 Krutky, Matthew A., Jillian L. Atherton, Spence Smith, Frederick A. Dodge, and Robert B. Barlow Do the properties of underwater lighting influence the visually guided behavior of Limulm? 178 Hale, Melina E. Startle responses of fish without Mauthner neurons: escape behavior of the Inmpfish (Cydopterus lii>njni\) ISO Epstein, David A., Herman T. Epstein, Frank M. Child, and Alan M. Kuzirian Memory consolidation in Hermissenda crassicornis . . . 182 Abenavoli, A., L. Forti. and A. Malgaroli Mechanisms of spontaneous miniature activity at CA3-CA1 synapses: evidence for a divergence from a random Poisson process 184 /•/..I I'I'RKU i,} A\/> BIOCHEMISTRY The Editors Introduction to the featured report. On mapping odor quality' 161 Wachowiak, Matt, Michal Zochowski, Lawrence B. Cohen, and Chun X. Falk The spatial representation of odors by olfactory re- ceptor neuron input to the olfactory bulb is concen- tration invariant 162 NEUROBIOLOGI Landowne, David Heavy water (D.,O) alters the sodium channel gating current in squid giant axons 164 White, Thomas H., Harris Ripps, Midiituru Srinivas, and Roberto Bruzzone Voltage gating properties of channels formed by a skate retinal connexin 165 Molina, Anthony J.A., Peter J.S. Smith, and Robert Paul Malchow Hydrogen ion fluxes from isolated retinal horizontal cells: modulation bv glutamate 168 Wang, Jing W. Odor-induced oscillatoiy activity in l)i»\npliill,>;n,n 1*9 Asokan, Rengasamy, Margaret T. Armstrong, and Peter B. Armstrong Association of a.,-macroglobulin with the coagulin clot in (he American horseshoe crab, I.iimilin pnlyplii'iinn. a potential role in stabilization Irom pro- teolvsis 190 Kuhns, William J., Max M. Burger, Mohan Sarker, Xavier Fernandez-Busquets, and Tracy Simpson Enzymatic biosynthesis of N-linked glycan bv the' ma- rine sponge M/inii/inir/ /nnli/mi 192 Armstrong, Peter B., and Rengasamy Asokan A Ca4 "'-independent cytolytic system from the blood of the marine snail liii\\n»i canaliculum 194 Heck, Diane E., Lydia Louis, Michael A. Gallo, and Jeffrey D. Laskin Modulalion <>l the development of plutei bv nitric oxide in the1 sea urchin .\ilnmn /niiirliilnln 195 CONTENTS: VOLUME 199 Jung, Sung-Kwon, Katherine Hammar, and Peter J.S. Smith Development of self-referencing oxygen microsensor and its application to single pancreatic HIT cells: effects of adenylate cyclase acdvator forskolin on ox- ygen consumption Hanselmann, Rhea, Roxanna Smolowitz, and Daniel G. Gibson Identification of proliferating cells in hard clams. . . Brothers, Christine, Ernest Marks III, and Roxanna Smolowitz Conditions affecting the growth and zoosporulation of the protistan parasite QPX in culture 199 200 Burgos, Mario H., Makoto Goda, and Shinya Inoue Fertilization-induced changes in the fine structure of stratified Arbariti eggs. II. Observations with electron microscopy 213 Gould, Robert M., Concetta M. Freund, John Engler, and Milan G. Morrison Optimization of homogenization conditions used to isolate mRNAs in processes of myelinating oligoden- drocytes 9)5 ECOLOGY, BIOGEOCHEMISTRY, A\D POPULATION BIOLOGY CELL BIOLOGY Sandberg, Leslie, Phillip Stafford, and George M. Langford Effects of myosin-II antibody on actin-dependent ves- icle transport in extracts of clam oocytes 202 Stafford, Phillip, Jeremiah Broun, and George M. Langford Interaction of actin- and microtubule-based motors in squid axoplasm probed with antibodies to myosin V and kinesin 203 Tran, P.T., V. Doye, F. Chang, and S. Inoue Microtubule-dependent nuclear positioning and nu- clear-dependent septum positioning in the fission yeast, Saccharomyces pombe 205 Crawford. Karen The role of microtubules during blastodisc forma- tion of the squid Loligo pealei 207 Weidner, Earl Cytoplasmic proteins on the surface of discharged microsporidian sporoplasms 208 Mackenzie, Roger, David Newman, Max M. Burger, Rene Roy, and William J. Kiihns Adhesion of a viral envelope protein to a non-self- binding domain of the aggregation factor in the marine sponge Mnrorionn fm>lifii~ri 209 Goda, Makoto, Mario H. Burgos, and Shinya Inoue Fertilization-induced changes in the fine structure of stratified Arbacia eggs. I. Observations on live cells with the centrifuge polarizing microscope 212 Kirkby, Ryan, Luc Claessens, Charles Hopkinson, Jr., Edward Rastetter, and Joseph Vallino Modeling the effects of land-use change on nitrogen biogeochemistry in the Ipswich watershed, Massachu- setts 218 Perring, Anne. Michael Williams, Charles Hopkinson, Jr., Edward Rastetter, and Joseph Vallino Solute dynamics in storm flow of the Ipswich River Basin: effects of land use 219 Westgate, Elizabeth J., Kevin D. Kroeger. Wendy J. Pabich, and Ivan Valiela Fate of anthropogenic nitrogen in a nearshore Cape Cod aquifer 221 Denault, Michelle. Erica Stieve. and Ivan Valiela Effects of nitrogen load and irradiance on photosyn- thetic pigment concentrations in Cladophora vaga- lni>idaa.nd Gmcilaria tikvahiaem estuaries of Waquoit Bav 223 Greenbaum, Adena, and Anne Giblin Differences in properties of salt marsh sediment be- tween hayed and reference sites 225 Chikarmane, Hemant M., Alan M. Kuzirian, Robbin Kozlowski, Mark Kuzirian, and Tony Lee Population genetic structure of the goosefish, Lo- pluus amrricanus 227 OIUL PRESENTATIONS Published bv tide onlv. . . No. 3, DECEMBER 2000 EVOLUTION NEUROBIOLOGY AND BEHAVIOR Rinkevich, B. A critical approach to the definition of Darwinian units of selection 931 Leise. Esther M., and Michael G. Hadfield An inducer of molluscan metamorphosis transforms activity patterns in a larval nervous system 241 Finley, Luke, and David Macmillan The structure and growth of the statocyst in the Australian crayfish Cherax destructor 251 CONTENTS: VOLUME 199 Jury, Steven H., and Winsor H. Watson III Thermosensitivity of the lobster, Homarus americanus, as determined bv cardiac assay 257 Cromarty, S. I., J. Mello, and G. Kass-Simon Molt-related and size-dependent differences in the escape response and post-threat behavior of the American lobster, Homarus americanus 265 CELL BIOLOGY Baghdasarian, Garen, and Leonard Muscatine Preferential expulsion of dividing algal cells as a mech- anism for regulating algal-cnidarian symbiosis 273 DEVELOPMENT AND REPRODUCTION Vickery, Minako S., and James B. McClintock Effects of food concentration and availability on the incidence of cloning in planktotrophic larvae of the sea star Pisaster ochraceus 298 Dickinson, Amanda J. G., Roger P. Croll, and Elena E. Voronezhskaya Development of embryonic cells containing seroto- nin, catecholamines, and FMRFamide-related pep- tides in Aplysia californica 305 RESEARCH NOTE Takeda, Naokuni Development of a penis from the vestigial penis in the female apple snail, Pomacea canaliculata 316 Kominami, Tetsuya, and Hiromi Takata Cellular basis of gastruladon in the sand dollar Scaphechmus mirabilis 287 Index for Volume 199 321 xii ERRATA The Biological Bulletin Volume 199, Number 2 In the article by P. T. Tran, V. Doye, F. Chang, and S. Inoue, which appeared on pages 205 to 206, the genus name Saccharomyces is in error. The correct genus name, in all instances, is Schizosacchromyces. Cover legend: The organism described as Saccharomyces pornbe should be Schizosacchromyces pombe. The editors regret these errors. Notice to Subscribers 2001 SUBSCRIPTION RATES FOR THE BIOLOGICAL BULLETIN Per year (six issues, two volumes): Per volume (three issues): Back and single issues: (subject to availability) Libraries $235.00 $117.50 $ 40.00 Individuals $95.00 $47.50 $20.00 For additional information, please contact our subscription manager at the Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543; tel: (508) 289-7402; e-mail: cmarrama® mbl.edu. Reference: Biol. Bull. 199: 231-240. (December 2000) A Critical Approach to the Definition of Darwinian Units of Selection B. RINKEVICH Israel Oceanographic and Limnological Research, National Institute of Oceanography, P.O. Box H030. Haifa 31080, Israel Abstract. What are the biological units of selection? In fact, the notion of "unit of selection" (UOS) is blurred by ambiguity and controversy. To further evaluate the biolog- ical entities that are the objects of natural selection, three novel conceptual criteria (holism, minimalism, functional- ism) are critically applied: they reveal, in addition to the self-evident case of the "individual," at least six distinct types of UOSs. These UOSs do not always have a defined structural organization; they can be parts of a living organ- ism, a cohesive group of conspecifics, a multiunit entity, a totipotent cell, a DNA fragment, or a whole organism. UOS types diversify by amalgamation or parcelation processes of apparent entities. Therefore, previous attempts to character- ize the UOSs solely on some morphological levels (gene, individual, group) without applying stringent criteria have failed to cope with the structural variations of natural phe- nomena and have led to the ambiguity of terms used. Introduction Much of the ambiguity, confusion, and controversy en- gendered by the concept of the "unit of selection" (UOS) seem to arise from a failure to identify the biological entities upon which natural selection operates (Sober and Wilson, 1994; Mayr. 1997; Gould and Lloyd, 1999, and literature therein). Along with the debates about the three to four possible organizational levels of selection (gene, individual, group, and metapopulation). the objections to the hierarchi- cal theory of selection (Wilson and Sober, 1994; Michod, 1997; Gould. 1998; Gould and Lloyd. 1999), and the dis- tinction between transmitted units and those which transmit (Wynne Edwards, 1962; Lewontin. 1970; Mayr. 1970. 1997: Dawkins. 1976; Hull. 1980; Gliddon and Gouyon, Received 17 August 1999; accepted 11 September 2000. E-mail: buki@ocean.org.il 1989; Sober and Wilson. 1994; Wilson and Sober. 1994; Williams. 1996; Gould. 1998). "metaphors have replaced the empirical world as foci for discussion while precise meanings and derivatives have been forgotten in the pro- cess" (Slobodkin. 1986). Even the basic term "unit of se- lection" is under dispute (Wilson and Sober, 1994), bearing polemic aspects (Mayr, 1997) as do other terms in this discipline (Gould and Lloyd, 1999). One approach to clarifying such an ambiguous field is a critical evaluation of the arguments and definitions used (Hull, 1980; Sober and Wilson. 1994; Mayr, 1997; Gould and Lloyd. 1999). Such a reevaluation process might ger- minate a novel idea or might help dispel excessive ambigu- ity. On the other hand, anathematized concepts could reap- pear, revealing further ill-considered definitions (Gould and Lloyd, 1999) or adding additional ambiguities. An alterna- tive approach is to envisage the main controversial issues through an untraditional analysis. In this essay, such an untraditional approach is used to examine the biological entities that are the objects of natural selection. By adapting the unbiased principle that any living thing can be all or part of a potential UOS, we can critically evaluate organisms — regardless of their level of morphological organization — on the basis of a few conceptual criteria. Criteria for Analysis of UOSs Three conceptual criteria guide this examination: Holism Genes and soma are not necessarily independent. The distinction between the terms "interactor" and "vehicle," as opposed to "replicator" and "gene" (Dawkins, 1976; Hull, 1980) is central in the debate over UOSs (Hull. 1980; Sober and Wilson. 1994; Mayr. 1997). The use of these terms to 231 232 B. RINKEVICH identify different units of selection evolved from the a priori rationale that living organisms are made of at least two distinct types of evolutionarily selected units. Addition- ally, the notion of the UOS has become ambiguous because it was used to refer to either replicators or vehicles, depend- ing on the choice of the author (Wilson and Sober, 1994). I suggest that this rationale is false and misleading, that it artificially distinguishes between "genes," "information," and "replication" on the one hand, and "soma," "vehicle." and "interactor" on the other (Lewontin. 1970; Dawkins, 1976: Hull. 1980; Buss, 1982; Gliddon and Gouyon, 1989; Sober and Wilson. 1994; Mayr. 1997). The genes in any organism have a fate in common with their amalgamated soma (Sober and Wilson, 1994, and literature therein). They are part of a whole; they are not completely independent (with the exception of specific cases as outlined in the next section), but rather functionally integrated within the soma. In physics, light and mass are regarded as two facets of energetic matter. Similarly, in biology, genes and soma should be regarded as two facets of an organic entity that constitutes a living organism. Even the term "unit" (Oxford Dictionary) embraces this metaphysical concept of holism. A unit is a thing (individual, person, group, etc.) that is complete or distinctive and that has the characteristics of the complex whole. Following this rationale, the so-called rep- licators and interactors of each entity are intermingled to form, tor each UOS, its idiographic (its own peculiar) entity, which is presented to natural selection as a coherent whole. This is in contrast to the acknowledgement of recent years that interactors, not replicators, constitute the causal unit of selection" (Gould and Lloyd. 1999). Minimalism Ignore complex cases; choose the simplest ones. Addi- tional ambiguity is caused by different hypotheses for the UOS that deliver opposing predictions about the traits that have evolved (Sober and Wilson, 1994; Wilson and Sober. 1994). In such cases, a search for the simplest manifestation of the system, the minimalist approach (Slobodkin, 1986), has been suggested to be the most useful in maintaining clarity. This approach has been characterized as "the pro- cess of deliberately choosing to work in the simplest pos- sible mode that is still recognizable as part of an existing professional field." Slobodkin ( 1986) has also discussed the main objection against this approach as the claim for un- critical acceptance of standards. However, this objection may not be the case in the controversy over the UOS, where metaphors, rather than empirical themes, dominate the sci- entific discipline (Wilson and Sober. 1994; Gould and Lloyd, 1999). When employing the minimalist approach (Slobodkin, 1986), or the very similar "back to basics" (Sober and Wilson, 1994) treatment, complex cases (such as the situations illustrated in Wynne Edwards, 1962) are left aside for future analyses when the field will presumably be more formally organized. Therefore, we must accept the idea that the UOS theory, almost three decades after it was first elaborated (Lewontin, 1970), should still be conceptu- alized through the clearest examples. Functionalism UOSs function in vivo. A unit of biological organization upon which selection might act should be both an autono- mous functional entity and physically and structurally co- herent, even if it is in the form of a gene. It cannot be in the form of "information" or "avatar" (Gliddon and Gouyon, 1989: Tuonii and Vuorisalo. 1989a) or "anything in the universe of which copies are made" (Dawkins, 1989). A UOS must function, because functionalism is the primary focus of natural selection. Functionalism, therefore, does not rest upon an active maintenance of distinctive properties (Gould, 1998), but evaluates the general sum of independent activities presented by a UOS. At this point, the existence of only a single functional level or of several functional levels (in hierarchical order. Tuomi and Vuorisalo, 1989a: Gould and Lloyd, 1999; or not) will not be discussed. Only a holistic unit (possessing cohesive structural and information properties) may reveal the capacity for functionalism. Therefore, previously distinguished UOSs such as replica- tors, interactors. vehicles, memes. etc.. that are literally not holistic, are excluded from being real UOSs. They remain as highly justified theoretical paradigms that characterize only components of holistic and functional units of selection. The three conceptual criteria (holism, minimalism, func- tionalism) provide enormous flexibility for analysis and circumvent the use of ill-defined issues and debatable argu- ments. These criteria have been used to scrutinize different potential types of UOSs that are presented by a variety of organismal entities. The term "organism" refers here to "any biological entity whose parts have evolved to function in a harmonious and coordinated fashion" (Wilson and Sober, 1994). This analysis has revealed several types of UOSs; of these, one traditional and six new characteristic types (Table 1. Nos. 1-7) are briefly described below. Seven Types of Units of Selection / inn — n/hl /n/rt of me ;'.v // Molecular sequences may themselves be UOSs. "Doctor there is a fly in my genome" was the title chosen by the journal New Scientist (Vol. 149, p. 16, 1996) for an article about a tiny fragment of an insect genome (called mariner. a jumping gene first discovered in the fruit fly Dro.wphila) that is embedded in human chromosome 17. This location directly coincides with a recombination hot spot and has been associated with distinct hereditary neurological syn- dromes (Reiter et ed organisms, monozygotic twins. ripedia anisms mous number of documentations that eukaryotic and pro- karyotic cells carry foreign DNA molecules of various types (plasmids, B chromosomes, t haplotypes. retroviruses, and more), as well as diverse mobile DNA sequences (such as transposons, retrotransposons, LINEs, SINEs. mobile in- trons) that are transmitted vertically or horizontally within genomes (Zeyl and Bell, 1996; Flavell. 1999) and may be regarded as real UOSs. These DNA sequences have func- tional and holistic properties: they are characteri/.ed by a discrete organismal realm, function in a coordinated fash- ion, and are clearly subject to natural selection forces. Many examples now point to real UOSs situated within the ge- nomes of other UOSs. A few will be outlined below. One well-studied group is the B-chromosomes. a variety of germ-line parasites described from more than a thousand species of plants and animals. These small chromosomes do not contribute to the regular functions of the host, and their numbers per cell vary even within the same host organism. More important, although they share the same nucleus with regular chromosomes, they have evolved peculiar charac- teristics of their own. By various non-Mendelian systems of biased transmission and by their ability to move specifically to one of the two products of the first meiosis division (such as by avoiding penetration into the polar body during oogenesis), they increase their representation in the germ- line nuclei. The B-chromosomes in the wasp Nasonia. which are transmitted solely through sperm, are a represen- tative case. The entire parental set of chromosomes in an infected zygote becomes condensed and is lost, leaving a haploidized animal that develops as a male, transmitting the B-chromosome to all its gametes (citations in Bell and Burt, 1990). Such functionalism of the parasitic entity reveals distinct host and parasitic units of selection. Within this context, I am reluctant to consider the B-chromosomes as selfish chromosomes. They are distinct molecular UOSs. The mouse t haplotypes (each extending over the proxi- mal half of chromosome 17) also have developed the ability to propagate at the expense of the wild-type homolog from heterozygous males. These entities probably evolved from a wild-type form of chromosome 17. Genes that were re- cruited later on, together with the addition of accompanying inversions, all increased the survival rates of the t haplo- types, until finally these entities started "taking on a life of their own" (reviewed in Silver, 1993). Not only a whole piece of chromosome may be counted as a UOS; even transposable genetic elements, gene size segments of DNA. may be so considered. This field is too broad to be even partially covered here, so only the most relevant features of these mobile elements will be discussed. Many transposable elements have the ability to jump from place to place on the chromosomes; they can behave like new introns creating novel intron processing patterns; they may spread vertically and horizontally within host organ- isms; and they can promote their own replication (the func- tionalism component). With time, the mobile elements be- come domesticated through full integration into the host's genome. A good example is the mariner which, by being functional in both germ lines and somatic cell lines, could infect many organisms, crossing several phyletic borders (arthropods, platyhelminths, nematodes, chordates), proba- bly by splicing into viral or other pathogenic genomes. During each introduction into a new host species, the mar- ine'/- transposon was probably highly mobile and signifi- cantly disruptive. With time, more and more defecting transposons with mutations that disabled the cut-and-paste enzyme were accumulated, littering eukaryotic genomes 232 identify different units of priori rationale that living two distinct types of evolutic ally, the notion of the UOS it was used to refer to either ing on the choice of the autl suggest that this rationale artificially distinguishes be and "replication" on the on and "interactor" on the otl 1976; Hull. 1980; Buss, 19! Sober and Wilson, 1994; I> organism have a fate in cc soma (Sober and Wilson, IS are part of a whole; they ; (with the exception of speci section), but rather function In physics, light and mass energetic matter. Similarly, .„ ,,_,, ^ „ should be regarded as two facets of an organic entity that constitutes a living organism. Even the term "unit" (Oxford Dictionary) embraces this metaphysical concept of holism. A unit is a thing (individual, person, group, etc.) that is complete or distinctive and that has the characteristics of the complex whole. Following this rationale, the so-called rep- licators and interactors of each entity are intermingled to form, for each UOS, its idiographic (its own peculiar) entity, which is presented to natural selection as a coherent whole. This is in contrast to the acknowledgement of recent years that interactors, not replicators, constitute the causal unit of selection" (Gould and Lloyd, 1999). Minimalism Ignore complex cases; choose the simplest ones. Addi- tional ambiguity is caused by different hypotheses for the UOS that deliver opposing predictions about the traits that have evolved (Sober and Wilson, 1994: Wilson and Sober. 1994). In such cases, a search for the simplest manifestation of the system, the minimalist approach (Slobodkin, 1986), has been suggested to be the most useful in maintaining clarity. This approach has been characterized as "the pro- cess of deliberately choosing to work in the simplest pos- sible mode that is still recognizable as part of an existing professional field." Slobodkin (1986) has also discussed the main objection against this approach as the claim tor un- critical acceptance of standards. However, this objection may not be the case in the controversy over the UOS, where metaphors, rather than empirical themes, dominate the sci- entific discipline (Wilson and Sober. 1994; Gould and Lloyd. 1999). When employing the minimalist approach (Slobodkin. 1986). or the very similar "hack to basics" (Sober and Wilson, 1994) treatment, complex cases (such as the situations illustrated in Wynne Edwards, 1962) are left activities presented by a UOS. At this point, the existence of only a single functional level or of several functional levels (in hierarchical order. Tuomi and Vuorisalo, 1989a; Gould and Lloyd. 1999; or not) will not be discussed. Only a holistic unit (possessing cohesive structural and information properties) may reveal the capacity for functionalism. Therefore, previously distinguished UOSs such as replica- tors, interactors. vehicles, memes. etc.. that are literally not holistic, are excluded from being real UOSs. They remain as highly justified theoretical paradigms that characterize only components of holistic and functional units of selection. The three conceptual criteria (holism, minimalism, func- tionalism) provide enormous flexibility for analysis and circumvent the use of ill-defined issues and debatable argu- ments. These criteria have been used to scrutinize different potential types of UOSs that are presented by a variety of organismal entities. 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Oliver and Boyd, Edinburgh. Zeyl, C., and G. Bell. 1996. Symbiotic DNA in eukaryotic genomes. Trends Ecol. Evol. 11: 10-15. Reference: Bio/. Bull. 199: 241-250. (December 2000) An Inducer of Molluscan Metamorphosis Transforms Activity Patterns in a Larval Nervous System ESTHER M. LEISE1-2'* AND MICHAEL G. HADFIELD1 lKewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Street, Honolulu. Hawaii 96813: and Department of Biology. University of North Carolina Greensboro, Greensboro, North Carolina 27402-6 J 74 Abstract. Larvae of the nudibranch mollusc Phestilla si- bogae metamorphose in response to a small organic com- pound released into seawater by their adult prey, the scler- actinian coral Porites compressa. The transformations that occur during metamorphosis, including loss of the ciliated velum (swimming organ), evacuation of the shell, and bodily elongation, are thought to be controlled by a combi- nation of neuronal and neuroendocrine activities. Activation of peripheral chemosensory neurons by the metamorphosis- inducing compound should therefore elicit changes within the central nervous system. We used extracellular recording techniques in an attempt to detect responses of neurons within the larval central ganglia to seawater conditioned by P. compressa, to seawater conditioned by the weakly induc- tive coral Pocillopora damicomis. and to non-inductive seawater controls. The activity patterns within the nervous systems of semi-intact larvae changed in response to both types of coral exudates. Changes took place in two size classes of action potentials, one of which is known to be associated with velar ciliary arrests. Introduction For a number of molluscan larvae, specific chemical compounds from the juvenile environment can act as che- mosensory cues and trigger metamorphosis. For example, inductive compounds may be given off by the adult prey Received 18 January 2000; accepted 7 September 2000. * To whom correspondence should be addressed. Present address: De- partment of Biology. University of North Carolina Greensboro, Greens- boro, NC 27402-6174. E-mail: Esther_Leise@uncg.edu Abbreviations: ASW. artificial seawater; CAS, ciliary arrest spike; CNS, central nervous system; FSW. 0.2-jM.m-filtered natural seawater; ISW. Pon'to-conditioned seawater; PSW. PonV/o/wra-conditioned seawater: SU. smaller units. (Hadfield and Karlson. 1969; Hadfield. 1977, 1978; Chia and Koss, 1978, 1988; Lambert and Todd, 1994; Avila et al, 1996; Lambert e tal., 1997), by adult conspecifics (Pech- enik, 1980: McGee and Targett. 1989; Pechenik and Gee, 1993), by bacteria associated with adult conspecifics (Fitt et al.. 1990; Tamburri et al., 1992), and by the algal food of the juveniles (Scheltema, 1961; Kriegstein et al., 1974; Switzer-Dunlap and Hadfield, 1977; Morse et a!., 1979; Levantine and Bonar, 1986; Morse, 1990; Boettcher and Targett, 1996; Leise et al.. 1996). In gastropods, sensory neurons that may mediate the induction of settlement and metamorphosis occur on the head, between the ciliated velar lobes (Bonar, 1978; Chia and Koss, 1982, 1984; Wodicka and Morse. 1991; Baxter and Morse, 1992; Uthe. 1995; Marois and Carew, 1997; Kempf et al.. 1997), and on the foot (Chia and Koss, 1989). Our understanding of how these neurons function is still limited. Observations of Morse and colleagues (Trapido-Rosenthal and Morse, 1985; Baxter and Morse, 1987, 1992; Morse, 1990; Wodicka and Morse, 1991 ) strongly imply that receptors for lysine, an amino acid that modifies inducer reception, lie on chemosensory cilia in the apical sensory organ of larval abalone. If pre-competent nudibranch and abalone larvae are exposed to an inducer substance, they display habituation — that is, decreased rates of metamorphosis — when they reach competency (Hadfield, 1980; Hadfield and Scheuer, 1985; Trapido-Rosenthal and Morse. 1986; Avila et al., 1996). Habituation is thus a phenomenon associated with the morphogenetic pathway that directly initiates metamorphosis. More recent studies are beginning to elucidate further internal mechanisms that are downstream from the chemo- sensory processes. These include changes in gene expres- sion (Degnan and Morse. 1993, 1995; Degnan et al.. 1997), protein synthesis, and second messenger levels (Inestrosa et 241 242 E. M. LEISE AND M. G. HADFIELD al.. 1993). Although the cellular circuitry that actually drives metamorphosis is still unknown, recent pharmaco- logical studies have revealed some attributes of this path- way. Serotonin, which occurs widely in larval molluscan nervous systems (Goldberg and Kater. 1989; Marois and Carew, 1997: Kempf et til., 1997). apparently acts as a neurotransmitter or neuromodulator that promotes meta- morphosis in the mud snail Ilytnuissa obsolete! (Couper and Leise, 1996). The neurotransmitter dopamine appears to be necessary for metamorphorphosis in the nudibranch Phes- tilla sibogae and the slipper limpet Crepidula fornicata, whereas norepinephrine may endogenously inhibit this pro- cess in Crepidula (Pires ct til., 1996, 2000). Nitric oxide appears to be yet another endogenous inhibitor of metamor- phosis, as shown by studies on Ilytnuissa (Froggett and Leise, 1999). Yet, even with these recent advances, we still have much to learn about the integrative mechanisms that follow the reception of chemosensory information to pro- duce, ultimately, a juvenile organism. Electrophysiological studies conducted on a variety of molluscan veligers have also provided some insight into their neural activities. Rapid and coordinated velum-wide ciliary arrests are driven by action potentials in the ciliated cells of the preoral band (Mackie et til., 1976; Arkett et til., 1987), and ramp depolarizations can slow ciliary beating on a more localized level (Arkett et al., 1987). Thus, metachro- nal beating appears to be controlled by the relative depo- larization of the ciliated cells and is modulated by excitatory neuronal input, presumably from the brain ganglia (Carter, 1926; Mackie et til., 1976; Arkett et til., 1987). These mechanisms are likely to be involved in the cessation of ciliary beating that accompanies larval settlement and crawling, behaviors that often precede metamorphosis. Bar- low (1990) demonstrated that the ciliated velar cells in abalone larvae change their spiking activity only as an indirect response to the presence of the inducer substance. They do not act as sensory receptor cells. Arkett et al. (1989) recorded depolarizing receptor potentials from sen- sory neurons in nudibranch larvae in response to a settle- ment-inducing substance, although the use of cobalt anes- thetic in their experiments limits the conclusions that can be drawn from their electrophysiological traces. Larvae of sev- eral molluscan species can be induced to metamorphose by an increase in external potassium ion concentration (Baloun and Morse, 1984; Yool et til., 1986; Pechenik and Heyman, 1987; Todd et til., 1991 ; Inestrosa et til.. 1992; Pechenik and Gee, 1993), a classical method for depolarizing nerve cells (Nicholls et al.. 1992), which again suggests that the pe- ripheral nervous system, the larval central nervous system (CNS), or both are active during the initial phases of meta- morphosis. If so, changes in the activity of central neurons, as well as in peripheral sensory receptors, should be detect- able as they respond to a natural inducing substance. The full range of metamorphic phenomena will most likely be controlled by neuroendocrine products as well as by classical synaptic interactions (Scheltema, 1974; Schacher et al., 1979). but molluscan metamorphosis in- cludes at least two relatively rapid events that may be under direct neuronal control. These are loss of the velum, a process common to all molluscan veliger larvae, and shell dehiscence, which occurs in many opisthobranchs (Bonar and Hadtield. 1974; Hadfield, 1978). These events, in ad- dition to the chemosensory initiation of metamorphosis, could involve neuronal networks within the CNS that drive appropriate effector organs. Indeed, Hadtield (1978) sum- marized data in support of the hypothesis that the nervous system was the most likely and sufficient regulatory system underlying all facets of metamorphosis in molluscs. To learn more about the role played by the nervous system during the metamorphosis of marine invertebrates, we used larvae of a nudibranch mollusc, Phestillti sibogae, to study the response of the CNS to a natural metamorpho- sis-inducing compound. The scleractinian coral Porites compressa is the major prey for adult P. sibogae in Hawaii. A small organic compound that is a natural exudate from live P. compressa induces metamorphosis in developmen- tally competent larvae (Hadfield and Karlson, 1969; Had- field, 1977; Hadfield and Pennington, 1990). Our extracel- lular recordings from the exposed dorsal surface of the brain ganglia provide evidence that activity patterns in the CNS change in the presence of the coral extract. We propose that the electrical changes we observed are associated with the initiation of metamorphosis, and that some of them are specific responses to larval exposure to P. compressa. Materials and Methods Veliger larvae of the nudibranch Phestillti sibogae Bergh were cultured in the laboratory in 0.2-/xm-filtered natural seawater (FSW) using previously described methods (Miller and Hadfield, 1986; Pires and Hadfield. 1991). During ini- tial experiments, insufficient electrical activity was recorded from the epidermal surfaces of intact larvae, so we used an //; vitro reduced preparation to maximize our ability to record spiking activity. To facilitate access to the larval brain, larvae without shells were used in all electrophysio- logical experiments. Deshelled larvae settle and metamor- phose normally, although they do not undergo shell dehis- cence (Pennington and Hadfield, 1989). Larval shells were decalcified by culturing about 100 larvae in a slender dish in 30 ml of artificial seawater (ASW) (Cavanaugh, 1956) lack- ing the usual 2.14 X 10~3 M sodium bicarbonate and buffered instead with 0.01 M Tris to pH 7.0 (Pires and Hadfield. 1993). Nine-day-old larvae were kept in ASW overnight so that metamorphically competent, shell-less, 10-day-old larvae were available as experimental subjects. About 70% of larvae cultured in this fashion had no shells 14 h after immersion. Deshelled larvae were rinsed in six ACTIVITY CHANGES IN A LARVAL CNS 243 changes of FSW over the following 2 h to reacclimate them to normal seawater (pH 8.3) before experimentation began. Isolated larval heads (Fig. 1) were produced by chilling 20-25 individuals in FSW in a small petri dish in an ice water bath. Larvae became immobile as the FSW tempera- ture approached 0°C. Small knives made from broken razor blades (Pires and Hadfield. 1993) were used to remove the visceral mass and foot from these cold, anesthetized larvae. This cut (line A in Fig. 1 A) exposed the dorsal surface of the brain for extracellular recording, although it may have also eliminated pan of the pedal ganglia. The eyes and statocysts remained in this isolated head preparation. We also conducted experiments on animals from which only the visceral mass was removed (head-foot prepara- tions). Results were similar, but we have chosen to leave those data unreported because fewer controls were con- ducted. Initial activity patterns in all experiments were B Figure 1. (A) Drawing of a deshelled larva (after Rasmussen, 1444) showing approximate location of the cut used to remove the visceral mass and foot from the head. Grey area represents approximate extent of the brain. The upper lobe containing the eyespot is likely to be a fusion product of the cerebral and pleural ganglia and may also contain elements of the parietal and buccal ganglia (Tardy. 1970). The region below the statocyst corresponds to the pedal ganglion. (B) Isolated head on the end of a suction electrode. The micropipette tip shown here is smaller than that typically used for recording purposes, to make the head more visible. The left eyespot is at arrow; right eyespot is visible through the transparent neural tissue within the open tip of the electrode. Velum is at arrowhead. For recording purposes, micropipettes were sized appropriately so that the entire cut surface could be contained by the electrode. >' 1 7X somewhat varied (Fig. 3A. C. E, G). so data from different dissected veliger heads were not pooled. Immediately after being cut, the chilled, isolated heads were transferred to fresh FSW at room temperature, where- upon they recovered normal metachronal beating of the velar cilia. Electrical recordings were made with a fire- polished glass micropipette suction electrode with an inner tip diameter of 40 to 50 ju.ni. The suction electrode was appressed to ihe exposed dorsal surface of the brain and gentle suction was applied to maintain contact between the electrode and the larval tissue. Larvae were exposed to one of three experimental solu- tions: FSW. FSW containing the natural metamotphosis- inducing compound produced by Porites compressa Dana (ISW), or a similar exudate from the relatively non-induc- tive coral Pocilloponi Jciiiucornis (PSW). PSW induces less than 30% metamorphosis compared to 90% induced by ISW (Hadfield. 1977). Adult P. siho<>ae do not use Pocilloponi as prey (Hadfield, 1977). ISW and PSW were prepared by placing about 22 g of living coral into 250 ml of aerated seawater in a covered beaker. Coral tips were used to maximize the ratio of living tissue to skeleton. The coral was removed after 48 h and the resulting conditioned sea- water passed through a 1.2-/j,m filter. ISW and PSW were stored in the refrigerator and used within 48 h of production. Freshly made ISW normally induces more than 92% of 10-day-old intact larvae to metamorphose within 24 h. If the coral showed signs of ill health during preparation of ISW or PSW, the coral and solutions were discarded. Assays for the metamorphosis-inducing capabilities of ISW and PSW were compared to FSW controls and conducted with intact larvae as previously described (Pennington and Hadfield, 1989). Assays were examined at 24 and 48 h and scored for number of larvae, juveniles, and empty shells. We also tested 34 isolated heads for their ability to metamorphose. These heads were cultured under sterile conditions for 48 h as previously described (Pires and Hadfield. 1993). then examined for loss of ciliated velar cells. Electrophysiological data were recorded for 5-10 mm before and after the addition of experimental solutions. The decision to expose each head to control or experimental solutions was made before recordings were initiated. Ex- periments were conducted in 35 X 10 mm plastic petri dishes in about 6 ml of FSW. Changes to bath solutions were made manually: 4 ml of the bath solution were ex- changed four times over the course of 1-3 min. during which time recording continued. Solution changes some- times introduced mechanical artifacts, so results are re- ported for spiking activity occurring after solution changes were complete. Changes in spiking activity typically began 2-3 min after solutions first contacted the larval head. Data were collected from a new isolated head for each experi- ment, amplified through a differential AC amplifier (A-M Systems, Inc.), and recorded in digital format on videocas- 244 E. M. LEISE AND M. G. HADFIELD 5s Figure 2. Representative trace from an isolated head in FSW. Large spikes are truncated and correlate with spontaneous velar ciliary arrest. No stimulus was used to elicit these large ciliary arrest spikes. Bottom trace is manually controlled cue (event marker) on the PCM data recorder. Cue was depressed, yielding an upward deflection, whenever spontaneous velar cilia were observed to cease beating. Audio monitor was turned off to avoid biasing the observer. Cessation of ciliary beating coincides with the largest spikes. sette tape through an Instrutech VR-100 PCM (pulse code modulation) device. This device has a manually operated event marker, or "cue" switch. When depressed, a positive 2.5-V deflection from ground is recorded on a separate channel on the videotape. Data were played back directly onto a Western Graphtek thermal chart recorder or. alterna- tively, collected on a 486 Insight computer and analyzed with the Enhanced Graphics Acquisition and Analysis (EGAA) software programs, ver. 3.50.02 (RC Electronics, Goleta, CA). Action potentials of different magnitudes were identified and counted using the EGAA Waveshape Recog- nition program, which stores start and stop times in digital data tiles. As necessary, files were converted to standard ASCII text format and analyzed further with Microsoft Excel 97 (Microsoft Corp.). Traces with relatively few spikes were analyzed directly from chart recorder records or the EGAA display screens. Two-sample analyses (two- tailed ; tests) were conducted with Statgraphics Plus ver. 7.1 (Manugistics, Inc.. Rockville, MD) or GB-STAT 6.0 (Dy- namic Microsystems. Silver Spring. MD). Results were graphed with DeltaGraph 4.0 (SPSS, San Francisco, CA). Results Extracellular recordings from the dorsal surfaces of brains in isolated heads of competent veliger larvae dis- played two general sizes of spiking units in FSW (Fig. 2). Continuous recordings were made while the preparations were exposed to the various experimental solutions. The largest spikes, between 200 and 500 /xV, were associated with partial or velum-wide ciliary arrests that occurred spontaneously in all preparations (Figs. 2. 3; Mackie ct til.. 1976; Arkett ct til., 1987). No stimulation was needed to elicit this activity. Initial patterns of activity in FSW were varied, but we recorded spontaneous ciliary arrest spikes (CASs) in all preparations (Fig. 3A. C. E. G). CAS activity typically occurred tonically. as relatively regular trains of single action potentials at 1 Hz or less. Spikes from smaller Figure 3. Representative 64-s traces, taken about 3 min before (A. C. E. G) and I mm after (B. D. F, H) addition of experimental solutions, demonstrate induced changes in spiking activity. Traces A. C. E, and G. under the heading "before." all illustrate activity in tillered seawater (FSW). Trace B. a sham experiment, shows activity after the addition of FSW. Traces D and F show activity in seawater conditioned by the presence of the inductive coral l'i>rnc\ . .™/>n -w/ (ISW), while trace H shows activity after the addition of seawater conditioned by the presence of /Ji>c///<>/>»™ iltiniicunux (PSW). In all traces, most velar ciliary arrest spikes (CASs) are truncated and were maximally 200 ^V in C and D and 500 ,uV in all oilier traces. Traces A and B from Expt. 90-60b, iraces C and D from expt. 41-21, traces E and F from e\pt. 40-M. (races G and H from e\pt. 41-22. (A) Note relative lack of activity in small units (SU). (B) Addition of FSW did not significanlly change ihe firing rates of CASs when averaged over 5 min (]|r| = 0.80] < f,, „.„,,.„ = 2.31 ). Low activity levels in SUs were likewise unaffected (Figs. 4A. 5A|. (C) Note the variable firing patterns of SUs in FSW. (D) Addition of ISW larrowl significantly increased activity of SUs ([\t = 3.14] > r.,,,5,,,.,4 = 2.15, Fig. 5B). but did nol affect aclmty of \elar arrest spikes (Fig. 4B|. (E) Note variable firing pattern of SUs. (F) In this experiment, addition of ISW did not significantly change activity in large or small units (Figs. 4B. 5B), but produced a qualitative change in the firing paltern of CASs. We recorded short bursts of 2 — t spikes during ihe 10 min after ISW addition. Longer hursts, with spike frequencies at or above I H/ (asterisks I. coincided with a contraction ot the velar lobes and cessation ol ciliary beating. (G) Note variable firing patterns of SUs. (H) Addition of PSW again produced no significant changes in average number of spikes/minute in large or small units, but induced an increased variability m the firing pattern of CASs (Figs. 4C, 5C). ACTIVITY CHANGES IN A LARVAL CNS 245 before FSW 50 10s ISW 20 10s after B ISW 50 10s PSW 50 n 10s * * H 246 E. M. LEISE AND M. G. HADFIELD units (20-100 jiiV) also occurred spontaneously, but with less regularity (Fig. 3 A, C, E, G). Ciliary arrest was often accompanied by a contraction of the entire velar lobe; during prolonged arrest periods the cilia and velar tissue were held in an upright position. At CAS frequencies below 1 Hz. velar cilia resumed beating between arrest spikes (Figs. 2; 3A, B). During spiking activity at frequencies above 1 Hz. cilia remained relatively motionless (Fig. 3F). We compared firing rates of CASs and the smaller units (SUs) before and after addition of experimental and control solutions to 13 isolated heads. In one experiment, addition of FSW elicited statistically significant changes in firing frequencies of both CASs and small spikes (Figs. 4A, 5A). In the remaining two experiments, as expected, no statisti- cally significant differences were seen in spiking activity after the addition of FSW (Figs. 3 A. B; 4A; 5A). In contrast to larval heads that were exposed to FSW. those exposed to ISW exhibited some type of statistically significant change in firing pattern, in either CASs, SUs, or both, in 6 of 7 experiments (Figs. 4B, 5B). In only one experiment. #90-61 (Fig. 3E. F), did we fail to observe any statistically significant differences in spiking activity in response to ISW. However, in this experiment, after the addition of ISW, CASs tended to occur in short bursts of 2-4 spikes (Fig. 3F). Short bursts of spikes elicited longer periods of ciliary arrest than did single CASs, and were often accompanied by contractions of the velar lobes. We observed similar results from preparations with an intact foot on several occasions (data not shown). In 4 of the 7 experiments, addition of ISW elicited a significant decrease in the frequency of CASs (Fig. 4B) and a change in the spiking activity of SUs (Fig. 5B). The addition of PSW to isolated heads elicited no statis- tically significant changes in firing rates (Figs. 4C, 5C), but in all cases, PSW elicited a qualitative change in CAS activity. With PSW. the firing pattern of the CASs became irregular (Fig. 3H). which accounted for the significant increase in variance that occurred in all experiments (Fig. 4C). No such increase in variance was detected for the firing rates of small spikes. Finally, we tested 34 isolated heads for their ability to metamorphose. The results were equivocal: four (12%) lost velar cilia, suggesting that isolated heads may be able to detect and respond to ISW. depending, perhaps, upon the amount of intact central nervous tissue. Because a large proportion (56%) died within 48 h, we cannot make a definitive conclusion about the metamorphic capabilities of isolated heads. Discussion Metamorphosis in the nudibranch Pliestillu siho^ue is triggered by a chemosensory event, namely, the perception by a competent larva of a small organic compound given off by its adult prey, the coral Porites compressa (Hadfield and Scheuer, 1985; Hadfield and Pennington, 1990). In 6 of 7 experiments, we recorded statistically significant changes in electrical activity from in vitro heads of larval P. sibogae shortly after the addition of a metamorphic inducer. In 3 of the 4 experiments in which spiking activity in small units changed, activity increased. In 4 of the 7 experiments with ISW. firing rates of velar ciliary arrest spikes decreased. Although we did not record consistent responses from all preparations, it is clear that long-lasting changes in electri- cal activity are initiated within minutes of initial exposure to the coral inducer. Competent larvae of P. sibogae display a rapid behav- ioral response to ISW that can be reliably observed under laboratory conditions (Koehl and Hadfield. unpubl. obs.). These larvae, which are negatively buoyant, stop swimming and rapidly sink when encountering ISW (Hadfield, unpubl. data). In the field, such a response would increase the chances of a larva contacting its adult food source. External signs of metamorphosis occur only 18-20 h after larvae have been exposed to an inducer substance for at least 4-6 h (Hadfield. 1977; Hadfield and Pennington, 1990). During this delay period, crucial physiological transformations and biochemical pathways must be activated as a prelude to the more obvious morphological transformations of metamor- phosis. The reduced preparation that we used may have produced neural activity different from that which occurs in an intact organism. The isolated heads retained most of the brain ganglia as well as intact velar lobes, eyespots, and stato- cysts. However, central circuits may have been damaged by a loss of gangliunic tissue, resulting in decreased connec- tivity and insufficient afferent information. This in turn may have led to unusual patterns of activity. Because we are reporting results from a relatively small number of experi- ments with a limited number of controls, we cannot fully explain the variability in endogenous activity, nor the vari- ability in our results. The responses to Porites compressa that we recorded in four experiments would lead to an increase in larval sinking, but not to a complete cessation of ciliary beating, as seen in the behavioral responses men- tioned above. This suggests that the isolated heads are not responding in a completely normal fashion. Larval Phestilla can apparently differentiate between their adult prey and at least one other coral species in their reef habitat. In addition to positive metamorphic responses, negative responses to unfavorable or even potentially lethal juvenile environments have been reported for other inver- tebrates, including several polychaete species (Woodin, 1986. 1991; Woodin el al., 1993; Walters el ai, 1996), bryozoan larvae (Walters et al.. 1996), and veligers of the gastropod ll\anassa obsoleta (Leise et al.. 1996). The abil- ity of Plu'stilla larvae to respond differentially to species of ACTIVITY CHANGES IN A LARVAL CNS 247 Porites and Poci/lo/xirn is thus not without precedent. How many coral species these small larvae can distinguish re- mains to he investigated. Beat frequency of the velar cilia is modulated hy excita- tory neural input in veliger larvae of the snails Mangelia nchiila (Mackie ct ai. 1976) and Callioswnm ligutiim (Ar- kett et til.. 1987) and the abalone Haliotis rufescenx (Bar- low. 1990). Velum-wide ciliary aiTests are caused hy an action potential that propagates throughout the velar ciliated cells. The large action potentials we recorded were always D. w 60 40 20 0 FSW n=6 B Q. CO 60 40 20 0 90-60a 90-60b 90-6 1a Experiment number r n=7 I 60 tfl 40 20 f ra 0) 0 90-58 90-60d 90-61 91-05 91-11 91-18 91-21 Experiment number PSW 91-02 91-19 91-22 Experiment number Figure 4. Mean number of velar ciliary arrest spikes recorded per minute before and after addition of experimental solutions. ± one standard deviation. Asterisks (*) indicate mean firing rate is significantly different from initial conditions (P < 0.05) after addition of control or experimen- tal solution. Experiments 90-60 (a. b) incorporated different isolated heads. Means were averaged from 10 min of continuous recordings whenever possible. Exceptions are noted on graphs as n = .\ number of minutes. (A) In one experiment, addition of FSW elicited a significantly slower rate of firing of CASs ([|f | = 4.85] > r,,,,<,:,.,: = 2.18). (B) Addition of ISW elicited a significant decrease in the firing rate of CASs by 40% or more in 4 ol the 7 experiments (e.g.. expt 91-18. [|r| = 4.16] > /„„..;, 2 >.,„ = 2.10). (C) No change in mean number of arrest spikes per minute was recorded from isolated heads after addition of PSW (e.g., expt 91-19, [|r| = 1.96] < /„ = 2.30). However, addition of PSW elicited a significant increase in the variance in all experiments (e.g.. expt 91-22, [F = 11.4] > Fo.05,2,.9.9 = 4-03). FSW • before D after 90-60a B 90-60b Experiment number ISW 1 600 0> 1 400 CO i 200 OJ Mean Spikes/Mir 60- 40- 20- 0- 90-58 90-60d 90-61 91-05 91-11 91-18 91-21 Experiment number PSW n=5 91-02 91-19 91-22 Experiment number Figure 5. Mean number of spikes per min recorded from smaller units before and after addition of experimental solutions, ± standard deviation. Means calculated from 10 min before and after addition of experimental solutions, except as indicated on graph (H = A number of minutes). Asterisks (*) indicate that mean hring rates before and after addition of experimental solution were significantly different. (A) Addition of FSW in one experiment elicited a significant increase in the number ot SUs ([\t\ = 4.16] > rn - 2.10). (B) Activity levels of SUs were highly variable both before and after addition of ISW. Firing rate of SUs increased significantly after addition of ISW in three experiments {e.g., expt 91-21. [|f = 3.13] > ',,05,1, ,4 = 2.15). but decreased in one experiment. (C) Addition of PSW elicited no change in tiring rates of SUs. Variances were similar in all of these experiments, both before and after PSW addition (cf. Fig. 4C). associated with ciliary arrests and were smaller than, hut similar to. the signals recorded from the velum of Mangelia and Calliostomti (Mackie ct til., 1976: Arkett et ai. 1987). The exact origin of the large spikes in Phestilla is unclear: they may be the propagated action potentials of the ciliated cells, or a combination of these spikes plus the summed output of central activity that drives ciliary arrests. In her work with larval abalone. Barlow (1990) found that expo- sure to an inducer substance increased the likelihood and duration of ciliary arrests. In our experiments, we mostly observed a decrease in firing frequency of the CASs, which 248 E. M. LEISE AND M. G. HADFIELD would lead to fewer, not more, ciliary arrests. Only the qualitative change to short bursts ot'CASs, as seen in some experiments (e.g., #90-61) would lead to longer ciliary arrests. The behavioral relevance of the spiking activity in the smaller-sized units is unknown. We do not know if their activity arises from circuits that detect environmental odor- ants or drive motor activities, such as crawling or changes in swimming speed or direction. As elicited by ISW. the bursts of smaller action potentials are irregular, unlike bursts from any of the well-known molluscan motor systems (e.g.. Get- ting and Dekin, 1985) or recently described olfactory cir- cuits (Gelperin and Tank, 1990; Gelperin ft at., 1993, 1996; Laurent and Davidowitz, 1994; Laurent et ai, 1996; Delaney et ai, 1994). Activity in the smaller larval units was also quite variable, with firing rates ranging from a few spikes per minute to hundreds per minute. We have no explanation for such variability, beyond suggesting that the amount of SU activity may reflect the amount of tissue lost during dissection. We also have no explanation for the increase in SU activity seen in one control experiment (Fig. 5 A). Extracellular recordings from distal stumps of either the rhinophoral or oral-tentacle nerves of adult P. sibogae display changes in firing activity of small units in response to Porites compressa that are similar to the changes we record from SUs in response to ISW ( Boudko and Hadricld, unpubl. data). We can only speculate that the SUs recorded from larval P. sibogae might indicate olfactory activity. The high mortality rate that occurred in experiments on the metamorphic capabilities of isolated heads does not allow us to make a definitive statement about their ability to metamorphose. Isolated velar lobes do not metamorphose — that is, they retain their ciliated velar cells in the presence of ISW — but such lobes lack the neural apparatus that can respond to a metamorphic inducer (Pires and Hadfield, 1993). Although our results support the idea that larval perception of an inducer substance depends upon peripheral chemosensory neurons and central processing circuitry, an additional caveat is warranted. Suction electrodes do not provide a tight seal against passage of fluid between the bathing medium and the core of the electrode. Thus, in our experiments, ISW in the bath seawater could have been interacting directly with neurons of the CNS as well as with epidermal sensory neurons. Thus, the neural activity we recorded in response to ISW may or may not duplicate neural activity occurring within intact larvae at the initiation of metamorphosis. 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Biol. 126: 383-393. Wodicka, L. M., and D. E. Morse. 1991. cDNA sequences reveal mRNAs for two G« signal transducing proteins from larval cilia. Biol. Hull. 180: 3I.S-327. \Voodin. S. A. 1986. Settlement of infauna: larval choice'1 Bull. Mm: Sci. .V): 401-407. \\oodin, S. A. 1991. Recruitment of infauna: positive or negative cues? Am. Zool. 31: 797-807. \\oodin, S. A., R. L. Marinelli. and D. E. Lincoln. 1993. Alleloehemi- cal inhibition of recruitment in a sedimentary assemblage. J. Chcin. Ecol. 19: 517-530. Yool, A. J., S. M. Grau, M. G. Hadtield. R. A. Jensen, D. A. Markell. and I). E. Morse. 1986. Excess potassium induces larval metamor- phosis in four marine invertebrate species. Biol. Bull. 170: 255-266. Reference: Bio/. Bull. 1W: 251-256. (December 2000) The Structure and Growth of the Statocyst in the Australian Crayfish Cherax destructor LUKE FINLEY AND DAVID MACMILLAN* Department of Zoolog\, University of Melbourne, Pcirkville, Victoria 3052, Australia Abstract. The morphology of the statocyst of the Austra- lian crayfish Cherax destructor was examined using scan- ning electron microscopy. It resembles in general structure, size, and position the statocysts of crayfish described pre- viously, and the size and distribution of the fields of setae on the floor of the capsule are similar but not the same. Over the size range examined, the relationship between the car- apace length, the length of the basal antennular segment, the diameter of the statocyst capsule, and the total number of setae are all linear. The number and position of setae on the floor of the statocyst capsule were mapped for animals in two size classes (small, ca. 20 mm; large, ca. 50 mm) to test for changes in their arrangement during growth. The change in the ratio of setal number to statocyst size between the two size classes was about three times greater for the anterior setal field than for the other fields. We propose that differ- ential development of the setal fields may be related to changes in the force-monitoring requirements of the animals as they increase in size, but this remains to be experimen- tally tested. Introduction Many decapod crustaceans have paired equilibrium or- gans called statocysts in the basal segment of each anten- nule. Statocysts monitor spatial orientation and movement (Cohen, 1955; Schone and Neil. 1977; Sekiguchi and Tera- zawa, 1997). Each statocyst is a sac-like epidermal invagi- nation of cuticle with a number of mechanosensory setae inside, mainly on the ventral floor. These are typically associated with a dense mass of sand, the statolith. The setae can be adjacent to the statolith and free to move, adjacent and touching, or cemented to the sand grains of the statolith. Received 14 October 1999; accepted 31 August 2000. * To whom correspondence should be addressed. d.macmillan@zoology.unimelb.edu.au E-mail: When the statolith deflects a seta it stimulates the neurons innervating it, and setae can differ in their physiological responses to stimulation (Cohen. 1955. 1960; Breithaupt and Tautz. 1988; Gate and Roye, 1997). The position and movement of the animal determine the pattern of setal stimulation, which in turn determines the form of compen- satory movements made by the appendages and body (San- deman and Okajima. 1972; Schone and Neil. 1977; Patton and Grove, 1992b). The morphology and spatial arrangement of setae within the statocyst vary between species (Cohen, 1955; Kovalev and Kharkeevich, 1993: Sekiguchi and Terazawa, 1997). and it has been suggested that groups of features may be associated with higher taxonomic groupings (Sekiguchi and Terazawa, 1997). In the statocyst of the crayfish Orconectes limosits, Hertwig et al. ( 1991 ) identified four separate fields of innervated setae: a lateral group of two semicircles, an approximately fusiform medial group with its axis roughly parallel to the long axis of the statocyst, and a single row of proximal setae. These setae appeared to be morphologically identical internally, but they differed in length and diameter in different parts of the field. Whether they differ in their physiological responses has not been tested. Although both structure and function of crustacean sta- tocysts are well understood, their growth has not been described as it has for other cuticular sensors on the crayfish and lobster tailfan (Letourneau. 1976; Schmitz. 1992: Stuart and Macmillan. 1997) and other appendages (Sandeman and Sandeman. 1996; Macmillan et al.. 1998; Steullet et al., 2000). Growth in crustaceans occurs by periodic shedding of the cuticle, a process known as ecdysis. or molting, the body increasing in size with each molt. As the body grows, the sensory representation from the integument may need to change to maintain appropriate sensory input and function. As new sensory structures can only be added to the cuticle when the animal molts, a comparison of sensory structures 251 252 L. FINLEY AND D. MACMILLAN in sequential molt stages reveals the order in which ele- ments develop. Because of their accessibility, setae on the telson have been the subject of a number of developmental studies based on this principle. Letourneau (19761 found. for example, that the order of addition of sensory setae to the telson of Procambarus clarkii is a function of the growth of the animal. Schmit/. 1 1992) described four func- tionally distinct setal types that are added at different rates. "Short smooth hairs" and "guard hairs" increase rapidly in number throughout development, whereas the number of two types of "feathered hydrodynamic hairs" remains rela- tively constant. We describe here the basic structure of the statocyst in the Australian crayfish C. destructor, and the relationship be- tween body size, basal antennal segment size, and statocyst capsule size over the size range of animals examined. We report the first data on the pattern of addition of setae within the capsule as the animal grows by comparing the statocysts from small and larse individuals. large foreign particles, effectively forming a closed capsule (Fig. IB). The cavity itself is oval and slightly pointed posteriorly (Figs. 1C. 2 A). The ventral floor of the cavity has an oval depression (Fig. 2 A, B). and setae project dorsally through the cuticle adjacent to this. A statolith composed of fused sand grains sits in the depression (Fig. 20. Relationships between size of animal and size of antennule and ca\'it\ The length of the basal segment of the antennule corre- lates closely with the carapace length (n -- 39; R2 0.971 1; P < 0.001; Fig. 3A), so we were able to collect data on both body size and statocyst parameters from scan- ning micrographs of the local area. The length of the stato- cyst capsule increases linearly as a function of the size of the basal segment of the antennule (n = 26, R~ ~ 0.9546: P < 0.001: Fie. 3A) and hence of the size of the animal. Materials and Methods Individuals of Che rax destructor were obtained from a commercial hatchery at Bendigo, Victoria, Australia. They were kept in 50 X 20 X 120 cm aquaria under constant temperature with a normal 12-hour light/dark cycle, and were fed dried pellet food weekly. Specimens with carapace lengths from 20 to 50 mm were examined. The animals were anesthetized by chilling in crushed ice for 30 min and were then decapitated. Statocysts were dissected from the dorsal surface of the basal segment of the antennules, and any extraneous tissue or adhesions were removed from around the cuticle of the statocyst with a fine paintbrush. The preparations were dehydrated in a series of ethanol solutions before being transferred to 1009r ethanol for 12 h. After an additional 24 h in a desiccator, conducting graphite paint was used to glue the preparations to a scanning electron microscope stub. They were sputter coated with gold, and examined with a Phillips 505 scan- ning electron microscope. The images were processed using Adobe Photoshop Version 4.0. Measurements of carapace, basal segment of the antennule, and statocyst diameter were recorded for body index relationships, and comparisons were made using SYSTAT 6.0 for Windows. Results Location and general structure of the statoc\st The statocysts of Che rax destructor are in the dorsal region of the basal segment within the antennules (= first antennae; Fig. 1 A, B). The statocyst is a cup-like invagina- tion of the cuticle forming a cavity with a triangular, ante- riorly facing opening on the dorsal surface. The opening is covered with a dense mat of setae that prevents entry of Arrangement of setae and changes in distribution during growth All of the setae on the base of the statocyst capsule of C. destructor, except those in the anterior part of the anterior setal field, are bound to the statolith (Fig. 2C). All setae that could be seen in scanning micrographs, because they were not obscured by the statolith. appeared to have the same external morphology (Fig. 2E), even though they varied in size. Because of the close association between the setae and the statolith, the process of removing it to examine the base of the capsule usually removed not only the setae but all associated tissues, including the tissues passing through the holes in the floor of the capsule. Remnants of these re- mained in a number of our preparations, however: these demonstrated that at least some of the setae are innervated through the holes in the base of the capsule (Fig. 2F). The presumption is that the holes represent innervation chan- nels, as they do in other species (Hertwig et al., 1991 ). The holes indicate the precise position of each seta on the floor of the capsule (Fig. 2A. B). Their disposition around the depression that normally holds the statolith resembles that in Orcont'ctc'fi liniosim (Hertwig et al., 1991). and direct correspondence with three of the four setal fields they described and named is apparent. A curved field made up of an inner double row and an outer single row forms a semicircle around the medial and posterior rim of the central depression. On the lateral side, this merges into the narrow end of a large triangle of setae occupying the area lateral to the rim of the depression. Opposite this large field, on the medial side of the depression, is a smaller triangular field. In an adult animal of around 50-mm carapace length, these fields are composed of about 68. 135. and 36 setae, respec- tively (Fig. 2D, Fig. 4) The total number of setae increases CRAYFISH STATOCYST GROWTH Anterior 253 5mm Anterior BS 2 mm Figure 1. Morphology of antennular region and statocyst of the crayfish Clicnn tlcMiiiflnr. (A) Dorsal view of the basal segment (BS) of the antennule. and the locution of the statocyst opening (SO). The rostrum and eyes have been removed. The position occupied by the rostrum is indicated by dotted lines. (B) Higher magnification of the basal segment (BS) of the antennule showing the dense screen of setae (H) that covers the statocyst opening (SO). (C) The statocyst capsule viewed through a window cut in the dorsal cuticle of the basal segment (BS) of the antennule to reveal the setae (SS) projecting upwards from the ventral floor (F) of the capsule. The statolith. with which all but the anterior setae make contact, has been removed. linearly with the size of the animal (n = 24: R~ - 0.8663; P < 0.005: Fig. 3B). To examine the way in which this increase occurs, we counted the number of setae in a group of animals with a basal antennule length of 1.97 mm (SD = 0.19) ("small") and compared the result with a sample of animals with a basal antennule length of 5.75 mm (SD = 0.27) ("large"). The results of the survey are shown in Figure 4. A two- factor analysis of variance on the data testing for setal field type and size of animals showed that the large animals have significantly more setae in each field than the small animals (F -- 322.6. P < 0.01 ). the number of setae in the three fields is significantly different (F( 2 50) — 848.9. P • 0.01 ). and the si/.e of the difference varies between fields 0.1mit.20.1 kU 166«m 6094X01 Figure 2. Scanning electron micrographs showing the statocyst of the crayfish Cherax destructor. (A) Dorsal view of the right antennule of an animal from the "small" group with part of the dorsal cuticle (C) cut away to reveal the floor of the capsule ot the statocyst (S). The basal segment of this animal was 1.99 mm long. (B) Dorsal view of the floor of the statocyst (S) from the left antennule of an animal from the "large" group. The basal segment of this animal was 5.7 mm long. The magnification is the same in A and B so that the large increase in the number of setae in the anterior lield is readily apparent. (C) Dorsal view of the statocyst capsule with part of the dorsal cuticle (Cl removed to reveal the sensory setae (SS) in contact with the statolith (SL). Note that many of the setae in the anterior field do not contact the statolith. (Dl Dorsal view of the ventral floor of the statocyst showing position of setae. The fields have been marked to correspond with the classification used previously in Onuih-ck^ /HHO.WI.V A Large anterior field 1 134 setae); curved field (69 setae): O, outer row (29 setae); + inner rows (40 setae); X. small field (4f> setae). (E) High magnification view of base of a seta from the outer curved held viewed from the dorsal aspect. (F) High magnification view of the ventral surface of the same statocyst base as in E, showing holes beneath each seta and remnants of the mechanical and neural connections broken during the statocyst removal and preparation process. CRAYFISH STATOCYST GROWTH 255 Carapace: FT = 0.9711 Statocyst: R2 = 0.9546 2345678 Basal segment of antennule (mm) B 300 250 200 150 100 50- = 24 01234567 Basal segment of antennule (mm) Figure 3. Statocyst size relationships. (A) Relationship between sta- tocyst diameter, carapace length, and length of the basal segment of the antennule. The bold and dotted lines are the linear regression lines. Note the high correlation for both body measurement indices. (B) Relationship between length of basal segment of antennule and total number of setae within the statocysts. Note the high level of correlation between the base of the antennule (and hence body size) and the number of setae. (F,-, 5()) = 69.2, P < 0.01). Tukey-Kramer pairwise comparisons between the three fields in both large and small animals showed that the number of setae is different in the three fields at the P < 0.01 significance level. Discussion The outcome of this work is straightforward. The result is a description of Statocyst morphology in a previously un- described crayfish species which permits some species com- parisons to be made. In addition, this is the first report on changes in the size and setal arrangements of the Statocyst with changing body size. The results therefore have impli- cations for comparative and developmental questions. In a mini-review, Sekiguchi and Terazawa (1997) com- pared information on statocysts across a range of crustacean species and found considerable morphological variation be- tween taxonomic groupings but some evidence of consis- tency within them. The number of examples available, however, is probably not yet sufficient for a firm conclusion on this issue. The general morphology of the Statocyst of Cheni.\ destructor does appear, however, to be closely similar to that of other crayfish species examined (Proctim- barus clitrkii: Takahata and Hisada, 1979; Orconectes li- mosus: Hertwig et m/i. Kiochem. Physid. 101A: 249-257. Patton, M. L., and R. F. Grove. 1992b. Slatolith hair movements and the regulation of tonic gravity reflexes in the lobster Homanis ameri- canus. Comp. Biochcm. Physid. 101A: 259-268. Sandeman, D. C., and A. Okajima. 1972. Statocyst-induced eye move- ments in the crab Scylla scmira. J. E\p. Biol. 57: 187-204. Sandeman, I). C., and R. E. Sandeman. 1996. Pre- and postembryonic development, growth and turnover of olfactory neurones in crayfish antennules. J. £v/>. Biol. 199: 2409-24 IS. Schmitz, B. 1992. Post embryonic development of the crayfish Procam- harus clarkii and its tailfan mechanosensory system. Pp. 69-90 in Nen'ous Systems,' Principles of Design and Function. P. N. Singh, ed. Wiley Eastern. New Delhi. Schone. H., and D. M. Neil. 1977. The integration of leg position- receptors and their interaction with statocyst inputs in spiny lobsters. Mar. Bclnn: Physiol. 5: 45-49. Sekiyuchi. H.. and T. Terazawa. 1997. Statocyst of Jasus edwardsii pueruli (Crustacea, Palinuridae), with a review of crustacean statocysts. Mm: Freshwater Res. 48: 715-714. Sleullet, P., H. S. Cate, and C. D. Derby. 2000. A spatio-temporal wave of turnover and functional maturation of olfactory receptor neurons in the spiny lobster. Pamitints argus. J. Ncnrosci. 20: 3282-3294. Stuart. T., and D. L. Macmillan. 1997. Development of sensory hairs on the telson of the Rock Lobster Jasus edwardsii. Aust. J. Zoo/. 45: 307-315. Takahata, M., and M. Hisada. 1979. Functional polarization of stato- cyst receptors in the crayfish Procamharus clarkii Girard. J. Comp. Plnsiol. 130: 201-207. Reference: Biol. Bull. 199: 2?7-264. (December 2000) Thermosensitivity of the Lobster, Homarus americanus, as Determined by Cardiac Assay STEVEN H. JURY* AND WINSOR H. WATSON 111 Zoology Department and Center for Marine Biology, University of New Hampshire. Durham, New Hampshire 03824 Abstract. It is generally accepted that crustaceans detect, and respond to, changes in water temperature, yet few studies have directly addressed their thermosensitivity. In this investigation a cardiac assay was used as an indicator that lobsters (Hoinarus americantts) sensed a change in temperature. The typical cardiac response of lobsters to a 1 -min application of a thermal stimulus, either warmer (;; = 19) or colder (n = 17) than the holding temperature of 15 °C, consisted of a short bradycardia (39.5 ± 8.0 s) followed by a prolonged tachycardia (188.2 ± 10.7 s). Lobsters exposed to a range of rates of temperature change (0.7, 1 .4, 2.6, 5.0 °C/min) responded in a dose-dependent manner, with fewer lobsters responding at slower rates of tempera- ture change. The location of temperature receptors could not be determined, but lesioning of the cardioregulatory nerves eliminated the cardiac response. Although the absolute de- tection threshold is not known, it is conservatively esti- mated that lobsters can detect temperature changes of greater than 1 °C, and probably as small as 0.15 °C. A comparison of winter and summer lobsters, both held at 15 °C for more than 4 weeks, revealed that although their responses to temperature changes were similar, winter lob- sters (n - 18) had a significantly lower baseline heart rate (34.8 ± 4.4 bpm) and a shorter duration cardiac response (174 s) than summer lobsters (n = 18: 49.9 ± 5.0 bpm, and 320 s respectively). This suggests that some temperature- independent seasonal modulation of cardiac activity may be occurring. Received 22 March 1999; accepted 14 August 2000. *Present address: Caribbean Marine Research Center. Tequesta, FL 33469. E-mail: sjury@cmrc.org Introduction Temperature is one of the most important and pervasive environmental influences on the American lobster, Homa- rus americanus (Cobb and Phillips, 1980; Aiken and Waddy, 1986; Factor. 1995). It is generally accepted that locomotory activity in this species is temperature dependent (McLeese and Wilder. 1958: Reynolds and Casterlin, 1979; Haakonsen and Anoruo. 1994) and that it carries out sea- sonal inshore to offshore migrations to gain the develop- mental benefits of warmer coastal temperatures in the spring and summer (Cooper and Uzmann, 1971; Pezzack and Dug- gan, 1986; Karnofsky el al.. 1989; Haakonsen and Anoruo, 1994; Factor, 1995; Watson el al, 1999). Laboratory studies have demonstrated that H. americanus has a thermal pref- erence of about 16 °C (Reynolds and Casterlin. 1979; Cros- sin et al., 1998). and it has been proposed that behavioral thermoregulation may allow members of the species to occupy thermal niches which maximize their metabolic or behavioral efficiency. The behavioral responses of lobsters to thermal gradients suggest they have some mechanism to sense temperature so that they may effectively respond to the thermal properties of their environment. Thermosensitivity in lobsters may be mediated by distinct thermoreceptors or thermosensitive neurons as in some other invertebrates (Prosser and Nelson, 1981; Mori and Ohshima, 1995). Although behavioral studies strongly sug- gest that H. americanus can sense temperature (Reynolds and Casterlin. 1979; Crossin et al., 1998), to our knowledge only one study has addressed how neurons respond to changes in temperature in this species. In that study, firing of cells associated with thoracic ganglia connectives gener- ally showed no spontaneous activity below 14 °C, but most became spontaneously active above this temperature. Inter- estingly, these cells "cycle reversibly from silent to contin- uously active to bursting and back as the temperature is 257 258 S. H. JURY AND W. H. WATSON III increased and decreased" (Konishi and Kravitz, 1978). Other than these cells, which may or may not play a role in thermally guided behaviors, we know little about the loca- tion of putative thermoreceptors, or the mechanisms used to detect temperature, in lobsters and most other crustaceans (Dorai Raj and Murray. 1962; Ache, 1982). In situations where the precise receptors have not been identified, or are not readily accessible to electrophysiolog- ical investigation, cardiac assays are a valuable tool for preliminary investigations of sensory capabilities (Larimer, 1964; Offutt, 1970; Florey and Kriebel. 1974; Dufort. 1997). For example, many crustaceans exhibit a drop in heart rate in response to novel stimuli (Maynard, 1960; Larimer, 1964; McMahon and Wilkens, 1972; DeWachter and McMahon, 1996). This cardiac response has been used to measure the ability of H. americanus to detect sound (Offutt, 1970) and salinity (Dufort, 1997). Although a num- ber of studies have addressed the effect of temperature on decapod heart rates at time scales ranging from hours to days (Ahsanullah and Newell. 1971; Florey and Kriebel, 1974; DeFur and Magnum, 1979; DeWachter and McMa- hon. 1996; DeWachter and Wilkens. 1996; Hokkanen and Demont. 1997). few have characterized the initial response (i.e., <5 min.) to brief changes in water temperature. The present study used a cardiac assay to demonstrate that American lobsters are consistently capable of sensing in- creases or decreases in temperature that are greater than 1 °C. The typical response elicited by both cold and warm stimuli was a brief slowing of the heart rate, followed by prolonged cardioacceleration. Winter and summer lobsters responded somewhat differently to thermal stimuli, suggest- ing some type of seasonal temperature-independent modu- lation of their responsiveness to thermal stimuli. Materials and Methods Animals Adult (82-92 mm carapace length), intermolt lobsters were held at 15 ± 1 °C (salinity 30 ± 1 ppt) for more than 4 weeks prior to use, and experiments were initiated at this temperature. All lobsters were captured from coastal New Hampshire waters, and experiments were conducted at the University of New Hampshire, Durham, New Hampshire. Experiments were carried out in both summer and winter under ambient light conditions. In the summer, the thermo- sensitivity of 18 lobsters was determined (cold stimuli, n = 9; warm stimuli, n = 9); in the winter, lobsters kept at the same temperature ( 15 °C) as summer lobsters were used in identical experiments (cold stimuli, n = 8; warm stimuli, ;; = 10). Recording of temperature and heart aciivit\ Small wire electrodes were inserted through the dorsal carapace above the heart and used with a UFI impedance converter (model #2991) to record heart rate (Dyer and Uglow, 1970). Because the impedance recording technique can be sensitive to temperature, the method was verified by using a second pair of electrodes and a Grass model 7D polygraph to simultaneously monitor the electrical activity associated with lobster heart contractions (see Watson and Wyse, 1978; Watson, 1980). External temperature was re- corded using a small (3 mm X 1 mm) thermistor (C & B Sciences/iWorx, Inc., Dover, NH) placed on the dorsal carapace. The thermistor was calibrated weekly over the range of temperatures used in the experiments. The time constant of the thermistor was 2.0 s (time to achieve 67% of the final response). The absolute resolution of the thermistor was ±0.15 °C, but it could accurately detect changes in temperature as small as 0.01 °C. However, because of turbulent mixing within the recording chamber, the slight time delay due to the time constant of the thermistor (Fig. 1 ). and the unknown location of temperature-sensitive neu- ral elements relative to the location of the thermistor, it was not possible to assess the thermal detection threshold with great accuracy. All temperatures presented are those re- corded by the externally located thermistor above the dorsal carapace. These should be interpreted conservatively, in the context of the methods used and the unknown location of the sensory receptors. Experimental chamber After insertion of the electrodes, lobsters were placed in a recording chamber consisting of an 18-cm-diameter PVC pipe covered on the top and bottom by perforated plates through which seawater (temperature 15 ± 1 °C) continu- ously flowed (Fig. 1). This arrangement kept lobsters rela- tively immobile and ensured that changes in temperature within the recording chamber were rapid and relatively homogeneous. The chamber was placed in an acrylic plastic insert (30 X 30 X 30 cm) that was immersed in a temper- ature-controlled 120-1 aquarium (the ambient bath). Ambi- ent seawater was continuously pumped (2 1/min) from the aquarium through the recording chamber, into the insert, and back to the aquarium. Thermal stimuli were delivered by switching the source of seawater from the ambient bath to the stimulus bath. This switching was accomplished by turning a stopcock and was considered the initiation of the stimulus (see arrows in Fig. 2). The stimulus bath was filled from the ambient bath to minimize novel chamber chemo- sensory cues (Fig. 1 ) and brought to the appropriate exper- imental temperature using aquarium heaters or cooling coils. The recording chamber was covered with black plastic to minimize visual disturbance, and the lobster was left in the experimental apparatus overnight before an experiment. Lobsters are much more sensitive to stimuli if allowed to recover from electrode insertion and become accustomed to the recording chamber (Larimer. 1964; Dufort, 1997). THF.RMOSHNS1TIVITY OF LOBSTERS 259 A. Heat/Cool Stopcock Animal i Chamber Heart Electrodes Stimulus ^V Ambient Balh Bath Pumps c. 50 100 150 200 250 Time (s) -1 1 23456 Time (s) Figure 1. Experimental apparatus used to record lobster cardiac re- sponses to changes in temperature. (A) Seawater (I? °C> flows continu- ously from the ambient bath into the animal chamber through perforated plates located above and below the lobster (direction of flow indicated by dark arrows). Heart rate is recorded before, during, and after exposure to a temperature stimulus. Switching the stopcock changes the source of sea- water from the ambient bath to seawater from the stimulus bath (direction of flow indicated by white arrows). A thermistor on the dorsal carapace is used to monitor temperature during each trial. (B) Rates of temperature change in a typical experiment in response to I min stimuli (turned on al time = 0 and off at arrow) of ± 0.7. 1.4. 2.6. and 5.0 °C warmer or colder than the ambient temperature. (C) Time constant of the thermistor when exposed to a step change in temperature of ± 2 °C (dotted line). The estimated time to achieve 67% of final temperature is 2.0 s. The following day, after basal heart rate was measured for at least 30 min, each animal was exposed for 1 min to a warm or cold stimulus that changed the temperature in the recording chamber at a rate of ±0.7 °C/min. This was followed by stimuli delivered at targeted rates of ±1.5 °C/min, ±2.5 °C/min. and ±5.0 °C/min for 1 min. Tem- perature was allowed to return to ambient (Fig. 2) between each treatment. Treatments were separated by at least 30 min. The temperature in the recording chamber was moni- tored with the dorsally located thermistor, and the actual mean rates achieved were 0.72 ± 0.04. 1.37 ± 0.06. 2.61 ± 0.10. and 4.95 ±0.16 °C/min. Thus, the average maximum warm stimuli after 60 s were 15.7. 16.4. 17.6. and 20.0 °C, and the maximum cold stimuli were 14.3. 13.6. 12.4. and 10.0 °C. Which stimulus (warm or cold) was tested on the first day was assigned randomly, and the other set of stimuli (warm or cold) were tested on the following day. A 25% change in heart rate — bradycardia (decrease) or tachycardia (in- crease)— was used as an indicator that lobsters sensed a change in water temperature (Offutt, 1970; Dufort. 1997). All records were digitized using a MacLab system (C & B Sciences/i Worx. Inc.) and were analyzed to determine the following: (1) delay to a response: (2) duration of brady- cardia, tachycardia, or both: (3) heart rate (bpm) during bradycardia: and (4) heart rate (bpm) during tachycardia. In addition, thermosensitivity thresholds were estimated from the water temperature measured above the dorsal carapace at the time of the initial cardiac response. Controls were conducted before any thermal stimuli were applied; the same protocol described above was followed, but without changing the temperature in the stimulus bath. Localization of punitive temperature receptors In an attempt to localize regions with putative tempera- ture receptors, lobsters missing antennae (n = 4) or missing antennae and antennules (n = 4) were tested for a response to a temperature change of +2.5 °C/min. Antennae or antennules were removed bilaterally at their base, the wounds were sealed with wax to prevent blood loss, and the lobsters were allowed more than 24 h to recover. -20-10 0 10 20 30 40 50 60 70 80 90 Time (s) 12 Time (s) Figure 2. Typical cardiac response to a change in temperature. (A) The top trace shows the typical response to a + 1 .4 °C/min stimulus: the lower trace is a plot of the temperature change during the 60-s trial. The dark closed arrow shows when the stimulus flow was turned on. and the white open arrow shows when it was turned off. (B) An enlargement of the hiahliahted area from (A), showing the time course of the bradycardia and associated temperature change. Note that the rapid response may be a result of the combination of the location of the thermistor relative to the location of the unknown temperature sensitive receptors and the slight delay due to the time constant of the thermistor. There was no response to controls when the flow was switched but the temperature was not changed. 260 S. H, JURY AND W. H. WATSON III To determine whether changes in cardiac activity were mediated by the cardioregulatory nerves, responses to ther- mal stimuli were measured before and after nerve lesions (n = 5). Changes in heart rate were initially recorded in response to thermal stimuli of +1.5 °C/min and -1.5 °C/ min. Then the cardioregulatory nerves were cut. and lob- sters were allowed at least 2 days to recover. Finally their cardiac responses were measured again in response to the same stimuli that were applied before the lesions. Lesions were made as described in Guirguis and Wilkens ( 1995). A small (3-cnr) rectangular piece of dorsal carapace just above the heart was removed, and superficial cuts were made with fine scissors through the connective tissue along the border of the opening. The shell was then replaced and fastened in place with tape. Sham-operated control animals (/; = 4) were treated in the same manner except that no cuts were made in the connective tissue. Statistical analysis Throughout the text, variation is presented as standard error of the mean (i.e.. mean ± SEM). A P value of <0.05 was considered to be significant for all statistical tests. Results Typical response to a change in temperature The typical cardiac response to both warm and cold stimuli consisted of a short bradycardia (39.5 ± 8.0 s). followed by a significantly (paired t test) longer tachycardia (188.2 ± 10.7 s; Fig. 2). In general, changes in heart activity were similar in response to both warm (n = 19) and cold (n - 17) stimuli. Although the intensity and duration of car- diac responses were similar for all temperatures tested (ANOVA, P > 0.05). some lobsters did not respond to slower rates of change (0.7 and 1.4 C/min). whereas almost all lobsters responded to the maximum rate of change (5.0 °C/min; Fig. 3). There was no cardiac response in control trials (H = 36). where temperature was not changed but ambient water was pumped through the chamber from the stimulus bath (Fig. 4). Sensitivity to warm and cult/ stimuli Lobsters were extremely sensitive to both warm and cold stimuli (Fig. 3). For example, when subjected to a + 2.6 °C/min stimulus, lobsters responded after just 3.8 ± 0.5 s, when the temperature in the chamber had changed by only 0.09 ± 0.04 °C. Lobsters exposed to the -2.6 °C/min stimulus responded after a drop of only 0.13 ± 0.09 °C. and the latency to respond (4.6 ± 1.8 s) was not significantly different (paired t test) than during a warm stimulus (Fig. 3). Temperature change measured al the initiation of a cardiac response by individual lobsters ranged from 0.01 0.50 0 1.0 2.0 3.0 4.0 5.0 Rate of Change (°C/min) Figure 3. Responses to thermal stimuli at different rates of change. (A) The thermal detection threshold, or the amount of temperature change required to elicit a cardiac response, was similar even when hot and cold stimuli were applied at different rates. (B) When thermal stimuli were applied at slow rates of change, the delay to respond was longer, especially in the case of cold stimuli. (C) Although lobsters responded similarly to thermal stimuli applied at fast and slow rates of change, some animals did not respond at all to slow rates of change, while all animals responded to hisiher rates ol chansic. to 0.79 °C. There were no significant differences (un- paired t test) between the sexes in the temperature change at initial response; when lobsters responded, they exhib- ited comparable thresholds, at all measured rates of change (Fig. 3. Kruskal-Wallis test). The average tem- perature-detection threshold, for all trials in which ani- mals responded, was 0.15 ± 0.03 °C. This is considered to be only an estimate because of the inherent time constant and resolution of the thermistor, the How of water in the chamber, and the location of the thermistor relative to the still unknown location of the receptors mediating the response. Nonetheless, this assay demon- strates that lobsters are sensitive to very small changes in temperature. THERMOSENSITIVITY OF LOBSTERS 261 A. 0) D response latency S bradycardia D tachycardia 50 100 150 200 250 300 350 Time (s) 80 - ' T fl Control rate D Initial rate £ 60 - D. £t 1 _ I T _ D Bradycardia D Tachycardia (I, X rt to- 4-J s I ~] ( = 2°- 0^ — - cold warm stimulus stimulus cold warm stimulus stimulus Summer Winter Figure 4. Responses to cold and warm stimuli by summer and winter animalv (A) The mean response latency, duration of bradycardia. and duration of tachycardia in response to ± 2.6 °C/min stimuli in both summer and winter lobsters. While the latency to respond, and thus thresholds, were similar between seasons, the duration of both bradycardia and tachycardia responses tended to be longer in summer animals. IB) Differences in the responsiveness of summer and winter lobsters. For all treatments, an application of control 15 °C stimuli to lobsters held at 15 C did not cause a statistically significant change in heart rate. Lobsters in both seasons responded to ± 2.6 °C/min stimuli with a short bradycardia followed by a longer tachycardia. The major difference between summer and winter lobsters, other than the duration of responses shown in A. was that animals in the winter had significantly lower initial heart rates. Thus, although all animals were held at 15 °C, there appears to be some temper- ature-independent seasonal modulation of heart rate. Localization of receptors All lobsters with ablated antennae (n - 4) or ablated antennae and antennules (n = 4) showed typical responses to a stimulus of +2.5 °C/min. There was no significant difference (unpaired t tests) in the duration of bradycardia and tachycardia, the thermosensitivity threshold, or the baseline heart rate when compared to intact lobsters (/; = 18). Prior to cardioregulatory nerve lesions, lobsters (n = 5} had a mean heart rate of 41 ± 2.9 bpm. Following recovery, their heart rate increased to 70 ± 5.3 bpm. which was significantly higher (paired t test) than the basal rate. This increase may not have been due solely to the lesion ot the cardioregulatory nerves: sham-operated controls (;i = 4) also had heart rates (48 ± 9.4 bpm) that were slightly higher than their pre-operation baselines (37 ± 5.4 bpm). although that difference was not significant (paired t test). In response to a hot or cold stimulus, all lobsters exhibited typical bradycardia and tachycardia responses before cardioregula- tory nerve lesioning. However, after lesioning none of the lobsters showed an increase or decrease in heart rate in response to thermal stimuli. In contrast, all of the sham- operated lobsters showed typical responses (100% showed both bradycardia and tachycardia) when exposed to identi- cal thermal stimuli. These data indicate that the change in heart rate elicited by warm and cold stimuli is mediated by the cardioregulatory nerves and not by the direct influence of temperature on the heart itself. The influence of season The characteristic cardiac response of lobsters to a change in temperature (a short bradycardia followed by a longer tachycardia) was similar for lobsters tested in the winter and those tested in the summer. However, the fol- lowing differences were apparent: ( 1 ) the mean baseline heart rate was significantly lower (unpaired t test) in winter lobsters (34.8 ± 4.4 bpm) than in summer lobsters (49.9 ± 5.0 bpm: Fig. 4): (2) the duration of bradycardia and tachy- cardia responses tended to be shorter in the winter lobsters (unpaired t tests. P < 0. 1 ); and (3) winter lobsters tended to respond to temperature changes with both tachycardia (83%) and bradycardia (72%). whereas only 50% of the summer lobsters responded with tachycardia, but 94% showed bradycardia. Thus, even though winter and summer lobsters were both held at 15 °C for at least one month and tested with identical warm and cold stimuli, they responded differently. This difference may be related to the observed seasonal differences in basal physiological state (Fig. 4). Discussion This study supports the findings of two previous behav- ioral studies which infer that American lobsters can sense changes in temperature (Reynolds and Casterlin. 1979: Crossin et ai. 1998). Assuming that the initiation of a cardiac response indicates detection of an environmental change, our conservative estimate is that lobsters can sense both increases and decreases in water temperature of greater than 1 °C (Fig. 3) and probably as small as 0.15 °C. Similar thermal sensitivity has been documented in a number of terrestrial arthropods (Murphy and Heath, 1983; Altner and Loftus. 1985). For example, the spider Cupieniws salei has a warm receptor with a detection threshold between 0.08 262 S. H. JURY AND W. H. WATSON III and 0.6 °C (Ehn and Tichy. 19%). Studies of thermorecep- tion in aquatic species are fewer, but are consistent with our findings. For example. Forward (1990) found that crab larvae (Rhithropanopeus Imrrinii and Neopanope sayi) as- cend or descend in a water column in response to absolute temperature changes of 0.29-0.49 °C, as long as the rate of change is fast enough (0.06-0.24 °C/min, depending on larval stage and species). Thus, the American lobster is probably not unusual in its ability to detect small changes in temperature, although the extent to which this level of thermosensitivity exists in other crustaceans remains to be investigated. Although several behavioral studies indicate that crusta- ceans are quite sensitive to changes in temperature, little is known about thermosensitivity in this large group of pri- marily aquatic invertebrates. A study of the thermal sensi- tivity of the dactyl receptors of Cancer imtewuirhts, C. antlwnvi. and Panidinis iiiterntptus strongly suggests that they possess a thermal sensory system capable of integrat- ing temperature information for use in thermally cued be- havior (Cook, 1984). However, the actual thermoreceptors have not been identified in these species. In lobsters, a number of neurons change their rate of firing in response to shifts in temperature, but it is not clear if these cells are actually serving the function of thermoreceptors. For exam- ple, intracellular recordings from cells of the thoracic gan- glia connectives of H. americanus show firing patterns that reversibly change from silent to continuously active to bursting over the range of 10-17 "C (Konishi and Kravitz, 1978). This is within the normal ecological range for this species, and while it is unknown what physiological or motor output results from this neuronal property, the corre- spondence to the behaviorally determined preferred temper- ature ( 16 "C; Crossin et til.. 1998) for this species is intrigu- ing. In the spiny lobster, Puniilinix japonicns, ligamental nerves innervating the pericardia! organ have also been reported to increase their firing in response to cold stimu- lation (Kuramoto and Tani. 1994). Once again, temperature stimuli were shown to have a direct physiological effect in viim. but it is unknown how. or if. this effect is related to the existence of thermoreceptors or behavioral thermoregula- tion. 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Al-Wassia, and E. W. Taylor. 1995. The effects of sudden changes in temperature on aquatic and aerial respiration in the lobster. Homarus gammariis (L.). Mar. Frc.shu: Behav. P/iys. 271 It: 13-28. Wood. I). E., R. A. Gleeson, and C. D. Derby. 1995. Modulation of behavior by biogenic amines and peptides in the blue crab. Callinectes sapuhis. ./. Comp. Plnsiol. A 111: 321-333. Reference: Biol. Bull. 199: 265-277. (December 2000) Molt-Related and Size-Dependent Differences in the Escape Response and Post-Threat Behavior of the American Lobster, Homarus americanus S. I. CROMARTY,* J. MELLO, AND G. KASS-SIMONt Biological Sciences Department, University of Rhode Island, Kingston. Rhode Island 02881 Abstract. Videotaped recordings of adult lobsters of dif- ferent molt stages were analyzed. The escape response of adults was compared with that of juveniles recorded in an earlier study. Juvenile lobsters always respond to a threat with escape behavior irrespective of their molt stage, but in adults the probability of eliciting a response was a function of molt stage: more hard-shelled (intermolt stage C) and (premolt stage D) animals tailflipped than did soft-shelled (postmolt stages A and B) animals. The number, frequency, and duration of tailflips, and the average distance swum by animals in each molt stage were measured for the entire escape response, for the initial power swim, and for the subsequent swims. These measure- ments were used to compute several parameters: velocity, acceleration, force, and work; average distance traveled in a tailflip for each kilogram of body weight (distance/kg/tail- flip): and average distance traveled for each bodylength (distance/bodylength). Among adults, intermolt (stage C) lobsters traveled sig- nificantly farther and faster than postmolt animals (stages A and B). Among juveniles, late postmolt (stage B) animals traveled farther. Among adults, although the total number of tailflips and the duration of the response were not signifi- cantly different among molt stages, the number of tailflips/ second (frequency) and distance traveled/kg/tailflip were greater for intermolt animals. In juvenile intermolts. how- ever, frequency and distance/kg/tailflip were markedly Received 22 October 1999; accepted 7 July 2000. * Present address: Department of Natural Sciences, Assumption Col- lege, 500 Salisbury Street, Worcester. MA 01609-1296. E-mail: scromart@eve.assumption.edu t To whom correspondence should be addressed. Biological Sciences Department. University of Rhode Island, 100 Flagg Rd.. Kingston, RI 02881-0816. E-mail: Kass.Simon@uri.edu lower than in the premolt stages. Although values were lower than intermolts and premolts, postmolt adults sus- tained their swimming frequency, distance/kg/tailflip, and distance/bodylength for the entire escape distance (as did postmolt juveniles). These parameters then dropped off sharply for both adult and juvenile intermolt and premolt animals in the second half of the escape distance. Post-threat behaviors reveal that stage D animals have the highest aggression index and often attack the presented stimulus, whereas stage A animals are the least likely to approach the stimulus and typically back away in a non- aggressive posture. Thus, although effects of the molt cycle on adult and juvenile escape behavior are similar in some ways, other physical characteristics of adults, such as weight, allometry. and physiology, seem to become important in determining the likelihood of escape behavior and the characteristics of the escape swim in each molt stage. Introduction The behavior of the American lobster, Homarus ameri- canus, varies (both in the laboratory and in the field) with sex and reproductive state (Cowan and Atema, 1990; Figler et al.. 1997, 1998: Cromarty et al, 1998; Mello et ai, 1999), relative size (Scrivener, 1971; Lang et al., 1977), time in residence (O'Neill and Cobb, 1979; Peeke et al., 1998; Cromarty et al., 1999), and dominance (Karnofsky and Price. 1989; Huber and Kravitz, 1995). However, molt- cycle-related behaviors have been rarely been studied — probably because the long-term approaches and experimen- tal designs needed are complex. The physiological transformations that occur in decapod crustaceans over the molt cycle are clearly profound; they include a variety of metabolic, neuroendocrine. and neuro- 265 266 S. I. CROMARTY ET AL. physiological changes (Knowles and Carlisle, 1956; Pas- sano, 1960; Kleinholz and Keller. 1979; Quackenbush, 1986) that could manifest themselves in distinctive molt- stage-related behavioral modifications. Specifically, the es- cape response behavior in decapod crustaceans is ideally suited for modulation because it is composed of multiple tailflips, or swims. The escape response consists of an initial power swim followed by a series of subsequent swims; in crayfish, the initial power swim is elicited by visual and tactile excitation in the front of the animal which is medi- ated by the medial giant neuronal system, while tactile excitation of the telson at the rear of the animal is mediated by the lateral giant system. The subsequent swims immedi- ately following the power swim are mediated by the non- giant system, which innervates the phasic flexor abdominal musculature (Wine and Krasne, 1972, 1982). Although the physiology of tailflip escape circuits has not yet been fully described in lobsters, the close similarity between the two species suggests that the innervation is similar. While studying confrontations between juvenile Ameri- can lobsters, Tamm and Cobb ( 1978) identified an increased probability- of eliciting an escape response in early postmolt stages (stages A and B). In contrast, the frequency of aggressive behaviors, in particular the meral spread, in- creased during mid-premolt stages (stages D, and D2). Hard-shelled lobsters tend to be aggressive, but soft-shelled lobsters tend to avoid confrontations. Stomatopods exhibit similar behavioral differences (Steger and Caldwell, 1983). These differences are understandable in view of the fact that in their postmolt, soft-shelled state, these animals are much more vulnerable to predation by predators and conspecifics than are hard-shelled animals, and they are less able to fend off attacks with aggressive behaviors, so that when threat- ened, they are forced to try to escape instead of mounting a defense (Tamm and Cobb. 1978; Atema and Cobb. 1980; Atema and Voigt. 1995). One might expect that a newly molted animal would have difficulty doing much of anything until the exoskeleton hardens. Although this is true for lobsters in very early stage A. our studies revealed subtle differences in escape behavior among juvenile lobsters over the molt cycle (Cromarty et a/., 1991; Cromarty. 1995). We found that overall, postmolt lobsters produced the more effective escape behavior. Soft- shelled, postmolt juvenile lobsters (stage B) traveled farther, produced more tailflips, and swam longer, at sustained velocity, than did premolt lobsters. Earliest postmolt (stage A) juveniles swam at a higher frequency. In contrast, pre- molt juveniles produced a quick, forceful initial power swim, followed by subsequent swims that rapidly decreased in velocity, acceleration, force, and work output (Cromarty et al.. 1991). The above studies focused on juvenile lobsters; even less information exists on molt-related changes in escape behav- ior in adult or larger animals. We know that the escape response occurs more frequently among juveniles and smaller adults than among large adults (Lang et al., 1977). and that the conduction time of medial giant impulses from the brain to the sixth abdominal ganglion increases greatly, causing an increase in the latency of the response. The relative ratio of abdomen length to carapace length de- creases with increased size, forcing the abdominal flexing muscles to propel a larger body mass (mostly claws). As with crayfish (Krasne and Wine, 1975), removal of the claws of a large lobster increases its propensity to tailflip (Lang ct nl., 1977), as we have also observed in these experiments. Since large lobsters are less apt to be preyed upon than small ones (Atema and Voigt, 1995), it is ex- pected that large soft- and hard-shelled adult lobsters would exhibit different but unique escape behaviors from one another. Because of the physical and behavioral differences be- tween adults and juveniles, on the one hand, and the phys- iological and behavioral differences among animals of dif- ferent molt stages, on the other, we wished to investigate whether adults and juveniles in the same molt stage differed in the measurable characteristics of the escape response. We therefore examined the escape response of adult male lob- sters of different molt stages in an experiment similar to the one we had designed for juvenile lobsters (Cromarty ct al.. 1991 ). We measured distance traveled (m); number of tail- flips (TO; duration of the response (s); frequency of tailflips (Tf/s); velocity (m/s), acceleration (m/s/s). force (N; kg • m/s/s) and work (J) of each tailflip; distance traveled in each tailflip for each unit of body weight (m/kg/Tf); distance traveled in each tailflip for each unit of bodylength (dis- tance/bodylength); and distance traveled in each tailflip for each unit of body weight (m/kg). In addition, we compared the escape thresholds of juveniles and adults. Our earlier work indicated that lobsters could show sig- nificant differences in post-stimulus behaviors towards the threatening object, as well as in the characteristics ot the escape behavior itself (Cromarty et al., 1999). Thus we also analyzed post-stimulus agonistic behaviors and now present evidence that these behaviors — like escape behavior — dif- fer significantly from one molt stage to another: premolt lobsters are more likely than postmolt animals to attack a threatening stimulus, and postmolt animals are more likely to back away from a stimulus with no display of aggression. Materials and Methods Procedures and experimental protocols are essentially the same as those described elsewhere (Cromarty et al.. 1991, 1998, 1999), but are summarized again here with relevant differences included. MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR 267 Animals Adult American lobsters (carapace length 74 to 90 mm) were obtained and housed as described previously (Cromarty el a!., 1999). Twenty-four hours prior to an experiment, an animal was moved to the Kingston campus of the University of Rhode Island, where it was placed in a holding tank (30 cnv1) and was not fed during this acclima- tion and experimental period. Isolation periods in the hold- ing tank were identical for all experimental animals. The tank had its own air supply. To avoid possible sex-related effects, only males were used in this study. Ten lobsters from each molt stage (A. B, C, and D) were randomly selected as they entered the stage. Lobsters weighed (in grams) an average of 451.4 ± 69.6 (mean ± SD) and had an average carapace length (in millimeters) of 81.3 ± 4.7 (mean ± SD). The experiments were performed randomly so that no molt-stage clustering occurred. A correlation statistic was run to check for molt stage and date of experiment. No correlation was found between the animals' molt stage, the time between the animals' capture and their use in the experiment, and the sequence of experiments (R2 < 0.18, F > 0.05). Lobsters were presented with the stimulus only once and were immediately sacrificed for identification of possible molt-related differences in the phasic flexor musculature system that is responsible for the escape response behavior. This required that over 200 animals be individually housed so that lobsters entering different molt stages could be selected. Freshly caught lobsters were continually added to the holding population to reduce "inactivity" and potential for increasing aggressive behavior (Cromarty et ai, 1999). Be- cause of possible seasonal differences in physiology and morphology such as those described in crayfish (Lnenicka and Zhao, 1991 ), experiments were conducted between June and October when Rhode Island waters maintain tempera- tures between 18°C and 23°C and similar conditions can be maintained in the indoor holding tanks. Again, no correla- tion was found between any of the significant parameters and the date of experiment. Experiments Each experiment was run between 1 200 and 1 500 hours in a 4000-1 tank filled with filtered recirculated seawater. The large amount of seawater held in the experimental tank made it impractical to drain the tank after each experiment, but carbon filters were continuously used throughout this experimental period to remove possible recognition odors originating from the lobsters' urine. Nevertheless, a corre- lation statistic was run to check for success of tailflipping and lobster order. No correlation was found between the order of experiments and the animals' success and failure of tailflipping (R2 C 0.13, F > 0.05). Salinity was kept between 29(7cr and 33^c. and adjust- ments (if any) were made before each experiment. One hour before an experiment, the physical condition of each animal was checked. Animals were used only if they moved around the tank or exhibited antennule flicking. Water temperature in the experimental tank was main- tained between 18°C and 20°C by a chiller. The experimen- tal area consisted of an open-ended tank ( 1.0-m L X 0.3-m W X 0.3-m H) immersed in a larger main tank (2.2-m L X 0.75-m W X 0.91-m H). A weighted wooden partition with a pulley acted as a blind (and separation to the main tank) at the open side of the experimental tank (Fig. 1 A). The experimental tank was designed with an open end so that a threatening stimulus could be introduced at that end. To ensure that lobsters were initially at the closed, non- stimulus end, a light was placed at the open end of the tank. The partition was raised once the lobster had reached the closed end. The light was then placed over the closed. non-stimulus end. This served to "push" the animal back towards the open (stimulus) end. Because adult lobsters did not respond to the stimulus that was used to induce an escape response in juveniles (a flat shiny and reflective mirror. 0.1 m2, housed in a wooden frame attached to a dowel stick), a piece of PVC tubing ( 15-cm L X 10-cm W) weighted with pebbles weighing 1.45 kg served as the threatening stimulus. The stimulus was raised above the open end. as depicted in Figure 1A. and was released into the water at a preset distance of 10 cm (measured from the open edge of the tank to the lobster's rostrum) whenever a lobster approached the open end of the tank. Cameras were placed in horizontal and vertical positions so that the experiments were simultaneously recorded on two video recording systems. Video recordings of each lobster were analyzed frame-by-frame. To measure distance traveled, a metric grid divided into 0.5-cm units was painted onto the side of the experimental tank. Distance traveled along the length of the tank was measured using the position of the tip of the lobster's rostrum as the point of reference. Time was automatically recorded on the videotape, and numbers of tailflips were counted in subsequent viewing of the recordings. After each experiment, the animal's molt stage was de- termined by examining cuticular and setal development in the pleopods (Aiken. 1973). Because animals become pro- gressively harder after ecdysis (stage E), we also determined postmolt periods by testing various carapace areas for ri- gidity (Aiken. 1980). Experimental animals were placed in the following categories: intermolt (stage C); premolt (stages D0, D,, D:. or D,); and postmolt (stage A up to 48 h following ecdysis and stage B from 48 to 96 h after ecdysis). Probabilities of an escape response were determined for each molt stage (n == 10) and statistically compared. The 268 S. [. CROMARTY ET AL Figure 1. (A) Diagram of the experimental tank (E). The stimulus was a weighted piece of PVC tubing filled with pebbles (A); the screen (B) was lifted while the lobster (Fl was at the opposite end of the tank. A light (D) at the closed end of the tank caused the animal to move towards the darker open end (K). The escape behavior of each lobster was recorded against a 0.5-cm metrically divided grid (G). The recording system consisted of vertical (C) and horizontal (H) cameras, a monitor (1). and a time-lapse VCR (J). (B) Schematic breakdown of a single tailflip as it was seen in the video analysis: (a) = beginning of swim; (f) = end of a single tailflip. (Drawings by K. Davignon, Graphics specialist. LIRI.) Previously pub- lished in The Biological Bulk-tin (Cromarty t-l til.. 1998). other characteristics of the escape response of animals that escaped were analyzed as in our earlier study of juvenile escape behavior (Cromarty et 450 g) lobsters responded to the stick stimulus, regardless of si/.e class or molt stage (see Fig. 2). Various stimuli (water injection over the lobster, a larger conspecific lobster, a predator (tautog), and bubbles blown over the lobster) also failed to elicit an escape response. However, a 15-cm-long piece of PVC tubing weighted with pebbles, dropped suddenly from above as the lobster ap- proached, caused both larger juvenile (±150 g) and adult (±450 g) lobsters to tailflip (Fig. 2). In the large-juvenile size class, 23 out of 34 tailflipped (stage A: 4 of 8; stage B: 1 of 8: stage C: 9 of 9: stage D: 9 of 9). In the large-adult size class. 15 out of 40 tailflipped (stage A: 2 of 10: stage B: 2 of 10; stage C; 5 of 10: stage D; 6 of 10) or 20% of soft-shelled (stages A and B) and 55% of hard-shelled lobsters (stages C and D: FEP. P = 0.01 ). When compar- ing the probability of eliciting an escape response for soft- shelled (stages A and B) versus hard-shelled (stages C and D) lobsters, soft-shelled postmolt lobsters were significantly (A) o (B) ^ °> i C V q> (C ) 0 < en W \ * 1) • 600 gram >600 gram STICK STIMULUS 15CMPVC STIMULUS 15CM PVC 30 CM PVC Figure 2. Percentage (%) of lobsters escaping at each weight and molt stage in response to stimuli of different sizes. The stimulus was (Ala stick to which a mirror was attached: (B) a 15-cm length of PVC weighted with stone weighing 1.45 kg: (C) two pieces of PVC tubing, one was 15 cm in length and weighing 1.45 kg. and the other 30 cm in length and weighing 2.45 kg (animals heavier than 600 g did not respond to the two stimuli ). Molt stages, weights of animals, and types of stimuli are below the .Y-axis. Ratios at top of each bar are the number of lobsters escaping to the total number of animals presented with the stimulus. 270 S. I. CROMARTY ET AL less likely to tailflip than hard-shelled premolt lobsters: 20% (4 of 20) compared to 55% ( 1 I of 20) (FEP. P = 0.01 ). Adult lobsters larger than 600 g did not tailflip, even when the size of the PVC tubing was increased from 15-cm L X 10-cm W. 1.45 kg, to 30-cm L X 10-cm W. 2.45 kg. Analvsis of escape behavior in adults A. Effect of weight on escape response in the various molt stages As had been shown by Lang ct al. ( 1977). size and weight have significant effects on a lobster's propensity to exhibit an escape response. Among the groups of animals tested, there were signifi- cant differences in weights (Table la). Stage B lobsters weighed significantly more than either stages C or D (ANOVA, F(3, 36) = 7.42, P == 0.0005). Stage B lobsters also had significantly larger carapace lengths than either stages C or D (ANOVA. F(3. 36) == 15.69. P = 0.0001). To determine whether any of the characteristics of the escape response were correlated with weight, linear regres- sions were calculated in which each of the following seven parameters were evaluated against weight, irrespective of molt stage: (1) probability of tailflipping: (2) duration of escape swimming: (3) tailflip frequency, (4) velocity, and (5) acceleration of the total escape swim; (6) force exerted during the swim; and (7) work performed. No correlation was found between the animals' weights and any of the parameters tested (R2 < 0.20; F > 0.05). Of the animals tested, only 15 out of 40 tailflipped. When the weights of animals that tailflipped (Table Ib) were compared, there were no significant differences among the four molt stages (ANOVA. F(3. 1 1 ) = 2.61 , P = 0.1 1 ). These animals were therefore subsequently used to analyze the characteristics of the escape response with respect to weight and molt stage. Among the animals that did not tailflip (Table Ic), there were significant molt-stage differences in the weights of the animals. Stages A and B weighed significantly more than stages C and D( ANOVA, F(3. 21) = 4.22, P = 0.002). More soft-shelled lobsters ( Id out of 20) than hard-shelled animals (9 out of 20) did not tailflip, suggesting that size in this weight class could determine whether an animal will tailflip; possible reasons for this are detailed in the discus- sion. B. Parameters of the athtlt escape response A summary of all the parameters tested, with means and standard deviations for each molt stage, are summarized in Table 2. Precision of measurements is a function of the number of video frames per second; therefore, since the power stroke and subsequent swims were separately ana- Ivzed, their mean values are not additive. Table 1 Weight (in grains) for adult lobsters in the four molt stages; values are mean ± standard error of the mean Softshelled premolt Hardshelled mtermolt Premolt STAGE A STAGE B STAGE C STAGE D (a) Combined wei ghts of all lobsters irrespective of escape behavior 514.3 595.1 420.3 372.1 474,2 512.1 421.0 403.4 470.0 582.0 373.0 440.2 592.2 358.6 456.0 326.2 401.0 544.2 435.6 424.5 497.0 441.4 417.4 484.9 400.0 487.6 438.7 396.0 535.1 438.0 390.6 360.4 476.2 513.4 433.5 378.0 420.8 596.3 369.0 4r,5.l 478.1 ± 19.3 506.9 ± 24.6 415.5 ± 9.2 405,1 ± 15.5 (b) Animals that tailflipped 470.0 441.4 420.3 403.4 420.8 513.4 421.0 326.2 456.0 484.9 435.6 396.0 438.7 360.4 378.0 445.4 ± 24.6 477.4 ± 36.0 434.3 ± 6.6 391.5 ± 21.8 (c) Animals that did not escape 514.3 595,1 373.0 372,1 474.2 512.1 417.4 440.2 592.2 358.6 390.6 424.5 401.0 544.2 433.5 465.1 497.0 582.0 369.0 400.0 487.6 535.1 438.0 476.2 596.3 486.3 ± 22.4 514.2 ± 29.7 396.7 ± 12.6 425.5 ± 19.7 /. Total escape response (initial power swim plus sub- sequent swims) Intermolt (stage C) animals tailflipped farther than either postmolt (stages AB) or premolt (stage D) animals (KW, X2 = 5.42, P = 0.046; Fig. 3A). Although the total time spent in the escape response was not significantly different among the three molt stages (KW, x2 = 2.58, P = 0.28; Fig. 3B), the velocity (distance/time) of the swim was also significantly higher for intermolt animals than for postmolt lobsters (KW, X2 = 5.94, P = 0.041; Fig. 3C). Although the apparently shorter duration of the swim for premolts was not significant, when time was used to calculate acceleration (velocity/time), the resulting value became significantly greater for premolt (stage D) lobsters (KW, x2 ' 6.76, P = 0.034; Fig. 3D). Neither the force (weight X accel- eration) exerted nor the work (force X distance) performed proved to be significantly different for the three molt stages MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR Table 2 «/ .\ixnitiiwtr differences iiiiii>iit> all components analyzed over the escape response 271 Molt Component stage Total escape response Initial power swim Total subsequent SU HI! Subsequent swims 1 (SSI ) Subsequent swims 2 (SS2) SSI vs. SS2 Distance (m) NA NA NA C > (AB = D) C > (AB = D) C > (AB = D) AB 0.29 ± 0.27 0.06 ± 0.01 0.25 ± 0.23 C 0.71 ± 0.10 0.17 ± 0.02 0.64 ± 0.08 D 0.39 ± 0.05 0.08 ± 0.02 0.35 ± 0.23 Duration (s) NS NS NS NS NS NS AB 1 .08 ± 0.53 0.29 ± 0.23 1.03 ± 0.47 0.35 ± 0.24 0.70 ± 0.39 C 1.32 ±0.29 0.13 ± 0.04 1.08 ± 0.39 0.38 ± 0.10 0.86 ±0.16 D 0.81 ±0.58 0.21 ± 0.13 0.75 ± 0.62 0.29 ± 0.25 0.61 ± 0.38 Velocity (m/s) C > (AB = D) NS NS NS NS NS AB 0.28 ± 0.22 0.63 ± 0.24 0.65 ± 0.26 0.90 ± 0.29 0.38 ± 0.08 C 0.56 ±0.14 0.33 ± 0.26 0.28 ± 0.28 0.51 ± 0.33 0.25 ±0.18 D 0.15 + 0.12 0.44 ± 0.46 0.37 ± 0.09 0.64 ± 0.29 0.27 ± 0.04 Acceleration (m/s/s) D > (AB) = C NS D > (AB) = C NS NS NS AB 0.27 ± 0.19 2.87 ± 3.06 0.22 ±0.16 1.77 ± 1.00 0.38 ± 0.26 C 0.46 ± 0.20 5.64 ± 3.16 0.36 ±0.12 2.66 ± 1.42 0.47 ±0.18 D 0.55 ± 0.20 4.12 ± 4.1 0.50 ±0.18 3.27 ± 1.79 0.66 ± 0.49 Force (N; (kg • m/s/s) NS NS NS NS NS NS AB 0.14 ± 0.11 1 .56 ± 1 .66 0.10 ± 0.06 1.02 ± 0.61 0.22 ± 0.16 C 0.17 ± 0.07 2.00 ± 0.88 0.14 ±0.04 0.95 ± 0.39 0.18 ± 0.07 D 0.22 ± 0.09 1.51 ± 1.39 0.20 ± 0.06 1.23 ± 0.69 0.25 ± 0.20 Work (J) NS NS NS NS NS NS AB 0.06 ± 0.09 0.16 ± 0.12 0.05 ± 0.04 0.04 ± 0.02 0.02 ± 0.01 C 0.12 ± 0.05 0.20 ± 0.06 0.11 ± 0.06 0.09 ± 0.04 0.02 ± 0.03 D 0.05 ± 0.03 0.15 ± 0.07 0.04 ± 0.07 0.03 ± 0.06 0.01 ± 0.03 Number of tailflips (Tf) NS NA NS NS NS NS AB 3.5 ± 1.9 2.5 ± 1.5 1.8 ± 0.9 0.9 ± 0.3 C 5.8 ± 1.8 4.8 ± 1.8 3.6 ± 1.0 1.3 ± 0.4 D 3.5 ± 3.1 2.5 ± 2.7 2.0 ± 2.0 1.6 ± 0.8 Frequency (Tf/s) (C = D) > AB NA (C = D) > AB (C = D) > AB NS Cl > C2 AB 3.05 ± 1.08 6.96 ± 1.69 3.63 ±3.17 3.33 ± 2.59 Dl > D2 C 4.52 ± 1.34 10.52 ±4.17 8.40 ± 3.54 2.12 ±0.91 D 3.94 ± 1 .02 10.78 ± 1.02 9.13 ± 2.40 1 .65 ± 1 .94 Distance/Weight (rn/kg) NS NS NS NA NA NA AB 0.52 ± 0.46 0.10 ± 0.03 0.41 ±0.50 C 1.67 ± 0.16 0.17 ± 0.03 1.52 ± 0.11 D 0.84 ± 0.72 0.21 ± 0.07 0.61 ± 0.72 DistanceAVeight/Tailflip (m/kg/Tf) C > (AB = D) NS C > (AB = D) NA NA NA AB 0.14 ± 0.07 0.10 ± 0.03 0.12 ±0.05 C 0.41 ± 0.09 0.17 ± 0.03 0.38 ± 0.06 D 0.23 * 0.07 0.21 ± 0.07 0.19 ± 0.05 Distance/Bodylength C >(AB = D) NS C > (AB = D) NA NA NA AB 3.5 ± 1.9 0.13 ± 0.03 3.1 ± 1.6 C 5.8 ± 1.8 0.10 ± 0.02 5.4 ± 1.7 D 3.5 ± 3.1 0.15 ±0.03 3.1 ± 2.8 A. B. C. and D represent the four molt stages. Mean ± SD of all components analyzed for three molt stages. Significant differences are indicated in boxes at the top of each column. Stages equated with those in the parentheses are not significantly different from them. AB. results of experiments with stages A and B were pooled due to only 4 animals that tailflipped. SSI. first half of the subsequent swimming distance; SS2. second half of the subsequent swimming distance. SSI versus SS2 compares the component in the two halves of the subsequent swimming distance. NA, not analyzed due to the experimental design (see methods). NS. no significant difference. 272 S. I. CROMARTY ET AL |A D C - A/B- 0.00 0.25 0.50 0.75 Total Distance (m) 1.00 D- C - A/B- 0.0 0.5 1.0 1.5 Total Time (s) 2.0 D- C - A/B- 0.0 0.2 0.4 0.6 Total Velocity (m/s) 0.8 D- C - A/B- 0 D- C - A/B- I, < 1 0 0.2 0.4 0.6 0. Total Acceleration (m/s/s) 1 I :: ' 1 ' D- C - A/B- 0.1 0.2 0.3 Total Force (kgm/s/s) 0.4 H 0.00 0.05 0.10 0.15 Total Work (J) 0.20 Figure 3. Parameters of the total escape response (initial power swim plus subsequent swims) for adult lobsters in all three molt stages. An asterisk (*) indicates significant differences. (A) Distance traveled in meters (in). (B) Time spent escaping in seconds (s). (Cl Velocity of tailflips in meters/second (m/s). (D) Acceleration in meters/second/second (m/s/s). (E) Force of tailflips in newtons (kg • m/s/s). (F) Work produced (force x distance) is measured in joules (J). (KW, x2 = 4.98. P = 0.083 and ,Y: = 2.15. P = 0.16: Fig. 3E and 3F, respectively). Although the total number of tailflips and the total time were not significantly different (KW. x2 z 4.20, P = 0.123 and x2 = 2.58, P = 0.275, respectively), swim frequency was significantly higher for intermolt and premolt lobsters, with stage C and D lobsters performing more tailflips per second than AB animals (KW, x2 = 6.93. P = 0.048; Fig. 4A). Distance traveled per lobster weight per tailflip was greater for intermolt (stage C) animals than for the other molt stages (C > (AB = D); KW, x2 = 5-98, P = 0.046, Fig. 4D), and distance traveled per bodylength was also greater for intermolt animals than for the other molt stages (stages (C > (AB = D); KW. x2 = 5.36, P = 0.047, Fig. 4F). 2. Initial power swim Except for distance traveled, none of the parameters were significantly different for the three molt stages at P < 0.05, no doubt because of the large variability among the animals that exhibited an escape response. However, trends in the tests suggest that intermolt and premolt lobsters executed a faster, more accelerating, and more forceful power swim than postmolt animals (0.05 < P < 0.10; KW). 3. Subsequent swiin\ a. Entire subsequent swim Of the original 15 animals that responded to the stimulus with escape swimming, only 12 executed subsequent swims. Of these. 3 were postmolts (stages A and B), 5 were intermolts (stage C). and 4 were premolts (stage D). The following parameters were statistically different: the fre- MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR 273 D- C- A/B- — D- C- A/B- H * • ' -H - i i i 01234567 ! Total Frequency (TF/s) . — ~ET] E A/B 5 10 15 20 o 5 10 15 Subsequent Swim Frequency (TF/s) ss Frequency j vs ss Frequency 2 D- C- A/B- D- C- A/B- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 «•» 0.1 0.2 O.J 0.4 0.5 0.6 Total Distance/Weight/Tailflip Subsequent Swim Distance/Weight/Tailflip D- C- A/B- D- C- A/B- 01234567 Total Distance/Bodylength 02468 Subsequent Swim Distance/Bodylength Figure 4. Mean frequency of tailflips (Tf/s) for adult lobsters for (A) total swim sequence; (B) the total subsequent swims frequency, and (C) the subsequent swims of both the first and second halves. Mean distance per weight per tailrlip (m/kg/Tf) for the total swim sequence (D) and for the total subsequent swims — that is. minus the power stroke (E). Mean distance per bodylength for total distance of swim sequence per bodylength (F) and for the total subsequent swims distance per bodylength (G). Stage AB (n = 3), stage C (;i = 5), and stage D (n = 4). An asterisk (*) indicates significant differences among the molt stages; a triangle (A) indicates significant differences between the two halves of the subsequent swims. quency of swimming was higher for stages C and D (KW X2 = 7.92, P = 0.037; Fig. 4B); the distance swum per weight per tailflip was higher in intermolt (stage C) animals (KW, x2 = 8.01, P = 0.028, Fig. 4E); and distance per bodylength was greater for intermolt (Stage C) animals than for the other molt stages (KW, x2 = 6.46, P = 0.046, Fig. 3G). b. Comparisons of SSI and SS2 As in the previous study (Cromarty et ai, 1991 ), the total distance traveled by each animal during the entire subse- quent escape swims was divided in half and then the swim- ming parameters were compared for each of the two halves across the molt stages and between the two halves of the swimming distance within each molt stage. SSI = the first half of the distance; SS2 = the second half. Comparison of SSI across molt stages. In the first half of the subsequent swims, stage C and D lobsters swam at a higher frequency than stages A and B (MANOVA. F( 1 , 9) = 23.18, P = 0.014; Fig. 4C). Comparison of SS2 across molt stages. No significant differences were found in any of the parameters for the second half of the subsequent swims among the molt stages (Table 2). Comparison of SSI and SS2 (SSj vs. SS,J within each mult stage. For the following parameters — subsequent swimming distance, duration, velocity, acceleration, force, work output, number of tailflips — no significant differences were found between the two halves of the subsequent swims within each molt stage. There was a significant drop-off in the frequency of swims between the first to the second halves of the subsequent swims for hard-shelled (stages C and D), while no differences between SSI and SS2 were 274 S. I. CROMARTY ET AL observed for soft-shelled (stages A and B) lobsters (MANOVA F(\. 9), P < 0.003; Fig. 4C). Comparison of SSI and SS2 across molt stages. Fre- quency of swimming was significantly different among the three molt stages (MANOVA. P < 0.026). Among hard- shelled lobsters (stages C and D). the frequency of swim- ming was greatly reduced in the latter half of the escape swims (Fig. 4C). C. Post-threat behavior There was a gradual increase in the aggression index of all lobsters in the experiment, such that stage A had an index value of 0.3 ± 0.5. while stage D had a value of 2.2 ± 1.4. The values for stage D were significantly greater than for molt stages A. B, and C (ANOVA F(3, 39), P < 0.0012: Fig. 5A). This is especially interesting given that stage B animals were significantly larger than stages C and D (see section A above), yet the post-threat aggression of the smaller hard-shelled lobsters was significantly higher. Among the animals that tailflipped, there were significant differences in the aggression index among the four molt stages (ANOVA F(3. 1 1 ). P < 0.02): soft-shelled lobsters (stages A and B) had very low or zero aggression towards the stimulus, whereas hard-shelled lobsters (stages C and D) had an overall aggression index of 1.4 ± .09 (Fig. 5B). Importantly, no weight differences were observed among the molt stages for the animals that did tailflip. When the post-threat behaviors of lobsters that did not tailflip were compared over the molt stages, a progression in the index was observed: starting with a value of 0.4 ± 0.5 for stage A lobsters, the index gradually increased until the index for stage D animals was 3.3 ± 0.5. Stage D lobsters had a significantly higher aggression index than molt stages A, B, and C (ANOVA F(3, 21). P < 0.0001: Fig. 5C). Although the soft-shelled animals were significantly larger than the hard-shelled ones (see section A), the smaller hard-shelled lobsters were more aggressive in their post- threat behaviors. Discussion In this study, we show that, like juveniles, adult male lobsters display significant molt-related differences in es- cape behavior. However, the escape behavior of adults, unlike that of juveniles (weight less than 100 g), is also influenced by physical factors. Thus, we have found that among animals that did not respond to a threat with an escape response, soft-shelled adults weighed significantly more than hard-shelled adults. This suggests that an animal's weight begins to modify the molt-dependent swimming response to threat. In our earlier experiments, all juvenile animals (both soft and hard-shelled; ±14 g) responded to a stimulus threat with escape swimming (Cromarty et «/.. 1991). No adults responded to the same stimulus that induced 14-g juveniles 3- 1- 3- •o C 1- •o C (10) (10) (10) do) T T Stage A Stage B Stage C Stage D (6) (5) (2) (2) Stage A Stage B Stage C Stage D D>(A=B=C) (4)T 3- (5) 2- (8) 1 - (8) I T fi Stage A Stage B Stage C Stage D Figure 5. Mean aggression index for post-threat behavior of adult lobsters for (A) all lobsters regardless of tailflipping; (B) lobsters that did tailflip; and (C) lobsters that did not tailflip. The numbers in parentheses represent the number of individuals in each molt stage; the statistical differences are displayed in a box in the upper left-hand corner of each graph. to swim. Indeed, adults failed to respond to a number of other stimuli (such as air bubbles, water injection, larger conspecifics) that were presented to them and ultimately responded only to a heavy weight (PVC tubing filled with pebbles and weighing 1.45 kg) dropped suddenly in their paths. There seems to be an inverse relationship between MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR 275 the probability of eliciting an escape response and the weight of an animal: 14-g juveniles tailflipped with a 100% probability; 450-g adults tailflipped with a probability of 50%, and 600-g adults failed to tailflip even when the stimulus size was doubled. Other workers have shown that lobsters weighing more than 600 g could be induced to tailflip only if their claws were autotomized (Lang et al., 1977), as we also have observed. It appears, therefore, that the effects of molt stage — that is, an animal's physiological condition, characterized by the hardness of its shell (Aiken, 1973: 1980). the flaccidity of its muscles (Passano, 1960), and the tilers of its hormones (Stevenson et al., 1979; Fadool et al., 1989; Snyder and Chang, 199 la, b) — begin to be modified by size and weight. That size and weight begin to modulate the molt-deter- mined characteristics of escape swimming can be seen if all our findings are taken into account. Among adults, as among juveniles, there was a significant drop off in the frequency and distance traveled/weight/tailflip during the second half of the subsequent swims for premolt hard- shelled animals, but not for postmolt animals. This suggests that escape swimming may have evolved as the primary survival strategy among soft-shelled juvenile animals, and that this strategy is retained in adults even as they become heavier; however, fewer large animals were likely to tailflip. perhaps because swimming becomes less energy-efficient (the heavier the animal, the more work is involved). Although size and weight appear to modulate the effects of molt stage on escape swimming, with larger adult soft- shelled animals not tailflipping, an inverse relationship to weight became apparent in the post-threat behaviors of our experimental animals. Regardless of whether the animals had tailflipped — and even when weight was taken into ac- count— the indices of aggression of the post-threat behav- iors increased incrementally from stages A and B (the largest animals) to stages C and D (the smallest animals). Weight and size appears to be of secondary importance in post-threat aggression; indeed, changes in aggression over the molt stages were the deciding factor, with the lobsters responding to a threat in accordance with the expected molt-related changes in aggressive behaviors (Tamm and Cobb. 1978). Undue significance should not be given to the inverse relationship of weight and size on aggression in general, however. In confrontations between lobsters in the same molt stage, the size of an opponent significantly af- fected the outcome of a bout (Scrivener, 1971 ; Mello et al., 1999; Bolingbroke and Kass-Simon, 2000). Overall, we found that among juveniles, soft-shelled an- imals were better swimmers than their hard-shelled coun- terparts, but among adults, hard-shelled premolt and inter- molt lobsters were the best swimmers. Thus juveniles of stage B outperformed stage C and D animals in the follow- ing parameters: distance traveled, number of tailflips pro- duced, distance/tailflip. time spent swimming, and velocity (Cromarty et al.. 1991). In contrast, among adults, hard- shelled premolt and intermolt animals outperformed soft- shelled animals in distance traveled, velocity, acceleration, frequency and distance traveled/lobster weight/tailflip. This would suggest that molt stage is the predominant determi- nant of the characteristics of escape behavior in smaller animals, while other physical factors such as weight and claw size may begin to dominate among adults. The physiological bases for the differences in adult and juvenile escape behavior over the molt cycle are likely to be manifold and varied. In addition to probable differences within the central nervous system, differences in endocrine, sensory, and motor systems are certain to exist. With regard to sensory systems (Watson. 1992). synaptic modulation has been described for mechanoreceptors (Pasz- tor and Bush. 1987) and stretch receptors (El Manira et al.. 1991). Studies by Coulter (1988) indicate that lobsters in stages C and D responded (with a meral spread) at different speeds to the presentation of an expanding black disc. Increases in lobster size have been correlated with a de- crease in the speed of an action potential traveling from the sensory system to the central nerve cord (Lang et al., 1977). It is possible that juvenile and adult lobsters perceive and respond to stimuli differently due to inherent age-related differences in sensory functioning. Other factors such as central (Kravitz et al., 1984; Krav- itz. 1988; Yeh et al., 1996. 1997; Horner et al.. 1997) and peripheral modulations (Kravitz et al., 1980; Kravitz, 1990; Schwanke et al., 1990) are likely to affect molt-cycle be- havior. We have recently found that 20-hydroxyecdysone (20-HE). the active steroid regulating the molt, also alters the neuromuscular properties of the claw-opener and phasic flexor systems in intermolt animals (Cromarty. 1995; Cromarty and Kass-Simon. 1998). in a way that is consis- tent with molt-determined behavioral differences (Tamm and Cobb. 1978; Cromarty et al., 1991). Our findings are consistent with the rise in the blood liter of 20-HE (Snyder and Chang, 1991a, b) when lobster aggression is beginning to peak. In our 20-HE experiments we found that 20-HE increases the size of the excitatory junctiona] potential (EJP) in the claw opener muscle, which is used in threat displays, and decreases the EJP amplitude in the abdominal phasic flexor, which is used in escape behavior. In crayfish. Cooper and Ruffner ( 1998) have found that EJP amplitude in the opener muscle of the walking legs is reduced; this keeps the dactyl from splaying and allows the animal to stand tall, as has been observed in dominant lobster displays (for recent review on modulation of aggressive behavior, see Kravitz, 2000). The effects of 20-HE on these above-mentioned tissues are consistent with the functions attributed to them during agonistic behavior. Our recent studies also show that when 20-HE is directly injected into the lobsters' hemo- lymph, aggressive behavior increases dramatically during agonistic encounters, although the probability of eliciting 276 S. I. CROMARTY ET AL escape swimming is unaltered (Bolingbroke and Kass-Si- mon. 2000). The sexual status of an American lobster may also alter its use of escape behavior. Intermolt gravid females that are presented with a startle stimulus do not tailflip. whereas intermolt males and non-gravid females tailflip readily (Cromarty et id., 1998): gravidity did not appear to affect escape behavior during a confrontation, but caused an in- crease in aggressive tail flipping (Mello et ai. 1999). It remains to be seen whether neuromuscular properties are modulated to reflect these sex-related behavioral differ- ences. Among juveniles, we have found that EJPs in the distal region of the muscle in soft-shelled stage B animals are larger and have a greater amplitude-duration integral than those of hard-shelled D stage animals (Cromarty et ul., 1995). Earlier studies by Schwanke ct ul. ( 1990) also found molt-related differences in the dactyl opener muscle. These findings correspond with the fact that stage B juveniles swim greater distances by covering more distance in each swim than do hard-shelled (stage C and D) juveniles. Fur- ther, although EJPs continue to be produced at frequencies up to 6 Hz in stage A and B juveniles, they begin to fail at 4 Hz in stage C and D animals. This also correlates with the fact that stage B juveniles swim longer and cover more ground, and that stage A animals are able to sustain swim- ming longer and at a higher frequency than stage C or D animals (Cromarty et ul., 1991: Cromarty et al.. 1995). Among adults, EJPs were found to fail at 4 Hz in soft- shelled postmolts but continued to 6 H/ in intermolt and premolt animals (Cromarty and Kass-Simon. 1994). As in juveniles, in adults, EJPs were largest and longest lasting in the distal region of soft-shelled postmolts. We have also found that EJPs in the proximal region of the abdominal phasic flexor muscles, the anchorage or insertion region of the contracting muscle, are significantly greater in adult C and D stages than in juvenile C and D stages (Cromarty and Kass-Simon. 1994). This coincides with their greater swim- ming ability and supports our present finding that escape swimming is used less as the animal grows, although hard- shelled animals retain it longer than soft-shelled animals do. This might be because escape swimming would be less effective in large soft-shelled animals — not only because of the large mass that would need to be propelled by the still-flaccid muscles, but also because the large transmitter output required by these muscles might not be sustainable for long periods at higher frequencies. Acknowledgments The authors thank Bill MacElroy for allowing us to collect animals while he was fishing offshore, and Tom Angell of the Rhode Island Department of Environmental Management for supplying us with additional lobsters. Thanks to Dr. Mike Clancy and Kathy Castro for help in lobster collection and maintenance. Drs. Stanley Cobb and Frank Heppner kindly provided laboratory space and equip- ment. We also thank Malia Schwartz for critiquing an earlier draft of the manuscript. This research was supported by a Whitehall Foundation grant to G.K-S. Grant-in Aid of Research from Sigma Xi and Lerner Gray Grants for Marine Research to S.I.C. Literature Cited Aiken, I). E. 1973. Proecdysis, setal development, and molt prediction in the American lobster (Hoiiuinis iimericaiuis). J. Fish. Res. Board Can. 30: 1334-1337. Aiken. D. E. 1980. Molting and growth. Pp. 91-163 in The Biology mid Management of Lobsters, vol. 1. J. S. Cobb and B. F. Phillips, eds. Academic Press. New York. 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The effect of social experience on serotonergic modulation of the escape circuit of crayfish. Science 271: 366-369. Yeh, S. R., B. E. Musolf, and D. H. Edwards. 1997. Neuronal adap- tations to changes in the social dominance stalus of the crayfish. J. Netomci. 17: 697-708. Reference: Bio/. Bull. 199: 278-286. (December 2000) Preferential Expulsion of Dividing Algal Cells as a Mechanism for Regulating Algal-Cnidarian Symbiosis GAREN BAGHDASARIAN* AND LEONARD MUSCATINE Department of Organismic Biologv, Ecology, and Evolution, University of California, Los Angeles, Los Angeles, California 90095-1606 Abstract. A wide range of both intrinsic and environmen- tal factors can influence the population dynamics of algae in symbiosis with marine cnidarians. The present study shows that loss of algae by expulsion from cnidarian hosts is one of the primary regulators of symbiont population density. Because there is a significant linear con-elation between the rate of algal expulsion and the rate of algal division, factors that increase division rates (e.g.. elevated temperature) also increase expulsion rates. Additionally, ?H-thymidine is taken up to a greater extent by algae destined to be expelled than by algae retained in the host cnidarians. Taken to- gether, data for rates of expulsion, rates of division at different temperatures, and uptake of H-thymidine suggest that dividing algal cells are preferentially expelled from their hosts. The preferential expulsion of dividing cells may be a mechanism for regulation of algal population density, where the rate of expulsion of algae may be an inverse function of the ability of host cells to accommodate new algal daughter cells. This kind of regulation is present in some cnidarian species (e.g.. Aiptasia pulchella. Pocillo- pura ilamicornis), but not in all (e.g., Montipora verrucosa, Porites compressa, and Fungia xctituriii). Introduction Algal-cnidarian symbioses are characterized by long- term stability wherein neither partner outgrows the other, and where algal population densities remain relatively con- stant (Drew, 1972; Pardy, 1974; Davies. 1984). During repopulation of aposymbiotic sea anemones (recovery from bleaching), symbiotic dinoflagellates grow at relatively high rates normally associated with log phase growth in culture (Berner et ai. 1993). If these high growth rates are sus- Received 2 August 1999; accepted 25 September 2000. * To whom correspondence should be addressed. tained. host fitness is reduced (Smith, 1992), leading to the eventual breakdown of the symbiosis (Neckelmann and Muscatine, 1983; Taylor et al.. 1989). Instead, as the size of the algal population reaches some optimum level, its growth rate decreases by a factor of 20 (Kinzie. 1974; Kinzie and Chee. 1979; Berner et al. 1993), and a "steady state" is achieved, in which the growth rates of the algae and the host cells are in dynamic equilibrium. Clearly, regulation of symbiont population density is essential in maintaining a stable symbiosis, yet little is known of the cellular mecha- nisms involved. A number of intrinsic and environmental factors can potentially regulate algal cell division and population growth. These factors could act pre- or post-mitotically (Hoegh-Guldberg and Smith, 1989); that is. population growth rate could be regulated either before or after algal cell division. Algal numbers could be regulated pre-mitoti- cally by limited nutrient availability (see, for example. Blank and Muscatine, 1987; Kolber et al., 1988; Falkowski ct ul.. 1993; Hoegh-Guldberg, 1994; Muller-Parker ct al.. 1994; Snidvongs and Kinzie. 1994); by density-dependent negative feedback by the algae themselves (Muscatine and Pool, 1979; McAuley and Darrah, 1990); by host-induced release of photosynthate from the algae (Muscatine, 1967; Cook, 1983; Douglas and Smith, 1984; Sutton and Hoegh- Guldberg, 1990; McAuley. 1992; Gates ct al.. 1995); or by factors manifested by the host cells that inhibit the algal cell cycle (Smith and Muscatine. 1999). Algal numbers could also be regulated postmitotically by degradation of algae in .situ (Muscatine and Pool. 1979; Titlyanov et ai, 1996; Jones and Yellowlees, 1997), by direct expulsion of excess algae (Hoegh-Guldberg and Smith, 1989: Stimson and Kin- zie, 1991; McCloskey et al.. 1996; Jones and Yellowlees, 1997), or by accommodation of excess algae by division of host cells (Muscatine and Pool, 1979; Neckelmann and 278 REGULATION OF ALGAL-CNIDARIAN SYMBIOSIS 279 Muscatine, 1983; Smith and Muscatine. 1986; Titlyanov et «/., 1996). Whereas previous studies have addressed pre-mitotic control of algae in symbiotic cnidarians. there is little in- formation on mechanisms involved in post-mitotic regula- tion, especially relating to loss of algae by expulsion. Jones and Yellowlees (1997) showed that the combined effect of changes in rates of algal division and loss are involved in repopulation of bleached corals and in the eventual regula- tion of steady-state algal-cnidarian symbioses. In a previous study, we observed that the Hawaiian sea anemone Aiptaxhi pulchellii maintained in the laboratory expels algae at a rate of about 0.046 d ' (Baghdasarian and Muscatine, unpubl. data), a value high enough to be a major factor in regulating algal densities. Studies of other cnidarians report not only that algae are lost by expulsion, but also that the expelled algae have higher mitotic indices than algae retained by the host (Suharsono and Brown, 1992; McCloskey etui., 1996). It has been hypothesized that the higher mitotic indices of expelled algae are due to release from some regulatory constraint by the hosts. In this study we test an alternative hypothesis, that higher mitotic indices of expelled algae are the result of preferential expulsion of dividing cells. It is important to note here that the release mechanism probably entails detachment of host cells (Gates et «/.. 1992), their disintegration, and release of algae. Whereas the mechanism of preferential detachment of host cells remains to be ad- dressed, here we focus on algal parameters. Materials and Methods Collection and maintenance of organisms Specimens of the sea anemone Aiptasia pulchella Carl- gren (1943) symbiotic with the dinoflagellate Symbiodinium pulchrorum were collected from Kaneohe Bay, Oahu, Ha- waii (spring and fall 1995/1996). The animals were trans- ferred by air to the University of California, Los Angeles, and maintained in natural seawater in 1 .5-liter glass bowls in a Percival model 1-35 VL incubator at 25°C on a 12-h light/dark cycle at irradiance levels of 80-100 jumole pho- tons m~2 s"1, from two Rainbow Lifeguard 40- watt Primetinic and two General Electric 40-watt Cool White light sources. The animals were fed twice a week in the evenings on Anemia sp. The morning after every feeding, the bowls were cleaned using cotton swabs, and the seawa- ter, collected from Redondo Beach, California, was re- placed. Prior to the experiments, sea anemones were starved for 24 h under the light and temperature conditions de- scribed above. The symbiotic corals Porites compressa Dana 1846, Montipora verrucosa Lamarck 1816, Pocillopora damicor- nis Linnaeus 1758, and Fungia scutaria Lamarck 1801 were collected from Kaneohe Bay, Oahu, Hawaii (October/No- vember 1996). The corals were transferred to the laboratory of the Hawaii Institute of Marine Biology, and were main- tained in running seawater and exposed to natural light levels. All experiments were performed within 24 h of the collections. Mitotic iin!c\ of algae Five A. pnlclwlUi were homogenized individually and their symbiotic algae isolated by repetitive centrifugation and resuspension (Steen, 1987). The technique was applied rigorously to eliminate the possibility of inclusion of algae still within host cells. The algal mitotic index (MI), defined as the percentage of cells with division plates (Wilkerson et ai, 1983) was determined by examination of at least 1000 algal cells under 400 X magnification using an Olympus BH-2 microscope. All additional microscopic observations were made under the same specifications. Incorporation of3H-thymidine by algae in situ Each of five A. pulchella specimens was incubated indi- vidually in 4 ml of a 2-^Ci/ml solution of 3H-thymidine (Sigma Chemical Company; Sp. act. 50 Ci per mmol) in filtered seawater (FSW) for 24 h under the same mainte- nance conditions as the stock animals. The sea anemones were then washed serially 10 times in FSW to remove unincorporated 3H-thymidine. Each wash consisted of add- ing fresh FSW, irrigating the coelenteron with a Pasteur pipette, and then waiting 3 min before changing the water again. Seven to nine washes were sufficient to remove unincorporated 3H-thymidine from the sea anemones and the incubation medium. Algae expelled during the labeling period were discarded. 3H-thymidine-labeled sea anemones were then incubated in FSW for an additional 1 5.5 h at 25°C in the light. At the end of the incubation period, both the algae released and those remaining in the hosts were iso- lated (using techniques described above on ice), adjusted to known volumes, and counted with a hemacytometer (Fisher Scientific). One-hundred-microliter samples of algae, along with 5 ml of Bio-Safe II biodegradable counting cocktail, were then added to plastic scintillation vials, and the incor- poration of 3H-thymidine by the algae was determined using an LKB Wallac 1214 Rackbeta liquid scintillation counter. Results were expressed as DPM * (106 algae)"1. To deter- mine whether the incorporation of 3H-thymidine was by the algae or by host cells that contain the algae and may have been released along with them, techniques described in Gates and Muscatine (1992) were used to stain cells with Hoechst 33258 to check for the occurrence of host cell nuclei. Rates of expulsion of algae Fourteen sea anemones were allowed to settle in 4 ml of FSW in 15-ml test tubes. Six of the sea anemones were 280 G. BAGHDASARIAN AND L. MUSCAT1NE incubated for 15.5 h at 25°C in the light, and the other eight were incubated for 15.5 h at 27.5°C in the light. After the incubation, algae in the incubation medium were recovered by centrifugation and set aside for analysis. Algae remain- ing in the animals were isolated by homogenization and centrifugation (Steen. 1987). Both the algae expelled into the medium and those retained by the hosts were counted, and the MI of the expelled algae was determined (Hoegh- Guldberg er al., 1987). Mitotic index and rate of expulsion of algae in corals Mitotic indices and rates of algal expulsion were also estab- lished for four species of scleractinian corals commonly found in Kaneohe Bay, Oahu, Hawaii. Six pieces of each coral type from different colonies (P. compressa, M. verrucosa, P. dami- comis, and in the case of F. scutaria, entire corals) were incubated in 30 ml of FSW at 27°C (seawater temperature in Kaneohe Bay) for 15.5 h. Next, to determine the effect of slight elevations of temperature on MI values and algal expulsion rates, six additional pieces from each coral type were incubated at 29.5°C for the same length of time. At the end of the incubation period, the corals were removed from the seawater. The released algae were collected by high-speed centrifugation of the incubation medium using a Damon IEC clinical centri- fuge, followed by resuspension in 5 ml of fresh FSW. Algae remaining in the corals were removed with a toothbrush. The product was collected in FSW. The algae were then cleaned of mucus and animal tissue by centrifugation and resuspension in 20 ml of FSW, and MI values and rates of algal expulsion were determined using the techniques described above. Results Mitotic index of algae: natural expulsion vs. mechanical isolation Algae naturally expelled from A. pulchella have a higher MI than the algae remaining in the hosts (Fig. 1; Wilcoxon signed rank nonparametric test: P = 0.001 ). To determine if the higher MI of the expelled algae is due to the absence of host-related regulation, algae were mechanically isolated from the sea anemones, and the MI was measured immedi- ately and after a 15.5-h incubation at 25°C in the light. No significant changes in MI were observed following the 15.5-h incubation period (Wilcoxon signed rank nonpara- metric test: P = 0.593). These data suggest that the higher MI of algae expelled by A. pulchella is not necessarily due to release from putative host-related regulation. Incorporation of H-thymidine b\ algae in situ To determine if the higher MI of expelled algal cells is due to preferential expulsion of dividing algae, sea anemo- nes were incubated with ^H-thymidine for 24 h, rinsed free of unincorporated 'H-thymidine, and then incubated in Figure 1. Mitotic index of the symbiotic alga Symbiodinium pulchro- nim after a 15.5-h incubation of the host sea anemone. Aiptasia pulchella, at 25°C. Comparison between expelled algae (H) and algae retained by the host (•). Error bars represent standard deviations of the mean. FSW for 15.5 h. Algae expelled and algae retained in the hosts were then assayed for incorporation of 3H-thyrnidine. Expelled algae had incorporated significantly higher levels of ?H-thymidine than cells remaining in the hosts (Fig. 2; Wilcoxon signed rank nonparametric test: P -- 0.043). Because released algae are often contained within host cells (Gates et al.. 1992), it was important to determine whether the ^H was associated with the algae or with the nuclei of host cells. This question was investigated by staining sam- ples of retained and released algae with Hoechst 33582 to detect host cell nuclei that might be associated with the algae. Using epifluorescence microscopy to analyze the cells (Gates et al.. 1992), we found no evidence of host cell nuclei (i.e.. host nuclear DNA contamination). Taken to- gether, these data suggest that the host preferentially expels algal cells that have entered S-phase of the cell cycle. Mitotic index and rate of expulsion of algae The correlation between the MI of the expelled algae and the rate of expulsion of these cells from the host could distinguish between expulsion of algae in random phases of the cell cycle YITMIX expulsion of algae in a preferred phase of the cell cycle. If expulsion of algae from the host is random, then there should be no correlation between division rate of the expelled algae (i.e., MI) and rate of expulsion. If. however, expulsion of algae is a function of cell cycle phase (more specifically, preferential release during late G-, or M phases), then a positive correlation between the two parameters would be expected, as illustrated REGULATION OF ALGAL-CNIDARIAN SYMBIOSIS 281 < ^o 6000- 5000- 4000- 3000 2000- 1000- •c c. Figure 2. 'H-Thymidine incorporation by the symbiotic alga Symhio- Jimum piilclirnniiii after a 15.5-h incubation of the host sea anemone. Ai/nusui pulchella. in tillered seawater. Comparison between expelled algae (M) and algae retained by the host (•). Sea anemones were initially incubated in a 2-/j,Ci/ml solution of 'H-thymidine for 24 h. Error bars represent standard deviations of the mean. theoretically in Figure 3. Figure 4 shows that there is a positive linear correlation between expulsion rate of algae and their MI. Effect of temperature on rate of algal expulsion If expulsion of algae is affected by algal division rate, then environmental factors (such as slight increases in sea- water temperature) that increase algal division rate (and hence MI) should also increase rate of expulsion of algae. Slightly elevated temperatures resulted in higher MI and expulsion rates (Fig. 5; Wilcoxon signed rank nonparamet- ric test; Expulsion rate: P == 0.034, MI: P == 0.050). Further, the ratio of algal expulsion to MI at 25°C = 2.2, and at 27.5°C = 2.4. The similarity of these two ratios suggests that the higher MI values due to slight elevations in temperature are concomitant with higher algal expulsion rates. Finally, at 27.5°C, the observed changes in rates of algal division and expulsion follow the same positive linear correlation associated with preferential algal expulsion as a function of their MI (Fig. 6). Rates of algal expulsion in corals The relation between algal expulsion rate and division rate was investigated in four species of Hawaiian corals. In general, the released algae had a higher MI than did the algae remaining in the hosts (Fig. 7; Wilcoxon signed Algal Expulsion Rate Figure 3. Hypothetical correlation between mitotic index of expelled symbiotic algae and their rate of expulsion from the hosts under conditions of preferential versus random cell expulsion. rank nonparametric test; significant differences seen in Pocillopora damicornis, Montipora vcrrucosa, Fungia scutaria: P = 0.028. but not in Porites compressa: P = 0.249). However, the linear correlation between algal 2.0-1 1.8- 1.6- T3 o = 1-4 o 1.2 i.o- <*• 0.81 0.6 0.00 0.02 0.04 0.06 0.08 0.10 -1. Algal Expulsion Rate (d ) Figure 4. Correlation between mitotic index and rate of expulsion of Synihiiiiliiiimn r>iilclir,iridiiiiuni imlchrnniin released from the symbiotic system. Error bars represent standard deviations of the mean. expulsion rate and MI, observed in A. pulchella, ap- pears to hold only for P. dainicornis (Fig. 8a): it does not hold for P. compressa. M. rernicosu. or F. xcutaria (Fig. 8b, c, d). 4.0n o re ac Q. X UJ 3.0- 2.0- •-S 1-0-1 0.0 0.00 0.05 0.10 0.15 0.20 -1 Algal Expulsion Rate (d ) Figure 6. Correlation between mitotic index and rate of expulsion of S\inhn ijinium pulchrorum under control and elevated temperatures. 25°C control (D). 27.5°C experimental (A). )' = -0.53328 + I7.244.V: R~ = 0.771. Figure 7. Mitotic indices of expelled algae (O) and algae remaining within host tissues (•) of different corals, following a 15.5-h incubation at 27°C. Error bars represent standard deviations of the mean. Discussion In algal-cnidarian symbioses, regulation of algal numbers is an essential part of the symbiotic relationship, both during "steady state" (Muscatine et id., 1975a, b; Trench, 1987) and during recovery of cnidarians from "bleaching events" (Gates, 1990; Hayes and Bush. 1990; Fitt et al.. 1993; Jones and Yellowlees, 1997). The present study has established that pref- erential expulsion of dividing algae contributes to regulation of algal-cnidarian symbiosis. If dividing algal cells are more likely to be expelled from the host, net algal population growth (within their hosts) will be effectively regulated. Mitotic index of algae expelled naturally versus isolated mechanically Algae naturally expelled from Aiptasia pulchella and other Hawaiian marine cnidarians have a higher MI than the algae remaining in their hosts (Figs. 1,7). Suharsono and Brown (1992) and McCloskey et al. (1996) have also observed this phenomenon in other cnidarian species. Citing the most parsi- monious explanation, these studies suggested that the increase in algal division rates is perhaps due to a lack of host regulation in the released algae. The present study tests this hypothesis by addressing an alternative hypothesis — that the higher MI of the released algae could be explained by preferential expulsion of dividing algal cells. These two possibilities were tested by separating the algae from the host and observing any changes in the algal MI. If the increase in MI is simply a function of a lack of host regulation, then artificially releasing the algae should result in an increase in MI. However, algae artificially 5.0-1 C 4.0 u .£ 3.0- D. X UJ o 2 2.0 •o c - 1.0 2 REGULATION OF ALGAL-CN1DARIAN SYMBIOSIS 4.0T 283 Pocillopora damicornis o.o- 0.00 0.02 0.04 0.06 Algal Expulsion Rate (d ) oo 3.0- T3 111 2.01 o X II •O £ 1.0- I 0.0 ^orites compressa 0.00 0.05 0.10 0.15 Algal Expulsion Rate (d ) 3.0- 01 r: CO 2.0- £ 1.0- 0.0 Montipora verrucosa 0.00 0.02 0.04 0.06 0.08 0.10 Algal Expulsion Rate (d '^ ) 3.0- 2.0- o X Ol •u £ 1.0- 0.0 Fungia scuta 0.0 0.1 0.2 0.3 Algal Expulsion Rate (d ) D Figure 8. Correlation between mitotic index and rates of expulsion for various hermatypic corals from Kaneohe Bay, Oahu. Hawaii. (A) Pocillopora Jamicornis (Y = 0.57457 + 54.019.Y: R~ = 0.869). (B) Ponies compressa (Y = 2.0435 - 3.7634X; R2 = 0.023). (C) Montipora verrucosa (Y = 1.5287 + 0.12587.Y: R2 = 0.000). (D) Fungia stntariu (Y = 1.3582 + 0.111')2X: R2 = 0.000). released from their host sea anemones (A. pitlchella) in this study showed no increase in MI. Incorporation of H-thymidine b\ algae in situ Uptake and incorporation of 3H-thymidine is an indicator of cells advancing through S-phase of the cell cycle. In A. piilchella. cells naturally lost from the symbiosis (following a preincubation in 'H-thymidine) incorporate more 3H-thy- midine (just prior to release) than did cells remaining in the hosts (Fig. 2). These data suggest that the released algae had been in S-phase during the 3H-thymidine incubation and, by inference, were growing and dividing. Therefore, it can be concluded that dividing cells are preferentially expelled from the population. 284 G. BAGHDASARIAN AND L. MUSCATINB This is the first study to successfully employ 3H-thymi- dine as an indicator of algal division in symbiotic dinoflagellates. Cheney ( 1974) observed uptake of ?H-thy- midine by the host cells of the coral Pocillopora damicor- nis, but not by the symbiotic algae. Absence of H-thymi- dine in the algae may have been due to ( 1 ) very low growth rates of the resident algae, resulting in minimal uptake of the 3H-thymidine; (2) uptake and retention of the 3H-thymidine by the host, thus minimizing availability of H-thymidine to the algae; or (3) a high percentage of substitution of the base thy mine by 5-hydroxymethyluracil in the algal DNA (Blank er nl.. 1988: Taylor, 1990), resulting in low affinity for the 3H-thymidine molecules in P. damicornis. The success of the present experiment may be attributed, in part, to the application of longer incubation times or higher doses of 3H-thymidine. 3H-thymidine is a tool that, in parallel with more classic approaches, may be useful in studying regula- tion of algal-cnidarian symbiosis; in this case, it has pro- vided evidence for preferential loss of dividing algae from symbiotic cnidarians. Mitotic index, algal expulsion, and effect of temperature Figure 4 shows a direct linear correlation between expul- sion and division rates of algae. That is. higher rates of division translate into greater expulsion of algae, supporting our suggestion of preferential loss of dividing cells (Fig. 3). Further, small increases in temperature, which result in slightly higher rates of division, also result in greater ex- pulsion of algae (Fig. 5). This increased expulsion follows the same linear correlation patterns (relative to division rate) observed previously (Fig. 6). The data therefore strongly support the interpretation that expulsion of algae is prefer- ential and related to the position of the algal cells within the cell cycle. Expulsion of algae as a mechanism for regulating algal- cnidarian symbiosis The mechanism involved in preferential expulsion of dividing cells is not clear, but appears to be related to the host's ability to accommodate algal growth. Whereas the expulsion rate of algae from /\. pnlchella in "steady-state" symbiosis is about 0.046 d ', expulsion of algae from re-infected aposymbiotic anemones during log phase re- population is negligible (pers. ohs.), confirming a previous study on repopulation of bleached individuals of the coral Acroporaformosa (Jones and Yellowlees, 1997). Therefore, it appears that algal cells are primarily expelled from the system when the host cells can no longer accommodate them. This process can act as a "fine tuning" mechanism for regulating a steady-state symbiosis, where expulsion of algae may be viewed as an inverse function of the host's ability to accommodate new algal cells. Further, this mech- anism could explain the differential expulsion of algae from tentacle versus body regions of the cnidarians, where dif- ferences in MI and algal densities would require different rates of expulsion from those regions (Muller-Parker and Pardy. 1987). Preferential expulsion of dividing cells can also play a stabili/ing role in algal-cnidarian symbiosis by dampening the effects of environmental conditions that can influence algal division rates. For example, whereas large increases in temperature can lower algal photosynthetic capacities, small increases in temperature may increase algal photosynthesis, metabolism, and thus growth and rates of division (Iglesias- Prieto ct al.. 1992). However, if rates of algal expulsion vary as a function of the environmentally induced changes in rates of division (Fig. 6), then effectively, by releasing the "excess" cells, the total number of algae within the host will be regulated. Preferential expulsion of dividing cells is not, however, the only means of regulating algal-cnidarian symbiosis. In case of the green hydra symbiosis, studies have already shown that algal ejection is not the normal mechanism for regulating population densities (McAuley. 1982). probably because of the higher numbers and growth rates of algae per host cell. Among Hawaiian anthozoans. although A. pulch- clla and P. damicornis do use preferential expulsion of dividing cells as a regulatory mechanism, other cnidarian species, such as P. compressa, M. vermcosa. and F. xcu- taria. may not (Fig. 8). This interspecific variability could be a function of the magnitude of "normal" algal growth rates at steady-state for each of the different species con- sidered. Species with higher steady-state growth rates might be more likely to depend on a system of preferential expul- sion of dividing cells for regulating their symbiosis. In contrast, among species that have very low daily rates of algal expulsion (such as Xenia macroxpiculata, Heteroxenia fiiscescens, Stylophora pistillata. and Millepora dichotoma; Hoegh-Guldberg et al.. 1987), the incidence of expulsion of algae would not significantly affect the regulation of popu- lation dynamics. Another possibility is that cnidarians with different algal clades may have evolved different mecha- nisms of regulation based on sensitivity to environmental factors or on the physiology of their hosts (Rowan et «/., 1997). In the case of M. vermcosa, P. compressa. and F. scutaria. the higher Ml values of the expelled algae relative to those remaining in the hosts (Fig. 7) may simply be due to a lack of host regulation of division following algal release (Suharsono and Brown. 1992; McCloskey et al.. 1996). A complete understanding of the role of expulsion of algal cells in regulating algal-cnidarian symbiosis requires further research into the release mechanisms involved. This study has shown that at least some symbiotic cnidarians preferentially expel dividing algal cells. In combination with other regulatory factors, this pattern of cell loss can REGULATION OF ALGAL-CNIDARIAN SYMBIOSIS 2S5 play a major role in regulating ulgal-cnidarian symbiosis in steady state. Acknowledgments This research was partially funded by various National Science Foundation grants to L.M. We thank two anony- mous reviewers for their comments on this work. Literature Cited Berner, T.. G. Baghdasarian. and L. Muscatine. 1993. Repopulation of a sea anemone with symbiotic dinoflagellates: analysis by in vivo fluorescence. J. Exp. Mar. Biol. Ecoi 170: 145-158. Blank, R. J., and L. Muscatine. 1987. How do combinations of nutri- ents cause symbiotic Chlorella to overgrow hydra? Symbiosis 3: 123- 134. Blank, R. 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Leletkin, J. Tsukahara, R. van Woesik, and K. Vamazato. 1996. Degradation of zooxanthellae and regulation of their density in hermatypic corals. Mar. Ecol. Prog. Set: 139: 167-178. Trench, R. K. 1987. Dinoftagellates in non-parasitic symbiosis. Pp. 530-570 in The Biology of Dinoflagellates. F. J. R. Taylor, ed. Black- well. Oxford. Wilkerson, F. P., G. Mullen-Parker, and L. Muscatine. 1983. Tem- poral patterns of cell division in natural populations of endosymbiotic algae. Limnol. Ocfumif-r. 28: 1009-1014. Reference: BioL Bull- 1ni.i pulchenimus and Scuphirhiiiiu mimhilis. Embryos of both species were cultured at 18°C and observed hourly. Embryos were embedded in Spurr resin. (A-F) H. pulcherrimus, 17-22 h. (G-L) S. mimhilis, 14-19 h. In H. pulcherrimus embryos, the primary and secondary invagination is clearly distinguished by the presence of a pause in the archenteron elongation (C-D. 1-2 hours). After the occurrence of the secondary invagination. the archenteron became slender. In S. mimbilis embryos, the archenteron invaginated contin- uously, and the diameter of the archenteron remained unchanged during gastrulation. The scale bar indicates 50 /xm. attachment. The processes of gastrulation were photo- graphed at intervals of 10 min. Results Morphological changes during gastrulation Figure 1 shows the processes of gastrulation in embryos of H. pulcherrimus (A-F) and S. mirabilis (G-L) kept at 18°C. In a regular echinoid, H. pulcherrimus, primary (Fig. 1 A-C) and secondary invagination (Fig. 1 D-F) were clearly distinguished by the presence of a time lag in archenteron elongation (1-2 h. Fig. 1C-D). On the other hand, the archenteron of S. mirabilis embryos elongated at a constant rate during the course of invagination (Fig. 1G-L). Besides archenteron elongation, several differences were noticed in the morphology of the embryos of these two species. In H. pulcherrimus, the height of the embryo in- creased to some extent during primary invagination. After the onset of secondary invagination, the embryos were shortened along the animal-vegetal axis (Fig. 2A). The width of the embryos increased as gastrulation progressed (Fig. 2B). This was caused by the expansion of the ecto- dermal layer, especially at the lateral blastocoel wall (Fig. 2C). In contrast, S. mirabilis embryos became shorter as invagination progressed (Fig. 2D). Though their width in- creased to some extent (Fig. 2E), the expansion of the ectodermal layer was not so conspicuous as in H. pulcher- rimus (Fig. 2F). Tracing of the vegetal blastomeres The dye-injected S. miruhilis embryos were examined to learn how vegetal cells move toward the blastopore during gastrulation (Fig. 3). Before the initiation of invagination, the boundary between labeled and nonlabeled cells was at about 50% of the distance from the vegetal pole to the animal pole (Fig. 3A, A'). The boundary gradually shifted toward the vegetal pole side as invagination progressed (Fig. 3B, B', C. C', D, D'). At the end of invagination, the 140-| A :i.W- 140- £ 120- TJ -; 110- IOOJ = u ro B 170 160- 150- 140 17 18 IV 20 21 22 15 16 17 IX 19 20 21 140 130 120-1 100 E 17 18 19 20 21 22 15 16 17 18 19 20 21 15 10 17 18 19 20 21 22 15 16 17 18 19 20 21 Time after fertilization (hour) Figure 2. Change in the height and width of the embryos, and in the thickness of the blastocoel wall (the lateral part of the embryo) during gastrulation. (A-C) Hemicentrotits pulcherrimus. (D-F) Scaphechinus mimhilis. Single arrows indicate the time of the initiation of gastrulation. Double arrows indicate the time of the onset of the secondary invagination in //. pulcherrimus embryos. Changes in the height of the embryo (A, D) show different patterns. In both species, the width of the embryo increased as invagination progressed (B, E). This is more evident in H. pulcherrimus than in S. mimhilis. The thickness of the blastocoel wall decreases during gastrulation. but the change is not conspicuous in S. mirabilis (C. F). 290 T. KOMINAMI AND H. TAKATA Figure 3. Movement of vegetal cells during gastrulation in Scaplifcliinnx mirabilis embryos: bright-field images (A-EK fluorescence images (A'-E1). (A. A') 14 h. (B. B'l 16 h. (C. C') 17 h. (D. D'l 18 h. (E. E'l 20 h. Distribution of labeled cells in invaginating gastrulae, which had been injected with Lucifer yellow CH into one of vegetal blastomeres at the 8-cell stage, was examined. As gastrulation proceeds, the boundary between labeled and nonlabeled cells moves downward. Arrowheads in A-E demarcate the boundary observed on fluorescent imaaes. The scale bar indicates 50 jxm. boundary was located at 10%- 15% of the embryo length from the vegetal pole (Fig. 3E. E'). This value corresponds to the thickness of the anal plate ectoderm. The relationship between the degree of invagination and the position of the boundary is shown in Figure 4. During the early stages of invagination. the position of the boundary shifted rapidly to the vegetal pole; during the later stages, the rate of the movement decreased gradually. The result clearly indicates that the involution of cells through the blastopore continues until the archenteron tip reaches the apical plate. 60- 50 ~ 20 §10 '"*-•-... * + •'""• 10 20 30 -4(1 511 70 SO 90 100 Degree of imagination (B/A, %) Figure 4. Change in the position of the boundary between animal and vegetal ectoderm during gastrulation. The degree of invagination (%) and the position of the boundary along the embryo axis (%) were sought using the parameters shown in the inset. The most fitting hyperbolic curve is also shown. The movement of the vegetal cells toward the vegetal pole con- tinues throughout the invagination processes, though the rate of the move- ment changes. Shapes of the ectodermal cells during early stages of gastrulation The shapes of the ectodermal cells during early stages of gastrulation were examined with SEM (Fig. 5). The cells were classified as columnar, skewed, wedge-shaped, and other, as described in the Materials and Methods. Among these types, wedge-shaped cells showed differences in their distribution between the two species of embryos. In H. pitlcherriiiins. two to three wedge-shaped cells were ob- served just at the bending point of the ectodermal epithe- lium (Fig. 5A-D, arrowheads). In S. mirabilis, such wedge- shaped cells were distributed more broadly apart from the blastopore (Fig. 5E-H. arrowheads). In addition, the num- ber of wedge-shaped cells was larger than in H. pulchcrri- mus. At the beginning of invagination, bottle-shaped (api- cally constricted and basally rounded-up) cells were frequently observed in the bending vegetal plate (arrows indicate bottle cells in Fig. 5D \H. /nilclicrriiinix] and 5G [S. mirabilis]'). Figure 6 shows the change in the ratio of these three types of cells during early stages of gastrulation. In both species, it takes 3 — 1 h to give rise to a short, stub-like gut rudiment after the first sign of invagination. These stages were di- vided hourly and designated Stages I-IV. In the animal halves, columnar and skewed cells were abundant (Fig. 6A, H. piilclieiriiiin.'i; 6C, S. mirabilis). In both species, the ratio of columnar cells increased as gastrulation progressed. In contrast, most cells in the vegetal half were skewed or distorted (Fig. 6B. H. piilcherrimus; 6D, 5. inimbilis). In H. pitlchernmns. wedge-shaped cells occupied nearly 40% of the total at the initial stage of invagination. but decreased to GASTRULATION IN SAND DOLLAR 291 Figure 5. Scanning electron micrographs of the cells in the ectoderm and invaginated archenteron during early stages of gastrulation: Hemicentrotus pulclierrimus (A-D): Scaphechinus mirabilis (E-H). In A-D, arrowheads indicate the bending point: in E-H they indicate the boundary between animal and vegetal cells. Arrows in D and G indicate bottle cells. Ectodermal cells of 5. mirabilis (F-H) were more elongated in the apico-basal direction than those of H. piilcherrimus (B-D). Columnar and skewed cells were frequently observed in both species. Wedge-shaped cells were also observed in both species, especially in the vegetal half. In H. pulcherrimus. two to three wedge-shaped cells were observed just at the bending point of the epithelium (B. C). Such wedge-shaped cells are distributed more broadly apart from the blastopore in S. mirabilis (F, G). The scale bar indicates 10 jum. about 209r as invagination progressed; in S. mirabilis, the ratio remained constant at a rather higher level. Columnar cells were barely observed in S. mirabilis (Fig. 6D), whereas this type of cell increased in H. pulclierrimus at the end of primary invagination (Fig. 6B). Secondary mesenchyme cells at the archenteron tip Figure 7 shows the secondary mesenchyme cells ob- served at the archenteron tip of the midgastrulae. In H. pulcherrimus, these cells were globular and formed long thin filopodia. Several SMCs were located between the archenteron tip and the future oral opening region. In 5. mirabilis, SMCs were flattened to some extent and formed broad ruffled membranes. No cells were observed between the archenteron tip and the future oral opening region. Although more than 200 gastrulating S. mirabilis embryos were examined, an image that showed direct contact be- tween the filopodia of the SMCs and the inner surface of the apical plate could not be obtained. Shape of archenteron cells during later stages of invagination Figure 8 shows cross fractures of the archenteron at later gastrula stages. SEM images of the archenteron at three levels along its axis (top, middle and bottom) are shown. The cells in the archenteron of H. pulcherrimus were cuboid and loosely in contact with each other (Fig. 8A-C). The numbers of cells observed in cross fractures increased from top (6-7) to bottom (about 12) of the archenteron. The archenteron cells had a rounded basal surface. In contrast, cells in the archenteron of S. mirabilis embryos were elongated along the apico-basal direc- tion (Fig. 8D-F). The numbers of cells observed in cross fractures were almost the same at the top ( 14 - 1 5 ), middle (13), and bottom (13) levels of the archenteron. As gastrulation proceeded, it became difficult to crack the archenteron along its long axis. The shapes of cells in the embryos at later stages of invagination were examined on histological sections. The stages shown in Figure 9 corre- spond to the secondary invagination in H. pulcherrimus embryos. As clearly shown, the cells in the archenteron of 292 T. KOMINAMI AND H. TAKATA 60 1 St. 1 Si. 2 SI. .1 SI. 4 SI. 1 SI. 2 Si. 3 Stage in early phase of imagination St. 4 Figure 6. Frequency of the appearance of columnar, skewed, and wedge-shaped cells during gastrulation: Hemicentrotus pit/cherriinus (A. B); Scai'hi'cliinin mirabilis (C, D). (A. C) Animal hemisphere. IB. D) Vegetal hemisphere. Columnar cells (solid lines) are more abundant in the animal hemisphere. Skewed cells (dotted lines) were observed more fre- quently in S. mirabilis. In both species, the population of columnar cells increased as the gastrulation proceeded (A. C). Wedge-shaped cells (tiro- ken lines) appear sparsely in the animal hemisphere. In contrast, the most abundant type of cells are wedge-shaped cells in the vegetal halves (B. D). Columnar cells were rarely observed in S. mirtihilis. but such cells in- creased in H. pulclierriinii* after the secondary imagination had started. H. /ntlclwrrinnis embryos were stretched along the axis of the archenteron (Fig. 9A-D). After the completion of the secondary invagination. the cells resumed a cuboid shape (Fig. 9E-F). In contrast, the cells in the archenteron of 5. mirabilis embryos were not stretched at any stage of later invagination (Fig. 9G-L). It should be noted that the cells near the blastopore were elongated along their apico-basal direction through all the stages examined. These changes in cell shape were quantified according to the methods described by Hardin (1988); two ratios, YIX (ratio of lengths along and perpendicular to the axis of the archenteron) and L/W (ratio of cell length and width) were obtained (Fig. 10). Both YIX and L/W increased during the secondary invag- ination in H. pulcherrimus embryos, and decreased to the initial level at the end of secondary invagination (Fig. 10A). On the other hand, the ratios did not change in S. mirabilis embryos through these stages of invagination (Fig. 10B). The result clearly shows that the archenteron cells in S. mirabilis embryos were not stretched along the axis of the archenteron. Attaching embryos to a glass disli coated with poly-L- Ivsine The obtained results suggest the ectodermal layer plays a role in the invagination process in S. mirabilis embryos. If Figure 7. Scanning electron micrographs of the secondary mesenchyme cells at the archenteron tip. Hemicciitronix puUlii'rriiiui\ (A): Scaphechiints mirahili.i (B). Insets in A and B show whole view of the mid-gastrula. Secondary mesenchyme cells in H. fiulchcrriimt* are globular in shape and form long thin filopodia. Several secondary mesenchyme cells (SMCs) are located between the archenteron tip and the inner surface of the future oral opening region. In contrast. SMCs are flattened and form ruffled membranes in S. minihili* gastrulae. No SMCs were observed between the archenteron tip and the future oral opening region. The scale bar indicates 10 /. c \~ 100 V t at 75 a SO 25 -2 o X 2S Envaginatlon L/W Y/X -2 17 IX 14 20 21 Time after fertili/.ation (hour) Figure 10. Change in the shape of the archenteron cells during later stages of gastrulation: Heinicciiiiniii\ />iilflifrriiiiii.\ (A): Scaphechinus mimhilis (B). Shape was expressed as two ratios: )7.V (length along the archenteron axis to length perpendicular to the axis) and L/W (cell length to width I. In H. pulcherrimus embryos, both ratios increased as secondary invagination progressed, up to at least 22 h. Then the ratios decreased to about 1.0. In S. mirabilis embryos, the ratios did not change significantly. though the degree of invagination increased. The Y/X ratio remained about 1.0, which indicates that the archenteron cells are not stretched along the axis of the archenteron. had finished primary invagination when attached (Fig. 11. embryos I and II). This suggests that the vegetal ectodermal layer moves toward the blastopore during primary invagination, and that the layer loses physical continuity from the gut rudiment after the completion of primary invagination. On the other hand, elongation of the archenteron was completely blocked in S. mirubilis if the embryos were attached to a coated glass dish, irre- spective of the degree of invagination (Fig. 13). This inhibitory effect of poly-L-lysine cannot be ascribed solely to the chemical toxicity of the drug, because the embryos restarted gastrulation soon after they detached from the glass dish (Fig. 12). We suppose that the ecto- dermal epithelium and the invaginated archenteron are physically continuous during the invagination processes and that the blockage of invagination is mainly due to physical constraint of the ectodermal layer attached to the glass dish. The precise mechanism by which elongation of the gut rudiment is blocked in S. niirnhilis embryos is unknown. The ectodermal layer seems to be more rigid in S. mimhilis embryos than in those of H. ptilclierrinn/s. because the former is thicker (Fig. 2F). In addition. S. ininibilis embryos retained normal configuration after they were fixed with 10% formaline, while the ectodermal layer of H. pulcher- riinus embryos was severely distorted when the fixative was applied. The ectodermal cells in S. mirabilis embryos are probably tightly connected with each other, forming a rigid structure over the entire vegetal ectoderm. Even if embryos are attached to the glass dish on one side of the body, such a rigid structure may be destroyed totally, resulting in a blockage of archenteron elongation. GASTRULATION IN SAND DOLLAR 295 1:10 B 3 70 0 60 | 5° "S 40 01 30 20 ° 10 O . IV o J 30 40 50 60 70 80 90 100 Time after the attachment (minute) Figure 11. Adhesion of Hemicentrotus pulcherrimus embryos to the glass dish coated with poly-L-lysine. The numeral at the top right corner in each photograph in A indicates the time after the attachment to the glass dish. Roman numerals (I-IV) in A and B indicate the same embryos. The embryos gastrulated almost normally if they had been attached after the primary invagination (embryo I and II). If the embryos had been just in the primary invagination when attached, the rate of elongation of the arch- enteron was slowed and the archenteron could not reach the apical plate (embryo III and IV). The scale bar indicates 100 /j.m. The initial phase of gastrulation In both species of embryos, bottle cells (Nakajima and Burke, 1996) were observed in the vegetal plate (Fig. 5D, G, arrows). The appearance of bottle cells in the vegetal plate may lead to the first step of invagination. if the archenteron cells retain the monolayer arrangement (Gustafson and Wolpert, 1963, 1967). Unlike the archenteron cells in H. puk-lieirimux embryos (Fig. 5C. D), those in S. mirabilis embryos were variable in shape and were not organized into a complete monolayer sheet (Fig. 5G, H). As a result, the force produced by bottle cells does not necessarily cause the bending of the vegetal plate. Other forces seem to be nec- essary to produce the invagination of the vegetal plate cells in S. iniruhilis embryos. In this study, several types of cells were observed on SEM images. The role of each type of cell is unknown. If cells are pulled apically or basally, they should become skewed, be- cause cells are connected with extracellular matrix ( Wessel and McClay, 1987; Burke et «/.. 1991: Berg et ai. 1996). If a monolayer cell sheet is bent, wedge-shaped cells should appear at the bending point. Thus, the shapes of cells are signs of the existence of the forces generated by surrounding tissues or by the cells themselves. In both species of embryos, the ratio of columnar cells in the animal hemisphere increased as invagi- nation progressed (Fig. 6A, C). On the other hand, most cells in the vegetal hemisphere were distorted (Fig. 6B, D). Espe- cially in 5. mimhilix embryos, columnar cells were barely observed through the stages examined. These results imply that i\ Figure 12. Adhesion of the Sctiphechiiius mirabilis embryos to the glass dish coated with poly-L-lysine. Embryo II was loosely attached to the glass dish, because its position changed during observation. In this embryo, invagination occurred almost normally. On the other hand, embryos I. 111. and IV were rather firmly attached to the glass dish and invagination of the 2ut rudiment were considerably delayed. Nonetheless, embryos III and IV reinitiated invagination when they detached from the glass dish. The scale bar indicates 100 /nm. 296 T. KOMINAMI AND H. TAKATA 1:0(1 I II HI V ..' .< 'i 0:20 B I 3- 70 £ 60 u C 50 S 1 40 •o 30 3 I 20 *o ! a 10 D terliolectz I. galbana D Mixed 15 20 25 Age (days) 30 Figure 6. Percentages of larvae of Pisaster ochraceus undergoing cloning when reared at 12-15°C and fed high levels of four different phytoplankton diets. Error bars represent mean values ± 1 SD. SEA STAR LARVAL CLONING 303 explanation for the high mortality is that the higher temper- atures triggered an increase in bacterial and microalgal growth in the cultures. In contrast, sea star larvae reared in the low-temperature treatments (7-10°C) showed no net positive growth, and in most cases, decreased in length, regardless of food avail- ability. These larvae also failed to attain the brachiolaria stage of development. It is unlikely these larvae would eventually become clonal because they continued to shrink in length over the course of the experiment. Decreased rates of growth and development at low temperatures may be related to decreased rates of larval metabolism (Boidron- Metairon, 1995). While low seawater temperatures have been suggested as an indirect cause of mortality in marine invertebrate larvae (Thorson, 1950). no studies of larval culturing have shown that low temperature can actually lead to a decrease in larval length as seen in the present study. The production of larval clones was greatest during phases of rapid larval growth in MT-HF condition. As P. ochraceus in the North Pacific spawns in the late spring, larvae typically encounter moderate seawater temperatures (12-15°C) and high phytoplankton availability (Cannon, 1978). Such conditions could be expected to enhance in situ rates of larval cloning. Further analysis indicated that pre- senting larvae with different levels and types of food under an optimal regime of seawater temperature had a pro- nounced effect on the initiation and rate of larval clone production. The greatest numbers of clones were produced by larvae in cultures presented a mixture of three single-celled algae. Although monospecific patches of single-celled algae are unlikely to exist in the natural environment, our use of monospecific algal diets simulated conditions in which nu- trient diversity might be limited. Thus some of the observed differences in growth (and cloning) rates among the larvae fed monoalgal diets may have resulted from differences in the nutrient content of the food rather than in the type of food, since the larvae were fed equal cell numbers of algae, not an equal nutritional content (Pechenik and Fisher. 1979). However, the amount of nutrients actually consumed by the larvae does not necessarily have any correlation with the nutrient content of the food presented, as some food types may be more palatable to the larvae than others. Future studies may shed more light on this subject. The important information gained from the food-availability ex- periment is that nutrient availability may be an important factor affecting larval growth and therefore the rate of cloning, as evidenced by the fact that growth rates among larvae fed a monoalgal diet of Isocluysis galbana were similar to those fed a diet of mixed algae, yet the larvae fed the mixed diet produced far more clones. In adult echinoderms. cloning (fission) is common and has been well described (Emson and Wilkie. 1980). Sea- sonal fluctuations in the incidence of cloning in adult sea stars, especially a high incidence in summer months, have been related to periods of maximum growth (Emson and Wilkie. 1980). This suggests that suitable biotic and envi- ronmental conditions such as abundant food and moderate temperatures may trigger cloning processes in adults just as they did in the larvae studied here. In some instances, more than 50% of the adults in a population were observed undergoing cloning (fission) when conditions were optimal (Emson and Wilkie, 1980). Cloning may serve as a mechanism to enhance recruit- ment in P. ochraceus and perhaps in other marine inverte- brates with planktotrophic modes of reproduction. Larvae dispersed across significant distances are likely to encounter a variety of environmental and biotic conditions, and our results suggest that those larvae encountering favorable conditions may be stimulated to reproduce by cloning, thereby possibly increasing the probability of successful larval metamorphosis and juvenile recruitment. Future stud- ies of the effects of larval cloning on larval survivalship and recruitment will provide more insight into the true impact of this phenomenon on the life history of sea stars with plank- totrophic larvae. Acknowledgments We thank Dennis Willows and Richard Strathmann of Friday Harbor Laboratories, University of Washington, for use of laboratory facilities and technical support. We are grateful to Al Schuetz of Johns Hopkins University for providing ovaries from Pisaster ochraceus. We thank Mi- chael Vickery for his assistance in collection and mainte- nance of larval cultures. 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(December 2000) Development of Embryonic Cells Containing Serotonin, Catecholamines, and FMRFamide-Related Peptides in Aplysia calif ornica AMANDA J. G. DICKINSON1'*, ROGER P. CROLL1. AND ELENA E. VORONEZHSKAYA' 1 Department of Physiology and Biophysics, Faculty of Medicine, Dallwusic Universitv, Halifax, Nova Scotia. B3H 4H7, Canada; and ' Institute of Developmental Biology, Russian Academy of Sciences, 117808 Moscow, Russia Abstract. This study demonstrates the presence of a rel- atively extensive but previously unrecognized nervous sys- tem in embryonic stages of the opisthobranch mollusc Aply- sia californica. During the trochophore stage, two pairs of cells were observed to be reactive to antibodies raised against the neuropeptides FMRFamide and EFLRIamide. These cells were located in the posterior region of the embryo, and their anterior projections terminated under the apical tuft. As the embryos developed into veliger stages, serotonin-like immunoreactive (LIR) cells appeared in the apical organ and were later observed to innervate the velum. Also, aldehyde-induced fluorescence indicative of cat- echolamines was present in cells in the foot, oral, and possibly apical regions during late embryonic veliger stages. Just before the embryo hatches as a free-swimming veliger. additional FMRFamide-LIR and catecholamine- Received 24 January 2000; accepted 2 October 2000. * To whom correspondance should be addressed. E-mail: ajdickin® is2.dal.ca Abbreviarions: ACP, 35 amino acid acidic peptide; CC-1. catecholamin- ergic central cell one; CC-2. catecholaminergic central cell two; CC-3, catecholaminergic central cell three; CNS. central nervous system; EFLRIamide, Glu-Phe-Leu-Arg-Ile-NH,; EDTA. ethylenediaminetet- raacetic acid; FC-1, FMRFamide central cell one; FC-2, FMRFamide central cell two; FC-3. FMRFamide central cell three; FITC, fluorescein isothiocyanate; F-/1, left FMRFamide-LIR posterior cell one; F-/2, left FMRFamide-LIR posterior cell two; F-H, right FMRFamide-LIR posterior cell one; F-r2, right FMRFamide-LIR posterior cell two; FMRFamide. Phe-Met-Arg-Phe-NH2; LIR, like-immunoreactive; PBS, phosphate-buff- ered saline; SEEPLY, 22 amino acid peptide SEQPDVDDYLRDVVLQ- SEEPLY; S-/1, left serotonin-LIR bilateral cell one. S-/2, left serotonin- LIR bilateral cell two. S-cl. right serotonin-LIR bilateral cell one; S-/-2, right serotonin-LIR bilateral cell two; SUM, serotonin-LIR unpaired me- dian cell. containing cells appeared in regions that correspond to the ganglia of what will become the adult central nervous system (CNS). Neurons and connectives that will contribute to the adult CNS appear to develop along the pathways that are pioneered by the earliest posterior FMRFamide-LIR cells. These observations are consistent with the hypothesis that, besides their presumed roles in the control of embry- onic behaviors, some elements may also guide the develop- ment of the CNS. Embryonic nervous systems that develop prior to and outside of the adult CNS have also been reported in pulmonate and prosobranch species of molluscs. Therefore, the demonstration of early developing neurons and their transmitter phenotypes in A. californica presents new opportunities for a better understanding of the ontog- eny and phytogeny of both behavioral and neuronal function in this important model species. Introduction The opisthobranch gastropod Aplysia californica. which has been studied extensively as a model for understanding the neuronal underpinnings of behavior (for review see Kandel [1979]), has also become an important model in the study of molluscan neurodevelopment. The development of the ganglia that constitute the central nervous system (CNS) has been studied in detail (Kriegstein, 1977; Schacher et ai, 1979; Kandel et al., 1981; Jacob, 1984). but more recent studies also report the presence of nerve cells that exist outside the boundaries of the developing ganglia that will constitute the adult CNS. For example, in an early embry- onic veliger stage, three serotonin-like immunoreactive (LIR) cells exist in the anterior apical organ: an unpaired median cell and a bilateral pair of cells (Croll and Voron- 305 306 A. J. G. DICKINSON ET AL ezhskaya, 1995; Croll and Voronezhskaya, 1996b; Marois and Carew, 1997a, b, c). Soon afterwards, these three cells are joined by another more lateral pair of apical serotonin- LIR cells. By the end of the embryonic period, as the veliger is about to hatch as a free-swimming larva, serotonin-LIR projections extend into the velar lobes, foot, and abdominal and visceral regions (Marois and Carew, 1997a, b, c). Kempt" el al. (1997) showed that such apical cells and processes appear to be general features of opisthobranch larvae. Similarly shaped and positioned apical cells have also been reported in larvae of other molluscan species (Bonar. 1978: Kulakovskiy and Flyachinskaya. 1994; Rai- neri and Ospovat, 1994; Raineri, 1995; Leise, 1996; Lin and Leise, 1996a, b; Dickinson el al, 1999; Voronezhskaya el al., 1999; Fames, 2000). In addition to the cells of an apical organ, other neuronal elements have been observed outside the boundaries of the developing adult CNS. Croll and Voronezhskaya (1996b) reported preliminary observations of elements containing peptides related to Phe-Met-Arg-Phe-NH2 (FMRFamide) in posterior regions of embryonic A. californica. Recent stud- ies also indicate the presence of similar neuronal elements in other molluscan species. For example, Croll and Voron- ezhskaya (1995; 1996a) identified neuronal elements in what corresponds to the trochophore or early veliger stage (Mescheryakov, 1990) of the pulmonate Lymnaea stagnalis. using antibodies raised against FMRFamide. These FMRF- amide-LIR cells develop in posterior regions of the embryo and send anterior projections that terminate in the regions of the future cerebral and pedal ganglia. A posterior FMRF- amide-LIR cell has also been observed in the early devel- opmental stages of the prosobranch Cre/'ithila fornicata (Dickinson et cil.. 1999). As in L sta^iuilis. this posterior FMRFamide-LIR cell also sends anterior projections that terminate in the region of the future cerebral and pedal ganglia. Therefore, cells expressing FMRFamide-like ini- munoreactivity appear to develop in a posterior-to-anterior sequence rather than the anterior-to-posterior development of the ganglia. In addition, these FMRFamide-LIR cells and their fibers seem to mark the pathways along which the adult ganglia and connectives develop, and therefore they may be involved in guiding the developing CNS. Additional peripherally located neurons in the foot and surrounding the mouth were revealed in the gastropods L. stn^nali.\ (Voronezhskaya et al.. 1999), C. fornicata (Dick- inson et al.. 1999), and Pliestilla sihoxae (Pires et al., 2000) and the bivalve Mytilus ednlis (Croll el til.. 1997), using techniques to localize catecholamines. The above descriptions of neurodevelopment in represen- tative species suggest the presence of a primary larval nervous system that appears earlier than and outside of the developing adult CNS. Morphological descriptions in other species also indicate that components of such primary larval nervous systems may either be incorporated into the adult ganglia or disappear. The present study investigated the early development of neurons that may compose a primary larval nervous system in A. califonuca. starting at the tro- chophore stage and continuing until the embryo hatches as a free-swimming veliger. We used immunocytochemical techniques to study the first cells expressing FMRFamide and related peptides, and we provide details of the morphol- ogy of these cells and the timing of their appearance, with comparisons to the earliest cells exhibiting serotonin-like immunoreactivity. We additionally used aldehyde-induced fluorescence, which has been previously applied to mollus- can tissues (Croll et til., 1997, 1999; Smith et al.. 1998), to examine cells containing catecholamines. This study shows that, as in these other molluscs, A. californica also has a nervous system first present in early embryonic stages. Furthermore, these observations suggest several new hy- potheses regarding the mechanism shaping the ontogeny of the nervous system in this well-studied species. Materials and Methods Aninuilx Adult specimens of Aplysiu californica were purchased from the Aplysia Resource Facility of the University of Miami and maintained in a salt-water aquarium. Egg masses were collected soon after oviposition and kept in separate containers of artificial salt water (Crystal Sea. Baltimore, MD) at 20°-22°C. Under these conditions, embryos re- quired about 9-10 days to develop from first cleavage to hatching. The hatched veliger required another few weeks before becoming competent to metamorphose into juvenile sea slugs (Kandel, 1979; Kandel et al.. 1981: Marois and Croll, 1992; Marois and Carew, 1997b). The developmental stages of A. californica were de- scribed previously (Kriegstein, 1977; Kandel, 1979; Kandel et til.. 1981; Marois and Croll. 1992; Marois and Carew, 1997b). In the present study, morphological and behavioral features were examined on each day from first cleavage to hatching, and the embryonic development was divided into three stages. During the trochophore stage (days 2.5-4) the embryo had a distinct apical tuft and a shell gland and began to move using the prototrochal cilia. On day 4 the body began to change shape as the rudiments of a velum, foot, and shell were observed. During the early embryonic veliger stage (days 5-7) the velum became bilobate and possessed long cilia along its edge. During the late embryonic veliger stage (days 8-10) the velum, foot, and shell enlarged and differentiated. Immunocytochemistry Immunohistological procedures were performed accord- ing to Marois and Croll (1992) and Marois and Carew ( 1997b). Egg ribbons were fixed in 47c paraformaldehyde in NEURODEVELOPMENT IN APU'SIA CALIFORNIA 307 phosphate-buffered saline (PBS; 50 mM Na:HPO4 • 7H2O and 140 mA/ NaCl in distilled water adjusted to pH 7.2) for 1 — I h at room temperature. Then embryos were removed from the capsules, washed in PBS, and stored in 70% ethanol at -18°C until further processing. For immunohis- tochemical processing, the stored embryos were first given two to three 5-min washes in PBS. The shells of older embryos (>day 4) were then decalcified with 10% ethyl- enediaminetetraacetic acid (EDTA) (Sigma Chemical Co., Mississauga. ON) in PBS for 30-45 min. Embryos were next washed for 2-3 h in 47r Triton X-100 in PBS. The embryos were then incubated in antibodies raised against FMRFamide, serotonin (both obtained from Diasorin, Still- water, MI), or antibodies (gifts from Dr. P. R. Benjamin, University of Sussex) against three FMRFamide gene en- coding peptides: the pentapeptide Glu-Phe-Leu-Arg-Ile- NH2 (EFLRIamide), the 22-amino-acid peptide SEQPDVD- DYLRDVVLQSEEPLY (SEEPLY), and a 35-amino-acid acidic peptide, SDPFFRFGKQQVATDDSGELDDEILSR- VSDDDKNI (ACP) (Santama et ul.. 1996). All these anti- bodies except anti-SEEPLY were diluted 1:500-1:1000 in PBS with the addition of 1.0% normal goat serum and 1.0% Triton X-100. The SEEPLY antibody was diluted 1:200 in a solution of 50 mM Tris base, 150 mM NaCl, pH 7.6. containing 0.25% w/v gelatin and 1% v/v Trition X-100 (Santama et al.. 1993). Incubation periods lasting 48 h at 4°C or 12 h at room temperature gave comparable results. The embryos were next rinsed three times (5 min each) with PBS and given a final wash for 1 h before incubating for 24-48 h in goat anti-rabbit antibodies conjugated to fluo- rescein isothiocyanate (FITC) or rhodamine (Bio/Can Sci- entific, Mississauga. Ontario) and diluted 1:50 in PBS with the addition of 1.0% Triton X-100. To localize FMRFamide-like immunoreactivity relative to external morphological structures at the trochophore stage, some embryos were double-labeled with monoclonal antibodies against *-tubulin (DM1 A clone from Sigma Chemical Co., Mississauga, ON) (Jackson et al., 1995). These embryos were first labeled, as described above, for FMRFamide-like immunoreactivity, then rinsed three times (5 min each) in PBS. Next, the embryos were incubated in anti-atubulin (diluted 1:500 in PBS) for 12 h at room temperature. The embryos were washed again three times in PBS before incubating in sheep anti-mouse serum conju- gated to FITC or rhodamine for 12 h at room temperature. These secondary antibodies were diluted 1:50 in PBS and 1% Triton X-100. Embryos processed for immunocytochemistry were mounted on glass slides in a 3: 1 mixture of glycerol to PBS for viewing on a Leitz Aristoplan microscope equipped for epifluorescence. FITC fluorescence was viewed using a 450-490-nm excitation filter and a 525/20-nm barrier filter; rhodamine fluorescence was viewed using a 530-560-nm excitation filter and 580-nm long-pass barrier filter. Em- bryos processed for FMRFamide- and *-tubulin-like immu- noreactivity were also viewed on a Zeiss Axiovert micro- scope equipped for confocal laser scanning (model LSM 410). As negative controls, embryos were processed without incubation in primary antibody; such specimens exhibited no detectable fluorescence. Positive controls involved par- allel processing of embryonic L. stagnalis that exhibited typical staining, as described elsewhere for serotonin and FMRFamide (Marois and Croll, 1992; Croll and Voron- ezhskaya, 1995; Croll and Voronezhskaya, 1996a). Catecholanune histo fluorescence The formaldehyde glutaraldehyde technique of Furness et al. (1977) was used to localize catecholamines. Embryos were incubated for at least 12 h in a solution consisting of 4% paraformaldehyde. 0.5% glutaraldehyde. and 35% su- crose in PBS. Similar results were also obtained when the embryos were stored in this solution for several weeks. The fixed embryos were decalcified in 10% EDTA in PBS for 45 min. Embryos were then placed on glass slides, air dried for several hours, and then mounted in a 3: 1 mixture of glycerol and PBS. These embryos were viewed and photographed through the Leitz compound microscope equipped with a 355-425-nm excitation filter and 460-nm long-pass barrier filter. Positive controls involved parallel processing of em- bryonic L. stagnalis that exhibited typical blue-green fluo- rescent staining, as described elsewhere for catecholamines (Voronezhskaya et til., 1999). Negative controls were per- formed by omitting the glutaraldehyde from the formalde- hyde glutaraldehyde solution, thus eliminating the charac- teristic fluorescent staining. Photography Most histological preparations were photographed on the Leitz compound microscope using Kodak TMAX 100 film: the negatives were digitally scanned. Photographs from the Zeiss confocal microscope were produced by superposition- ing stacks of 10-15 images obtained through stepped se- quences of focal planes at intervals of 1-2 ju.m. All the images were then assembled into plates and labeled using Photoshop 5.0 (Adobe Systems, Inc.. San Jose. CA). Con- trast and brightness of the images were adjusted to provide consistency within plates. Results Trochophore stage (days 2.5-4) Halfway through day 2, two bilaterally symmetrical pairs of FMRFamide-LIR posterior cells were observed (Figs. 1A. 2A). Fibers projected ipsilaterally and anteriorly from each cell on the right (F-rl and F-r2) and the left (F-/1 and F-/2) (Fig. 1 A). By day 3 these fibers terminated in a plexus 308 A. J. G. DICKINSON ET AL. A DAY 2.5 apical tuft B DAY 4 C DAYS DDAY9 prototroch 'telotroch Right Lateral Views prototroch Trochophore foot Veliger Figure 1. Schematic representations of FMRFamide-LIR cells and fibers in embryos of Aplysia californica. Top row: views from the right side and slightly superior to give a three-dimensional perspective; bottom row: dorsal views. Anterior is to the right in each figure. (A) Two pairs of posteriorly located FMRFamide-LIR cells with anterior projections observed on day 2.5. (B) FMRFamide-LIR cells and their processes observed on day 4. The FMRFamide-LIR processes reached the anterior region, where they formed a plexus under the apical tuft. (C) FMRFamide-LIR cells were no longer symmetrical by day 5; the cells (/I and 12} on the left appeared in a ventral position, and the cells on the right (/•! and i2) appeared more dorsally. (D) On day 9 additional FMRFamide-LIR cells (FC-1, FC-2, FC-3) appeared in the anterior region. of FMRFamide-LIR processes in the region beneath the apical tuft (see day 4, Figs. IB. 2B, D). Also by day 3, one to two additional FMRFamide-LIR fibers extended across the midline of the body just anterior to the somata of the FMRFamide-LIR posterior cells (See day 4, Figs. IB, 2D). Initially the pairs of FMRFamide-LIR cells were positioned symmetrically within the embryo, but they gradually be- came displaced and by the end of day 4 had all moved to the right side of the body (Fig. 1C). The FMRFamide-LIR cells and processes were also identified using antibodies against EFLRIamide (Fig. 2C). No immtmoreactivity was detected during the trochophore stage or any later stages with anti- bodies against SEEPLY and ACP. Etirly embryonic veliger staifc ( duyx 5-7) By day 5 the FMRFamide-LIR cells had assumed more anterior positions in the embryo. F-/-1 and F-/-2 moved apart from each other, with F-H occupying a more dorsal loca- tion. F-/1 and F-/2 remained close to each other and together assumed a central and ventral position (Figs. 1C, 3 A). Their anteriorly projecting fibers crossed the midline and formed a commissure in the anterior region. All cells and processes listed above were also identified using antibodies against EFLRIamide (Fig. 3B). Also by day 5, a serotonin-LIR unpaired median cell (SUM; see Marois and Carew [1997b]) appeared beneath the apical tuft. Soon afterwards, a pair of vase-shaped serotonin-LIR cells (S-rl and S-/1 ) were observed to the left and right of SUM. Short serotonin-LIR fibers projected ventrally from the SUM. S-rl, and S-/1 to form a plexus in the same region as the FMRFamide-LIR commissure (see day 9, Figs. 4A, 5A, B, also see Marois and Carew [1997b]). By day 7 the three serotonin-LIR cells (SUM, S-rl, and S-/1 ) were joined by a new pair of serotonin-LIR cells (S-r2 and S-/2) located slightly posteroventrally to S-rl and S-/1 on either side of the plexus (see day 9, Figs. 4A, 5A, B). Late embn-tmic veliger on prehutching stage (days 8-10) By day 8 the posterior FMRFamide-LIR cells and their fibers appeared just anterior to the midpoint along the an- teroposterior axis. F-/1 and F-/2 together assumed a ventral and central position, while F-rl and F-r2 each assumed more dorsal positions than previously (see day 9, Figs. ID, 3C). FMRFamide-LIR fibers extended ventrally from the NEURODEVELOPMENT IN APLYS1A CAUFORNICA 309 Figure 2. Aplvsia californica during trochophore stages. Anterior is to the right in each figure. (A) Dorsal view of an embryo on day 2.5 showing the two pairs of posteriorly located FMRFumide-LIR cells with anterior projections. Scale bar = 20 /mm. (B) Right lateral view of an embryo showing immunoreactivity for '^-tubulin on day 3. Displays the locations of the apical tuft, prototroch. telotroch, and anal cell in the trochophore. Scale bar = 25 /xm. (C) EFLRIamide-like immunoreactivity observed on day 3. showing F-rl, F-/1 and -12 (which are not in focus), and the plexus under the apical tuft (arrow). Scale bar = 25 ju.ni. (D) FMRFamide-like immunoreactivity observed in the same embryo as 2B showing F-rl, -r2, -1\ and -12, and plexus under the apical tuft. Scale bar = 25 /am. C and D demonstrate the similar pattern of immunoreactivity for EFLRlamide and FMRFamide. apical commissure toward the foot. Also by day 8, formal- dehyde glutaraldehyde-induced fluorescence, indicative of catecholamine-containing cells, was observed in the foot region. The catecholamine-containing foot cells were vase- shaped and appeared in two bilaterally symmetric groups of two to three cells on each side (see day 9, Figs. 4B, 6A, B). By day 9 additional FMRFamide-LIR cells were ob- served in positions consistent with the locations previously identified as the developing cerebral, pedal, and pleural ganglia of the future adult CNS in A. califoniicii (Krieg- stein, 1977; Marois and Carew, 1990; Marois and Carew, 1997b) and other opisthobranchs (Kempf et al.. 1997a). One of these FMRFamide central cells (FC-1) appeared near F-rl, another (FC-2) was located near F-r2, and a third (FC-3) was observed to the left of the apical commissure (Figs. ID, 3C). Also by day 9, the number of catechol- amine-containing cells increased to four to five cells on each side of the foot (Figs. 4B, 6A, 6B). Another pair of cate- cholamine-containing cells was located in the oral region. Catecholamine-containing fibers also extended from each 310 A. J. G. DICKINSON ET AL. A 5-HT B CA's Figure 3. FMRFamide- and EFLRIamide-LIR cells and fibers in repre- sentative embryonic veliger stages of Aplyxiu ciilifnrnica. Anterior is to the right in each figure. Arrows indicate the FMRFamide-LIR plexus. (A) FMRF- amide-like imnumoreactivity observed on day 5, showing the asymmetry of F-/1, F-/2, F-i-l, and F-cl. Scale bar = 15 /urn. (B) Montage of two photo- graphs showing the EFLRIamide-LIR cells and libers on day 7. Scale bar = 28 fim. (C) FMRFamide-like immunoreactivity on day 9. Two additional cells, FC-1 and FC-3, are shown in this focus. Scale bar = 2X /uni. Right Lateral View Right Lateral View Dorsal View Ventral View Figure 4. Schematic representations of serotonin-LIR and catechol- amine-containing neurons in Aplysia califomica during day 9. Top row: views from the right side and slightly superior to give a three-dimensional perspective. Anterior is to the right in each figure. (A) Serotonin-LIR cells (S-/1, S-/2. S-/'l, S-i'2, SUMl and fibers in the apical organ. Bottom row: dorsal view. (Bl Cells and fibers containing catecholamines were located in the foot (arrow heads), oral region (arrow), and the region of the future CNS (CC-1, CC-2, CC-3). Bottom row: ventral view. group of foot cells toward the region below the apical tuft where the FMRFamide-LIR apical commissure and seroto- nin-LIR plexus were located. In this region, three cate- cholamine-containing central cells were observed (Fig. 6B); two of these cells (CC-1, CC-2) were located on the right and another cell (CC-3) on the left. Unfortunately, the formaldehyde glutaraldehyde technique resulted in high background fluorescence, making it difficult to determine whether these cells were located in the apical organ or the developing cerebral or pedal ganglia of the future adult CNS. Also by this time, serotonin-LIR fibers could be seen projecting toward the velum, foot, and posterior region (Figs. 4A, 5 A). Discussion The current study offers evidence that a relatively exten- sive nervous system forms during embryonic development of the opisthobranch gastropod Aplysiti califomica. The early nervous system includes posterior FMRFamide-LIR cells that first appear during the trochophore stage. By the veliger stage, serotonin-LIR cells appear in the apical organ (Marois and Carew, 1997a, b, c), and shortly before hatch- ing catecholamine-containing cells appear around the mouth and in the foot. The first neurons within the developing NEURODEVELOPMENT IN APLYSIA CALIh'OKNICA 311 Figure 5. Serolonin-LIR cells and fibers in Aplyxia ciilifornicii during days 8-9. Anterior is to the right in each figure. (A) Right lateral view of an embryo on day 9 showing serotonin-LIR cells in the apical organ (S-/1, S-/2, S-rl. SUMl and fibers projecting into the foot (arrowhead) and velum (arrow). Scale bar = 30 /xm. (B) Dorsal view of an embryo on day S showing three of the serotonin-LIR cells (S-/I. S-rl. SUM) of the apical organ. The apical commissure is indicated by the arrowhead. Scale bar = 28 /urn. ganglia, which will eventually constitute the adult CNS, only begin to appear during late embryonic stages (Schacher ft nl., 1979). Such an arrangement of neuronal cells and fibers is similar to that found in representative pulmonate and prosobranch gastropod species (Croll and Voronezh- skaya. 1996a; Dickinson et al., 1999). Posterior FMRFumide-LlR cells Two pairs of FMRFamide-LIR cells appear in posterior regions and project anteriorly directed fibers in early tro- chophore stages of A. califarnica. FMRFamide-like immu- noreactivity has also been observed posteriorly in early embryos of the other gastropod molluscs; however, differ- ences were observed in the number and precise positions of these cells. Lyiwuiea stagwili.? (Croll and Voronezhskaya, 1995, 1996a) and Crepidula fornicata (Dickinson el . Mm: Biol. Ecol. 189: 77-9 1 . Bate, C. M. 1976. Pioneer neurons in an insect embryo. 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Post-translational processing of the alternative neu- ropeptide precursor encoded by the FMRFamide gene in the pulmonate snail, Lymnaea stagnalis. Eur. J. Neurosci. 8: 968-977. Schacher, S., E. R. Kandel, and R. Woolley. 1979. Development of neurons in the abdominal ganglion of Ap/vsia californica I. Axosomatic synaptic contacts. De\: Biol. 71: 176-190. Smith. S. A., J. Nason, and R. P. Croll. 1998. Distribution of cat- echolamines in the sea scallop, Placopecten mage/lanicus. Can. J. Zool. 76: 1254-1262. Syed, N. L, and W. Winlow. 1989. Morphology and electrophysiology of neurons innervating the ciliated locomotor epithelium in Lvnmaea stagnalis (L.). Comp. Biochem. Phvsiol. 93A: 633-644. Voronezhskaya, E. E., and K. Elekes. 1996. Transient and sustained expression of FMRFamide-like immunoreactivity in the developing nervous system of Lymnaea stagnalis. Cell Mol. Neurobiol 16: 661- 676. Voronezhskaya, E. E., and K. Elekes. 1997. Expression of FMRF- amide gene neuropeptides is partly different in the embryonic nervous system of the pond snail. Lymnaea stagnalis L. Neurobio/ogy 5: 91-93. Voronezhskaya, E. E., L. Hiripi, K. Elekes, and R. P. Croll. 1999. Development of catecholaminergic neurons in the pond snail, L\mnaea stagna/is I: Embryonic development of dopaminergic neurons and dopamine-dependent behaviors. J. Comp. Neurol. 404: 297-309. Reference: Binl. Bull 199: 316-320. (December 2000) Development of a Penis from the Vestigial Penis in the Female Apple Snail, Pomacea canaliculata NAOKUNI TAKEDA Brainway Group, Brain Science Institute. The Institute of Physical and Chemical Research, 2-1 Wako. Saitama 351-0198, Japan In the apple snail (Pomacea canaliculata), females have an undifferentiated mass of tissue near the anus. Although this mass is called the vestigial penis, there are no signs of a hermaphroditic gonad or any structure that represents a transition from one se.x to the other. Based on consider- ations of the steroid hormone theory of reproduction and in view of disruption of endocrine s\slems in molluscs by organotins, a study was made of the effects of tributyltin on female snails. Exposure to tributyltin resulted in the so- called imposex phenomenon, and both a penis and a penis sheath were newly generated from the so-called vestigial penis. The same phenomenon was also induced b\ testos- terone. Thus the vestigial penis, named more than one hundred vears ago, has been demonstrated for the first time to be a rudiment of the penis itself. The Ampullariidae (Gastropoda, Prosobranchia, Archi- taenioglossa) is a family of freshwater prosobranchs that are widely distributed in Asia, Africa, and South America ( 1 ). It is well known that some molluscs exhibit unusual sexual diversity and hermaphroditism (2, 3). One of the most interesting features of members of the Ampullariidae is that females in 5 of the 10 genera — namely, Pila (4, 5, 6. 7. 8. 9, 10, 11, 12), Pomacea (13, 14. 15. 16), Lunixtes (13, 17, 18), Afropomus (19), and Turbinicola (13) — have a so- called vestigial penis in addition to the normal reproductive system (Fig. 1). The vestigial penis was first described by Bouvier (4, 5), a skilled neuroanatomist. in Ampullaria (Pila) polita more than 100 years ago. Since then, many molluscan researchers have used that term for this tissue without further explanation. The question examined here is this: Is the organ historically described as a vestigial penis a remnant of a penis — a degenerate structure that lacks the Received 7 December 1999; accepted 10 August 2000. E-mail: takeda@brainway.riken.go.jp capacity to develop further — or is it a rudiment, or precur- sor— an incipient structure that, under the proper conditions, could develop into a penis? The vestigial penis is a tongue-like structure lying inside the elevated mantle skirt, near the anus (Fig. 2 A). Histolog- ically, it consists of connective tissue, and no differentiation of the structure is apparent during the life cycle of the female (Fig. 2B). To my knowledge, no experimental evidence has been pre- sented to justify the designation of the elevated tissue near the anus in some female snails as a "vestigial penis" (4, 5). No clear evidence of hermaphroditism has yet been shown in any extant species of Ampullariidae. In Pomacea canaliculata, the apple snail, the positions of the gonads are basically different: the testis is located at the tip of the spiral, and the ovary is spread over the surface of the hepatopancreas at a location similar to that of the testis. It has also been confirmed that there is no apparent precursor or vestige of a hermaphroditic condi- tion in any part of the reproductive system throughout the life history. As Andrews (13) stated, the copulatory apparatus appears to develop at the same rate in both sexes until the gonad becomes active, when its growth is arrested in the female. Andrews hypothesized that the gonad might produce a hormone responsible for the cessation of growth, but in the early 1960s, when this work was published, the chemical nature of reproductive hormones in molluscs had not yet been established. In 1991, Berthold (20) proposed that the so-called vestigial organ be designated an "oriment" — a term implying that the tissue is a precursor with the potential to develop into an adult organ. The question then arose as to whether such a designation might be appropriate. As a basis for such a designation, at the very least, some experiments involving implantation of testes into females should be performed to determine whether a true penis might develop from the tissue mass. 316 IMPOSEX IN THE APPLE SNAIL. P. CANALICULATA .7 317 Figure 1. General appearance of the vestigia] penis in a female apple snail (Pomacea canaliculata). The shell was removed and the region to the left of the head was dissected to reveal the vestigial penis ( 1 ), rectum (2), and oviduct (3), which are enclosed in a square. The siphon (4). pulmonary sac (5), albumen gland (6), and ovary (7) are also indicated. Scale bar represents 1 cm. As an approach to this problem, I examined the effects of an endocrine disruptor that appears to be associated with as yet unresolved environmental problems. Organotins, and in particular tributyltin, which is a component of some anti- fouling paints, induce a condition known as "imposex" in prosobranch gastropods. Imposex, in which a penis and vas deferens develop in females (21 ), has been widely observed in marine snails that belong to the Caenogastropoda; Nu- cella and Littorina are common examples (22). Females of these species lack a vestigial penis, but the capacity exists for induction of a penis and vas deferens. Pomacea canal- iculata has been proposed as a potential bioindicator for tributyltin (22), which is used as a biocidal agent against molluscs, in fungicides (23) and in anti-fouling paints in freshwater environments. Anticipating possible endocrine disruption by tributyltin, I examined its effects to see whether the vestigial penis in this species might develop further after female snails were exposed to this compound and, if such a penis did develop, how would it differentiate? Female specimens off. canaliculata were reared in water that contained 30 ng/1 tributyltin. About 3 months after the start of treatment, the outside of the elevated tissue of the vestigial penis began to form a long process that resembled a penis, and its interior developed as a thick mass. These structures grew gradually and reached a maximum size after about 6 months of treatment with tributyltin (Fig. 3A). Histological staining revealed that the inside tissue mass contained a penis; the cross section of a penis was also found within the tissue mass (Fig. 3B). Thus, the outside structure appeared to be a penis sheath. Within about one further month, a complete penis had developed from the tissue of the vestigial penis (Fig. 4). In P. canaliculata, the copulatory system of the male consists of a stout penis sheath and a long, slender penis 318 N. TAKEDA VP B Figure 2. (A) General appearance of the vestigial penis in a female apple snail (Pomacea canaliculata: control) reared by artificial mass cul- ture. Scale bar represents 1 mm. (B) Histological appearance of the vestigial penis in A. The regions containing a vestigial penis were fixed in Bouin's fluid and embedded in paraffin wax by the standard method. Sections were stained with hematoxylin and eosin. Scale bar represents 200 ;im. A. anus; C, ctenidium; OD, oviduct; VP. vestigial penis. within it (24). The penis and the penis sheath are located together to the left of the extreme right margin of the mantle cavity. In treated females, the arrangement of these male copulatory organs was similar but differed in the distance between the penis sheath and the penis: the penis sheath in females was located at the edge of the ctenidium at the mantle skirt, at a distance from the penis. It has been suggested that tributyltin inhibits cytochrome P450 aromatase, which converts testosterone to estradiol in females (25, 26). Inhibition of aromatase activity thus in- creases levels of testosterone which induces imposex, with the development of male copulatory organs. Development of the imposex phenomenon in P. canaliculata was also confirmed by direct treatment with testosterone. Female snails reared in water that contained 500 ng/1 testosterone exhibited changes similar to those induced by tributyltin, including the development of a penis sheath and a penis. Therefore, these observations support the proposed mecha- nism of action of tributyltin. It is difficult to explain the unusual phenomenon of a rudimentary penis in females; however. I propose the fol- lowing hypothesis. In the early stages of development, both sexual rudiments develop as an undifferentiated tissue mass. Once the sex of the snail is determined genetically (27), however, these rudiments differentiate in response to the secretion of specific sex steroid hormones. The undifferen- tiated tissue mass that develops into a penis in males is left as an arrested rudiment in females. The vestigial penis develops into a complete copulatory organ only if the anlage of the gonad becomes a testis. B Figure 3. (A) Morphology of the imposex induced by trihutyltin in a female apple snail (Pumacea canaliculata). Female snails (n = 100) for experiments were reared in a freshwater tank that contained tributyltin (Tokyo Kasei, Co. Ltd.. Tokyo, Japan) at 30 ng/l for about f> months. The state of imposex was checked at weekly intervals. Scale bar represents 1 mm. Similar results were also obtained in female snails (n = 100) reared with testosterone (Wako. Co. Ltd., Osaka. Japan) at 500 ng/1 for about 7 months. (B) Histological appearance of the vestigial penis in A. The arrowhead indicates the cross section of a penis. Hematoxylin and eosin stain. See legend to Fig. 2B for methods. Scale bar represents 200 JLUTI. P. penis; PS. penis sheath; VD. vas deferens (see legend to Fig. 2 for other abbreviations). 1MPOSEX IN THE APPLE SNAIL. P. CANALICULATA 319 Figure 4. (A) A penis that arose from the vestigial penis of a female apple snail (Pomacca canaliculate!) after treatment with tributyltin for about 7 months. The same phenomenon was also seen in female snails reared with testosterone for about 8 months. See legend to Fig. 3A for methods. (B) The extirpated penis from the vestigial penis. See legends to Fig. 2 and 3 for other abbreviations. Scale represents 1 mm. The "steroid hormone theory," which I proposed previously (28, 29) for the reproduction of terrestrial pulmonates, appar- ently also applies to prosobranch snails. This theory states basically that the development of accessory sex organs is controlled by steroid hormones secreted by the gonad. This concept of the effects of hormones on snail reproduction, together with the effects of endocrine disruption in molluscs, allowed me to demonstrate, for the first time, that the so-called "vestigial penis," named more than one hundred years ago (4. 5), is a rudiment of the penis itself. 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MALGAROLI, Mechanisms of spontaneous miniature activity at CA3-CA1 synapses: evidence for a divergence from a random Poisson process, 1 84 Actin, 202, 203 Action potential, 135 Adenylate cyclase, 197 Adhesion of a viral envelope protein to a non-self binding domain of the aggregation factor in the marine sponge Microciona prolifera, 209 Adhesive proteoglycans, 192 Aggregation factor, 192, 209 Aggression, 265 Aiptasia, 76 Allometric scaling in small colonies of the scleractinian coral Siderastrea siderea (Ellis and Solander), 21 Allometry, 21 cAMP. 197 Amphibian. 187 An inducer of molluscan metamorphosis transforms activity patterns in a larval nervous system, 241 Annual Report of the Marine Biological Laboratory, v. 199 ( 1 ), R 1 Anuran, 187 Apical organ, 305 Aplysia, 305 Arbacia eggs, 212, 213 ARMSTRONG. MARGARET T., see Rengasamy Asokan. 190 ARMSTRONG, PETER B., AND RENGASAMY ASOKAN. A Ca+:-independent cytolytic system from the blood of the marine snail Busycon cana- liciilum. 194 ARMSTRONG. PETER B., see John M. Harrington, 189; Rengasamy Asokan, 190 ASOKAN, RENGASAMY, MARGARET T. ARMSTRONG, AND PETER B. ARM- STRONG, Association of a,-macroglobulin with the coagulin clot in the American horseshoe crab. Limulus polyphemus: a potential role in stabilization from proteolysis, 190 ASOKAN, RENGASAMY, see Peter B. Armstrong, 194 Association of a-,-macroglobulin with the coagulin clot in the American horseshoe crab, Limulus polyphemus: a potential role in stabilization from proteolysis, 190 Asteroidea. 298 ATHERTON, JILLIAN L., MATTHEW A. KRUTKY, JAMES M. HITT, FREDERICK A. DODGE, AND ROBERT B. BARLOW, Optic nerve responses of Limulus in its natural habitat at night, 176 ATHERTON, JILLIAN L., see Matthew A. Krutky, 178 Axonal transport, 202, 203 Axoplasm, 203 B BAGHDASARIAN, GAREN, AND LEONARD MLISCATINE, Preferential expulsion of dividing algal cells as a mechanism for regulating algal-cnidarian symbiosis, 278 BAKER, SHIRLEY M., JEFFREY S. LEVINTON, AND J. EVAN WARD. Particle transport in the zebra mussel, Dreissena polymorpha (Pallas), 1 16 BARLOW, ROBERT B.. see James M. Hitt, 171; Jillian L. Atherton, 176; Matthew A. Krutky, 178 BARNES, DAVID K. A., AND MATTHEW H. DICK, Overgrowth competition between clades: implications for interpretation of the fossil record and overgrowth indices, 85 BENTLEY, M. G., see G. J. Watson, 50 BEZANILLA, FRANCISCO, see Joshua J. C. Rosenthal, 135 Biogeochemistry, 218 Biogeography, 126 Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt), 126 Biosynthesis, 192 Birefringence, 212 Bivalve, 29, 116, 144, 199 Blood clotting, 190 Boatwhistle, 173 BOLTON. TOBY F., FLORENCE I. M. THOMAS, AND CELERE N. LEONARD, Maternal energy investment in eggs and jelly coats surrounding eggs of the echinoid Arbacia punctulata, 1 Boron, 221 BROTHERS, CHRISTINE, ERNEST MARKS in, AND ROXANNA SMOLOWITZ, Con- ditions affecting the growth and zoosporulation of the protistan par- asite QPX in culture, 200 BROWN. JEREMIAH, see Phillip Stafford, 203 BRUZZONE, ROBERTO, see Thomas H. White. 165 Bryozoan. 85 BucKLAND-NlCKS, JOHN, AND ALAN N. HODGSON, Fertilization in Callo- chiton castaneus (Mollusca), 59 BURGER, MAX M., see William J. Kuhns, 192; Roger MacKenzie, 209 BURGOS, MARIO H., MAKOTO GODA, AND SHINYA INOUE, Fertilization- induced changes in the fine structure of stratified Arbacia eggs. II. Observations with electron microscopy ,213 BURGOS, MARIO H., see Makoto Goda, 212 BYRNE, R. A., see T. H. Diet?., 14 Ca2 + , 212 A Ca+2-independent cytolytic system from the blood of the marine snail Busvcon canaliculwn, 194 Cable properties, 135 Calcium imaging, 162 Camouflage, 6 Carotenoid, 223 Cassiopeia. 76 Cataract, 187 Catecholamines, 305 Cell contact, 287 motility, 202, 203 movement, 287 proliferating, 199 Cellular basis of gastrulation in the sand dollar Scaphechinus mirabilis, 287 Centrifugation, 212, 213 Centrifuge polarizing microscope, 212 CHANG, F., see P. T. Tran, 205 Channel, 165 Cherax destructor, 25 1 CHERRY, J. S., see T. H. Dietz. 14 CHIKARMANE, HEMANT M., ALAN M. KUZIRIAN. ROBBIN KOZLOWSKI, MARK KUZIRIAN, AND TONY LEE, Population genetic structure of the goose- fish, Lophius americamis, 227 CHILD. FRANK M., see David A. Epstein, 182 Chimerism, 231 Chlorophyll, 223 Circadian rhythms in the receptive fields of the Limulus lateral eye, 171 Clades, 85 Cladophora vagabunda, 223 CLAESSENS, Luc, see Ryan Kirkby, 218 321 322 INDEX TO VOLUME 199 Cleavage. 207 Cnidaria, 68, 278 COHEN, LAWRENCE B.. see Matt Wachowiak. \(i2 Colony. 231 Conditions affecting the growth and z.oosporulation of the protistan para- site QPX in culture, 200 Conduction velocity, 135 Connexin, 165 Copepod, 95 Coral, 21.68 CRAWFORD, KAREN. The role of microtubules during blastodisc formation of the squid Loligo pealei. 207 Crayfish. 251 A critical approach to the definition of Darwinian units of selection. 231 CROLL. ROGER P.. see Amanda J. G. Dickinson, 305 CROMARTY. S. I., J. MELLO. AND G. KASS-SIMON, Molt-related and size- dependent differences in the escape response and post-threat behavior of the American lobster, Hoinarus americanus, 265 Crustacea, 265 Cytoplasmic streaming. 207 Cytoskeleton. 144. 202. 203 D DACEY, JOHN W. H.. see Richard W. Hill. 108 DAVIS, THEODORE M.. see Inigo Novales Flamarique. 187 Denaturing gradient gel electrophoresis. 126 DENAULT, MICHELLE. ERICA STIEVE, AND IVAN VALIELA. Effects of nitrogen load and irradiance on photosynthetic pigment concentrations in Cladophora vagabiinJn and Gmcilaria tikvahiiie in estuaries of Wa- quoit Bay. 223 Denitrification, 221 Descending octaval nucleus. 173 Detritus. 225 Development. 68. 195 Development of a penis from the vestigial penis in the female apple snail, Ponutcea canaliculata, 316 Development of embryonic cells containing serotonin, catecholamines. and FMFRamide-related peptides in Aplysia califomica, 305 Development of self-referencing oxygen microsensor and its application to single pancreatic HIT cells; effects of adenylate cyclase activator forskolin on oxygen consumption. 197 DICK, MATTHEW H., see David K. Barnes. 85 DICKINSON, AMANDA J. G., ROGER P. CROLL, AND ELENA E. VORONEZH- SKAYA, Development of embryonic cells containing serotonin, cat- echolamines. and FMFRamide-related peptides in Aplysia califurnk n. 305 DIETZ, T. H.. A. S. UDOETOK. J. S. CHERRY, H. SILVERMAN, AND R. A. BRYNE, Kidney function and sulfate uptake and loss in the freshwater bivalve Toxolastna texasensist 14 Differences in properties of salt marsh sediment between hayed and ref- erence sites, 223 Differentiation. 41 Dimethylsulfoniopropionate, 1 1 18 Dimethylsulfoniopropionate in giant clams (Tridacnidae), 108 Disease, 199 Do the properties of underwater lighting inlluence the visually guided behavior of Linnilu\'!. 178 DODGE, FREDERICK A., see James M. Hut, 171; Jillian L. Atherton, 176; Matthew A. Krutky, 178 DOYE, V., see P. T. Tran, 205 Dreissenn, 116, 144 Drosopliila. 170 E Early development of zooxanthella-containing eggs of the corals Pocillo- pora verrucosa and P. eydtnixi with special reference to the distribu- tion of zooxanthellae, 68 Echinodermata. 298 Echinoid. 1 EDDS-WALTON, PEGGY L., see Richard R. Fay. 173 EDMUNDS, PETER J.. see Steven V. Vollmer. 21 EDWARD. AHS[ R. see Richard W. Hill. 108 Effects of food concentration and availability on the incidence of cloning in planktotrophic larvae of the sea star Pisaster ochraceus, 298 Effects of myosin-II antibody on actin-dependent vesicle transport in extracts of clam oocytes, 202 KttC'.ts of nitrogen load and irradiance on photosynthetic pigment concen- trations in Clutloptun'ii vagabunda and Grticiltiriu tikviihiti? in estu- aries of Waquoit Bay. 223 Electron microscopy. 213 Embryo, 305 Embryogenesis, 29 Endocrine disruption, 316 Endoplasmic reticulum, 212, 213 Endoscopy, 1 16 ENGLER, JOHN, see Robert M. Gould, 2 1 5 Enzymatic biosynthesis of N-linked glycan by the marine sponge Micro- fioiui prolifera, 192 Enzyme activity, 100 EPSTEIN. DAVID A.. HERMAN T. EPSTEIN, FRANK M. CHILD. AND ALAN M. KUZIRIAN. Memory consolidation in Hermissenda crassicornis, 182 EPSTEIN. HERMAN T., see David A. Epstein, 182 ER transport, 202, 203 Escape response. 265 Eye. 171. 176 Eyeshine, 6 Factors influencing spawning and pairing in the scale worm Hannothoe inihriiiiki, 50 FALK. CHUN X., see Matt Wachowiak, 162 FARMER, MARK A., see Timothy S. Wakefield, 76 Fate of anthropogenic nitrogen in a nearshore Cape Cod aquifer, 221 FAY, RICHARD R., AND PEGGY L. EDDS-WALTON, Frequency response of auditory brainstem units in toadfish Opsamm tan. 1 73 FERNANDEZ-BUSQUETS. XAVIER, see William J. Kuhns. 192 Fertilization, 59. 144. 212, 213 Fertilization in Callochilon castaneus (Mollusca). 59 Fertilization-induced changes in the fine structure of stratified Arbacia eggs. I. Observations on live cells with the centrifuge polarizing microscope. 212 Fertilization-induced changes in the fine structure of stratified Arbacia eggs. II. Observations with electron microscopy. 213 FINLEY, LUKE, AND DAVID MACMILLAN. The structure and growth of the statocyst in the Australian crayfish Cherax destructor. 251 Fish, startle response. 180 FISHER, CHARLES R., see Stephane Hourdez, 95 FMRFamide, 305 Forskolin. 197 FORTI. L., see A. Abenavoli. 184 Fouling organism, 189 Freeze substitution, 76 Frequency response of auditory brainstem units in toadfish Opsannx tan. 173" FREUND, CONCETTA M., see Robert M. Gould. 215 Frog Lim-1-like protein is expressed predominantly in the nervous tissue. gonads, and early embryos of the bivalve mollusc Mytilus gallopro- yiitciiili.i. 29 GALLO. MICHAEL A., see Diane E. Heck. 195 Gastropod. 241 Gastrulation, 287 GATEN. E.. see M. L. Johnson. 6 Gating current, 164 GALIDRON, S. M.. see G. J. Watson, 50 Gene, 231 activity, 100 expression, 215 INDEX TO VOLUME 199 323 Gene expression and enzyme activities of the sodium pump during sea urchin development: implications for indices of physiological state, 100 Giant axon. squid. 135 GIBLIN. ANNE, see Adena Greenbaum. 223 GIBSON. DANIEL G., see Rhea Hanselmann. 199 GIBSON. GLENYS D.. AND JENNIFER M. L. HARVEY. Morphogenesis during asexual reproduction in Pygospio elegans Claparede (Annelida. Polychaeta). 41 GODA, MAKOTO. MARIO H. BURGOS, AND SHINYA INOUE. Fertilization- induced changes in the fine structure of stratified Arbacia eggs. I. Observations on live cells with the centrifuge polarizing microscope, 212 GODA. MAKOTO, see Mario H. Burgos. 213 Gonad, 29 GOULD. ROBERT M., CONCETTA M. FREUND, JOHN ENOLER, AND HILARY G. MORRISON, Optimization of homogenization conditions used to isolate niRNAs in processes of myelinating oligodendrocytes, 215 Gracilaria tikvahiae. 223 GREENBAUM, ADENA. AND ANNE GIBLIN. Differences in properties of salt marsh sediment between hayed and reference sites. 223 Groundwater. 221 Growth. 251 H HADFIELD. MICHAEL G.. see Esther M. Leise. 24 1 HALE. MELINA E.. Startle responses of fish without Mauthner neurons: escape behavior of the lumpfish (Cyclopterus lumpus), ISO HAMMAR. KATHERINE. see Sung-Kwong Jung. 197 HANSELMANN, RHEA. ROXANNA SMOLOWITZ. AND DANIEL G. GIBSON. Iden- tification of proliferating cells in hard clams, 199 Hard clam. 199 Hannothoe imbricate, 50 HARRINGTON, JOHN M., AND PETER B. ARMSTRONG, Initial characterization of a potential anti-fouling system in the American horseshoe crab, Limiiliis polyphemus, 189 HARVEY. JENNIFER M. L., see Glenys D. Gibson. 41 Haying. 225 Hearing. 173 Heart rate. 257 Heavy water. 164 Heavy water (D,0) alters the sodium channel gating current in squid giant axons. 164 HECK. DIANE E.. LYDIA Louis, MICHAEL A. GALLO, AND JEFFREY D. LASKIN. Modulation of the development of plutei by nitric oxide in the sea urchin Arbacia punctulata, 195 Hemocyte proliferation, 199 Hemoglobin. 95 functional properties. 95 structure. 95 Hemoglobin from a deep-sea hydrothermal-vent copepod. 95 Hemolysis. 194 Htrinissenda, 182 HERRING, P. J.. see M. L. Johnson, 6 HIDAKA, M., see M. Hirose, 68 HILL, RICHARD W.. JOHN W. H. DACEY. AND AHSER EDWARD, Dimethyl- sulfoniopropionate in giant clams (Tridacnidae), 108 Hippocampus. 184 HIROSE. M.. R. A. KINZIE III, AND M. HIDAKA, Early development of zooxanthella-containing eggs of the corals Pocillopora verrucosa and P. evdouxi with special reference to the distribution of zooxanthellae. 68 " HITT. JAMES M.. see Jillian L. Atherton, 176 HITT. JAMES M., FREDERICK A. DODGE. EHUD KAPLAN. AND ROBERT B. BARLOW. Circadian rhythms in the receptive fields of the Limulus lateral eye. 171 HODGSON. ALAN N.. see John Buckland-Nicks. 59 Hoimmis. 257. 265 HOPKINSON. CHARLES. JR., see Ryan Kirkby, 218: Anne Perring, 219 Horizontal cell. 168 HOURDEZ, STEPHANE, JASON LAMONTAGNE, PAT PETERSON, ROY E. WEBER. AND CHARLES R. FISHER. Hemoglobin from a deep-sea hydrothermal- vent copepod. 95 Human immunodeficiency virus. 209 Hydrogen ion fluxes from isolated retinal horizontal cells, modulation by alutamate. 168 I Identification of proliferating cells in hard clams. 199 Immunity, innate. 189. 190. 194 Imposex, 316 Individual, 231 Initial characterization of a potential anti-fouling system in the American horseshoe crab, Limulus polyphemus, 189 INOUE. SHINYA. see P. T. Tran, 205; Makoto Goda. 212; Mario H. Burgos. 213 Interaction of actin- and microtubule-based motors in squid axoplasm probed with antibodies to myosin V and kinesis. 203 Intercellular communication. 165 Intrinsic membrane properties of laryngeal motoneurons that control sex- ually differentiated vocal behavior in African clawed frogs, Xenopus leavis, 175 Ipswich. 218 Irradiance. 223 Jelly coat. 1 JOHNSON. J. L., P. M. J. SHELTON, E. GATEN. AND P. J. HERRING. Relation- ship of dorsoventral eyeshine distributions to habitat depth and animal size in mesopelagic decapods. 6 JUNG, SUNG-KWON, KATHERINE HAMMAR, AND PETER J. S. SMITH. Devel- opment of self-referencing oxygen microsensor and its application to single pancreatic HIT cells; effects of adenylate cyclase activator forskolin on oxygen consumption, 197 JURY, STEVEN H., AND WINSOR H. WATSON III. Thermosensitivity of the lobster, Homarus americamis, as determined by cardiac assay. 257 K KACZMAREK, LEONARD K.. see Ayako Yamaguchi. 175 KAPLAN, EHUD, see James M. Hitt, 1 7 1 KASS-SIMON. G.. see S. I. Cromarty. 265 KELLEY. DARCY B.. see Ayako Yamaguchi. 175 KEMPF, STEPHEN C., see Timothy S. Wakefield. 76 Kidney function. 14 Kidney function and sulfate uptake and loss in the freshwater bivalve Toxolasma te.wsensis. 14 Kinesin, 203 KINZIE, R. A.. Ill, see M. Hirose. 68 KIRKBY. RYAN, Luc CLAESSENS. CHARLES HOPKINSON, JR., EDWARD RAS- TETTER, AND JOSEPH VALLINO, Modeling the effects of land-use changes on nitrogen biogeochemistry in the Ipswich watershed, Mas- sachusetts. 218 KOMINAMI, TETSUYA. AND HIROMI TAKATA, Cellular basis of gastrulation in the sand dollar Scaphechinus mirabilis, 287 KOZLOWSKI, ROBBIN. see Hemant M. Chikarmane, 227 KROEGER, KEVIN D., see Elizabeth J. Westgate. 221 KRUTKY, MATTHEW A.. JILLIAN L. ATHERTON. SPENCE SMITH. FREDERICK A. DODGE. AND ROBERT B. BARLOW. Do the properties of underwater lighting influence the visually guided behavior of Limulus?, 178 KRUTKY. MATTHEW A., see Jillian L. Atherton. 176 KUHNS. WILLIAM J., MAX M. BURGER. MOHAN SARKER, XAVIER FERNAN- DEZ-BuSQUETS, AND TRACY SIMPSON. Enzymatic biosynthesis of N- linked glycan by the marine sponge Microciona prolifera. 1 92 KUHNS, WILLIAM J.. see Roger MacKenzie, 209 KUZIRIAN. ALAN M.. see David A. Epstein. 182; Hemant M. Chikarmane. 227 KUZIRIAN, MARK, see Hemant M. Chikarmane. 227 324 INDEX TO VOLUME 199 Labynnthomorpha, 200 LAJEUNESSE. R. C, AND R. K. TRENCH, Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt), 126 LAMONTAONE, JASON, see Stephane Hourdez, 95 LANDOWNE, DAVID, Heavy water (D2O) alters the sodium channel gating current in squid giant axons, 164 LANOFORD, F. M., see G. J. Watson. 50 LANOFORD, GEORGE M.. see Leslie Sandberg, 202; Phillip Stafford, 203 LASKIN, JEFFREY D., see Diane E. Heck, 195 Learning, human, 182 LEE, TONY, see Hemant M. Chikarmane. 227 LEISE, ESTHER M., AND MICHAEL G. HADFIELD, An inducer of molluscan metamorphosis transforms activity patterns in a larval nervous system, 241 LEONARD, CELERE N., see Toby F. Bolton. 1 LEONG, PATRICK K. K., see Adam G. Marsh, 100 LEVINTON, JEFFREY S., see Shirley M. Baker, 1 16 Lim-1 protein, 29 Limulus, 171, 176. 178. 189, 190 Lobster, 257, 265 Loligo, 135 Lophius americanus, 227 Louis, LYDIA, see Diane E. Heck, 195 LYNN, J. W., see M. J. Misamore, 144 M MACKENZIE, ROGER, DAVID NEWMAN, MAX M. BURGER, RENE ROY. AND WILLIAM J. KUHNS, Adhesion of a viral envelope protein to a non-self- binding domain of the aggregation factor in the marine sponge Mi- crociona prolifera, 209 MACMILLAN, DAVID, see Luke Finley, 251 Macroalgae, 223 a:macroglobulin, 190 MALCHOW, ROBERT PAUL, see Anthony J. A. Molina, 168 MALGAROLI, A., see A. Abenavoli, 184 MANAHAN, DONAL T., see Adam G. Marsh, 100 Marine Biological Laboratory Annual Report, v. 199 (1), Rl General Scientific Meetings, Short Reports. 157 Marine invertebrate reproduction. 1 MARKS, ERNEST, III, see Christine Brothers, 200 MARSH, ADAM G., PATRICK K. K. LEONG, AND DONAL T. MANAHAN, Gene expression and enzyme activities of the sodium pump during sea urchin development: implications for indices of physiological state. 100 Maternal energy investment in eggs and jelly coats surrounding eggs of the echinoid Arbacia punclulahi. 1 Maternal investment, 1 Mauthner neuron, 180 MBL Annual Report, v. 199 (1), R I General Scientific Meetings, Short Reports, 157 McCLiNTOCK, JAMES B., see Minako S. Vickery, 298 Mechanisms of spontaneous miniature activity at CA3-CA1 synapses: evidence for a divergence from a random Poisson process, 184 MELLO, J., see S. I. Cromarty, 265 Memory blocking, 182 long-term, 182 short-term, 182 consolidation, 182 Memory consolidation in Hermissenda crassicomis, 182 MEPSC, 184 Mesopelagic ecology, 6 Microciona, 192, 209 Microsporidian, 208 Microtubule. 205, 207 Microtubule-dependent nuclear positioning and nuclear-dependent septum positioning in the fission yeast, S. pombe, 205 MIKHAILOV, ALEXANDER T., see Mario Torrado. 29 MISAMORE, M. J., AND J. W. LYNN, Role of the cytoskeleton in sperm entry during fertilization in the freshwater bivalve Dreissena polymorpha, 144 Modeling the effects of land-use changes on nitrogen biogeochemistry in the Ipswich watershed, Massachusetts, 218 Modular organisms, 21 Modulation of the development of plutei by nitric oxide in the sea urchin Arbacia punclulala, 195 MOLINA, ANTHONY J. A., PETER J. S. SMITH, AND ROBERT PAUL MALCHOW. Hydrogen ion fluxes from isolated retinal horizontal cells, modulation by glutamate, 168 Mollusc, 59, 194, 241 Molt cycle. 265 Molt-related and size-dependent differences in the escape response and post-threat behavior of the American lobster, Homarus americanus, 265 Monkfish, 227 Morphogenesis, 287 Morphogenesis during asexual reproduction in Pygospio elegans Clapa- rede (Annelida, Polychaeta), 41 Morphology, 251 MORRISON, HILARY G., see Robert M. Gould, 215 Mosaic, 231 Motoneuron, 175 mRNA transport. 215 MUSCATINE, LEONARD, see Garen Baghdasarian, 278 Myelination, 215 Myosin, 202. 203 N Nervous ganglion, 29 NEWMAN. DAVID, see Roger MacKenzie, 209 Nitrate, 221 Nitric oxide. 195 Nitrogen, 223 processing, 218 NOVALES FLAMARIQUE, INIGO, KRISTIINA OVASKA, AND THEODORE M. DAVIS. UV-B-induced damage to the skin and ocular system of amphibians, 187 Nuclear positioning, 205 Nudibranch. 241 o Odor recognition, 162 Odor-induced oscillatory activity in Drosophila CNS. 1 70 Olfaction, 170 Olfactory bulb, input to, 162 Oocyte, 202 Opisthobranch. 241 Optic nerve, 1 76 Optic nerve responses of Limulus in its natural habitat at night, 1 76 Optimization of homogenization conditions used to isolate mRNAs in processes of myelinating oligodendrocytes, 215 Organotin, 316 Oscillation, 170 OVASKA, KRISTIINA, see Inigo Novales Flamanque, 187 Overgrowth competition, 85 Overgrowth competition between clades: implications for interpretation of the fossil record and overgrowth indices, 85 Oxygen consumption, 197 microsensor. 197 PABLICH, WENDY J., see Elizabeth J. Westgate PAH clearance. 14 INDEX TO VOLUME 199 325 Pancreatic HIT cell. 197 Particle selection, 1 Id Particle transport in the zebra mussel, Dreissena polymorpha (Pallas). I Id Passive tracer, 221 PCNA. 199 PEG clearance, 14 PERRING, ANNE, MICHAEL WILLIAMS, CHARLES HOPKINSON, JR.. EDWARD RASTETTER. AND JOSEPH VALLINO, Solute dynamics in storm flow of the Ipswich River Basin: effects of land use, 219 PETERSON, PAT, see Stephane Hourdez. 95 pH. 200 Pheromone, 50 Phycoerythrin, 223 Pigment. 223 Pigment cell, 195 Planula, 68 Plume, 221 Polychaete, 41, 50 Polyplacophora, 59 Pomticeii ctiniilicitUihi, 316 Population, 227 Population genetic structure of the goosefish, Lophius americanus, 227 Preferential expulsion of dividing algal cells as a mechanism for regulating algal-cnidarian symbiosis. 278 Protease inhibitor, 190 Protein synthesis, 215 Proteoglycans, 192, 209 QPX. 199, 200 Quanta, spontaneous release. 184 Q R RAPD. 227 RASTETTER. EDWARD, see Ryan Kirkby, 218; Anne Perring, 219 Regeneration. 41 Relationship of dorsoventral eyeshine distributions to habitat depth and animal size in mesopelagic decapods, 6 Renal clearance. 14 Reproduction, 50 Retina, 165, 168 Revised description of the fine structure of in situ "Zooxanthellae" genus Symbiodinium, 76 RINKEVICH, B., A critical approach to the definition of Darwinian units of selection, 231 RIPPS, HARRIS, see Thomas H. White. 165 The role of microtubules during blastodisc formation of the squid Lo/igo pealei, 207 Role of the cytoskeleton in sperm entry during fertilization in the fresh- water bivalve Dreissena polvmorpha, 144 ROSENTHAL, JOSHUA J. C., AND FRANCISCO BEZANILLA. Seasonal variation in conduction velocity of action potentials in squid giant axon, 135 ROY. RENE, see Roger MacKenzie, 209 Salinity, 200 Salt marsh, 225 SANDBERG. LESLIE, PHILLIP STAFFORD, AND GEORGE M. LANGFORD, Effects of myosin-II antibody on actin-dependent vesicle transport in extracts of clam oocytes, 202 SARKER, MOHAN, see William J. Kuhns, 192 Scaling, 21 Sea star larval cloning, 298 Sea urchin, 195, 287 development, 100 Seasonal variation in conduction velocity of action potentials in squid giant axon. 135 Segmentation, 41 Self-referencing, 197 Septic, 221 Septum positioning, 205 Serotonin, 305 Sex steroid hormone. 316 Sexual differences. 175 SHELTON. P. M. J.. see M. L. Johnson. 6 SILVERMAN, H., see T. H. Dietz, 14 SIMPSON. TRACY, see William J. Kuhns, 192 Size-dependent behavior, 265 Skate. 168 SMITH. PETEK J. S.. see Anthony J. A. Molina, 168; Sung-Kwong Jung, 197 SMITH, SPENCE, see Matthew A. Krutky, 178 SMOLOWITZ, ROXANNA, see Rhea Hanselmann. 199; Christine Brothers, 200 SO4 clearance. 14 Sodium channel, 164 pump, 100 Solute dynamics, 219 Solute dynamics in storm flow of the Ipswich River Basin: effects of land use, 219 The spatial representation of odors by olfactory receptor neuron input to the olfactory bulb is concentration invariant, 162 Spawning, 50 Species diversity, 126 Sponge. 192, 209 Sporoplasm, 208 Squid. 135, 164. 207 SRINIVAS, MIDUTURU, see Thomas H. White. 165 STAFFORD, PHILLIP. JEREMIAN BROWN, AND GEORGE M. LANGFORD, Inter- action of actin- and microtubule-based motors in squid axoplasm probed with antibodies to myosin V and kinesis, 203 STAFFORD, PHILLIP, see Leslie Sandberg, 202 Startle responses of fish without Mauthner neurons: escape behavior of the lumpfish (Cyclopterus lum/nts), ISO Statocyst, 251 Stem cell, 231 STIEVE, ERICA, see Michelle Denault, 223 The structure and growth of the statocyst in the Australian crayfish Cherax destructor, 251 Sulfate transport. 14 Suppression subtractive hybridization. 215 Surface motor proteins, 192 Symbiodinium, 76, 108, 126 Symbiosis, 76, 126. 278 Symbiotic dinoflagellate, 278 Synapse, 184 Synchrony, 170 Tail flip, 265 TAKATA, HIROMI, see Tetsuya Kominami, 287 TAKEDA, NAOKUNI, Development of a penis from the vestigial penis in the female apple snail, Pomacea canaliculate, 316 Temperature. 200, 257 acclimation, 135 Thermosensitivity. 257 Thermosensitivity of the lobster. Homarus americanus, as determined by cardiac assay, 257 THOMAS, FLORENCE I. M., see Toby F. Bolton, 1 TORRADO, MARIO. AND ALEXANDER T. MIKHAILOV, Frog Lim-1-like protein is expressed predominantly in the nervous tissue, gonads, and early embryos of the bivalve mollusc Mytilus ga/loprovincialis, 29 Torus semicircularis, 173 TRAN, P. T., V. DOYE, F. CHANG, AND S. INOUE, Microtubule-dependent nuclear positioning and nuclear-dependent septum positioning in the fission yeast, S. pombe, 205 Transitivity index, 85 TRENCH, R. K., see R. C. LaJeunesse, 126 Tridacnidae, 108 : 326 INDEX TO VOLUME 199 u UDOETOK. A. S., see T. H. Diet/., 14 infrastructure. 76 Unionid, 14 Unit of selection, 23 1 UV-B radiation. 187 UV-B-induced damage to the skin and ocular system of amphibians. 187 VALIELA, IVAN, see Elizabeth J. Westgate. 221; Michelle Denault, 223 VALLINO, JOSEPH, see Ryan Kirkby. 218; Anne Perring. 219 Veliger. 241, 305 Vesicle transport, 202, 203 Vestigial penis, 316 VICKERY, MINAKO S., AND JAMES B. McCuNTOCK, Effects of food con- centration and availability on the incidence of cloning in planktotro- phic larvae of the sea star Pisasrer ochraceus, 298 Viral envelope, 209 Vision. 6, 171, 176, 178 Vocal behavior, 175 VOLLMER, STEVEN V., AND PETER J. EDMUNDS, Allometric scaling in small colonies of the scleractinian coral Siderastrea siderea (Ellis and Solander), 21 Voltage gating properties of channels formed by a skate retinal connexin, 165 VORONEZHSKAYA, ELENA E., see Amanda J. G. Dickinson, 305 Revised description of the fine structure of in situ "Zooxanthellae" genus Symbiodinium, 76 WANG, JIN W., Odor-induced oscillatory activity in Drosophila CNS, 170 WARD. J. EVAN, see Shirley M. Baker, 1 16 Wastewater, 221 Watershed modeling. 218 WATSON, G. J., F. M. LANOFORD. S. M. GUADRON, AND M. G. BENTLEY, Factors influencing spawning and pairing in the scale worm Har- mothoe imbricata, 50 WATSON, WINSOR H., Ill, see Steven H. Jury. 257 WEBER. ROY E.. see Stephane Hourdez, 95 WEIDNER. EARL, Cytoplasmic proteins on the surface of discharged mi- crospondian sporoplasms. 208 WESTGATE, ELIZABETH J.. DEVIN D. KROEGER, WENDY J. PALICH, AND IVAN VALIELA, Fate of anthropogenic nitrogen in a nearshore Cape Cod aquifer, 221 WHITE, THOMAS H., HARRIS RIPPS, MIDUTURLI SRINIVAS. AND ROBERTO BRUZZONE, Voltage gating properties of channels formed by a skate retinal connexin, 165 WILLIAMS, MICHAEL, see Anne Perring, 219 YAMAGUCHI. AYAKO, LEONARD K. KACZMAREK. AND DARCY B. K.ELLEY, Intrinsic membrane properties of laryngeal motoneurons that control sexually differentiated vocal behavior in African clawed frogs. Xeno- pus leavis. 175 W WACHOWIAK, MATT. MICHAL ZOCHOWSKI, LAWRENCE B. COHEN, AND CHUN X. FALK, The spatial representation of odors by olfactory receptor neuron input to the olfactory bulb is concentration invariant. 162 WAKEFIELD, TIMOTHY S., MARK A. FARMER, AND STEPHEN C. KEMPF, Zebra mussel, 116. 144 ZOCHOWSKI. 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