THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Associate Editors SEP 9 1998 ;, MA JAMES A. BLAKE, ENSR Marine & Coastal Center, Woods Hole Louis E. BURNETT, Grice Marine Biological Laboratory, College of Charleston WILLIAM D. COHEN, Hunter College, City University of New York CHARLES D. DERBY, Georgia State University SHINYA INOUE, Marine Biological Laboratory RUDOLF A. RAFF, Indiana University Editorial Board PETER B. ARMSTRONG, University of California, Davis ANDREW R. CAMERON, California Institute of Technology THOMAS H. DIETZ, Louisiana State University RICHARD B. EMLET, Oregon Institute of Marine Biology, University of Oregon DAVID EPEL, Hopkins Marine Station, Stanford Univer- sity DAPHNE GAIL FAUTIN, University of Kansas WILLIAM F. GILLY, Hopkins Marine Station, Stanford University ROGER T. HANLON, Marine Biological Laboratory GREGORY HINKLE, University of Massachusetts, Dart- mouth MAKOTO KOBAYASHI, Hiroshima University of Eco- nomics MICHAEL LABARBERA, University of Chicago DONAL T. MANAHAN, University of Southern California MARGARET MCFALL-NGAI, Kewalo Marine Laboratory, University of Hawaii MARK W. MILLER, Institute of Neurobiology, University of Puerto Rico TATSUO MOTOKAWA, Tokyo Institute of Technology YOSHITAKA NAGAHAMA, National Institute for Basic Biology, Japan SHERRY D. PAINTER, Marine Biomedical Institute, Uni- versity of Texas Medical Branch K. RANGA RAO, University of West Florida BARUCH RINKEVICH, Israel Oceanographic & Limno- logical Research Ltd. RICHARD STRATHMANN, Friday Harbor Laboratories, University of Washington STEVEN VOGEL, Duke University J. HERBERT WAITE, University of Delaware SARAH ANN WOODIN, University of South Carolina RICHARD K. ZIMMER-FAUST, University of California, Los Angeles Editor: MICHAEL J. GREENBERG, The Whitney Laboratory, University of Florida Managing Editor: PAMELA L. CLAPP, Marine Biological Laboratory AUGUST, 1998 Printed and Issued by LANCASTER PRESS, Inc. 3575 HEMPLAND ROAD LANCASTER, PA Cover Expression of endostyle-specific genes in a 1- month-old adult of an ascidian ( dona intesti- nalis ), revealed by in situ hybridization in a whole mount. Ascidians, or tunicates, are ubiquitous ma- rine animals; they are also primitive chordates and therefore share the characteristic features of the phylum Chordata with the cephalochordates and vertebrates. But the endostyle, a pharyngeal secre- tory structure associated with food collection, is also found in ascidians, amphioxus, and larval lampreys; moreover, the endostyle is transformed into the thyroid gland in the course of vertebrate evolution. Therefore, the endostyle-specific genes are key markers that should help to elucidate, not only the development of the organ itself, but also the evolution of the chordates. Michio Ogasawara and Noriyuki Satoh of Kyoto University are pursu- ing these matters; details of their work are reported in this issue. CONTENTS IMAGING AND MICROSCOPY PHYSIOLOGY Piston, David W. Concepts in Imaging and Microscopy. Choosing objective lenses: the importance of numerical aperture and magnification in digital optical microscopy RESEARCH NOTES Harosi, Ferenc I., lone Hunt von Herbing, and Jeffrey R. Van Keuren Sickling of anoxic red blood cells in fish Stuart, J. A., E. L. Ooi, J. McLeod, A. E. Bourns, and J. S. Ballantyne D- and t-/3-hydroxybutyrate dehydrogenases and the evolution of ketone body metabolism in gastropod molluscs Rodhouse, Paul G. Physiological progenesis in cephalopod molluscs . . 12 17 Leys, Sally P., and Henry M. Reiswig Transport pathways in the neotropical sponge Aplysma 30 Nair, P. Satish, and William E. Robinson Calcium speciation and exchange between blood and extrapallial fluid of the quahog Mercenaria mtrcenaria (L.) 43 Nakatani, Is. mm. Yoshinori Okada, and Takuji Kitahara Induction of extra claws on the chelipeds of a cray- fish, Procambarus clarkii 52 ECOLOGY AND EVOLUTION Ogasawara, Michio, and Noriyuki Satoh Isolation and characterization of endostyle-specific genes in the ascidian dona intestmalis 60 Maruyama, Tadashi, Masaharu Ishikura, Satoru Yama- zaki, and Satoru Kanai Molecular phylogeny of zooxanthellate bivalves ... 70 DEVELOPMENT AND REPRODUCTION Holm, Eric R., Brian T. Nedved, Eugenio Carpizo- Ituarte, and Michael G. Hadfield Metamorphic-signal transduction in Hydroides elegans (Polychaeta: Serpulidae) is not mediated by a G protein NEUROBIOLOGY AND BEHAVIOR Cobb, Christopher S., and Roddy Williamson Electrophysiology and innervation of the photosensi- tive epistellar body in the lesser octopus Eledone cirrhosa . 78 21 Annual Report of the Marine Biological Laboratory . . 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AH abbreviated components must be followed by a pe- riod, whole word components must not (i.e.. J. Cancer Res.) D. Space between all components (e.g., J. Cell. Comp. Physiol.. not J.Cell.Comp.Physiol.) E. Unusual words in journal titles should be spelled out in full, rather than employing new abbreviations invented by the author. For example, use Rit Vfsindafjelags 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 as the word order of the complete title (i.e., Proc. and Trans, placed where they appear, not transposed as in some BIOLOGICAL ABSTRACTS listings). H. A few well-known international journals in their pre- ferred forms rather than WORLD LIST or USASI usage (e.g.. Nature, Science. Evolution NOT Nature, Land., Science, N.Y.; Evolution, Lancaster, Pa.) 6. Reprints, page proofs, and charges. Authors of arti- cles in black and white (no color figures) receive their first 50 reprints (without covers) free of charge. Color reprints and additional black-and-white reprints may be purchased; authors will receive order forms. Reprints normally will be delivered about 2 to 3 months after the issue date. Authors (or delegates for foreign authors) will receive page proofs of articles shortly before publication. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). Other than these charges for au- thors' alterations. The Biological Bulletin does not have page charges. Reference: Biol. Bull. 195: 1-4. (August, 1998) Concepts in Imaging and Microscopy Choosing Objective Lenses: The Importance of Numerical Aperture and Magnification in Digital Optical Microscopy DAVID W. PISTON Department of Molecular Physiology and Biophysics, Vanderbilt University, Nit.slmlle, Tennessee 37232-0615 Abstract. Microscopic images are characterized by a number of microscope-specific parameters numerical ap- erture (NA), magnification (M), and resolution (R) and by parameters that also depend on the specimen for ex- ample, contrast, signal-to-noise ratio, dynamic range, and integration time. In this article, issues associated with the microscope-specific parameters NA, M, and R are discussed with respect to both widefield and laser scanning confocal microscopies. Although most of the discussion points apply to optical microscopy in general, the main application con- sidered is fluorescence microscopy. Introduction The objective lens is arguably the most important com- ponent of any light microscope (Keller, 1995). Advances in digital imaging have completely changed the way that optical microscopy is performed, and have also changed the relevant specifications for objective lenses. Although lens design, construction, and quality have improved to keep up with the requirements of modern light micros- copy, the markings on the lenses remain as they have been for decades. On the objective lens shown in Figure Received 13 February 1998; accepted 28 May 1998. E-mail: dave.piston@mcmail.vanderbilt.edu This is the second in a series of articles entitled "Concepts in Imaging and Microscopy." This series is supported by the Opto-Precision Instru- ments Association (OPIA) and was introduced with an editorial in the April 1998 issue of this journal (Biol. Bull. 194: 99). The first article in the series was written by Dr. Kenneth R. Castleman and appeared in the same issue (Biol. Bull. 194: 100-107). 1. the word "FLUAR" describes the type of lens design; although all manufacturers use similar types of designs, the nomenclature varies from company to company. The next most notable feature on the objective lens is the magnification (M), which in the illustration is 100X. It is written in the largest font of all the specifications, yet as is discussed here, it is not the most important parame- ter. This distinction belongs to the numerical aperture (NA), which is written next to the magnification, but in a smaller font, and in this case is 1.30. The immersion medium for this objective is also given. Below the magni- fication and numerical aperture, the tube length (^) and the coverslip thickness (0.17 mm) are given. Currently all manufacturers are offering infinity-corrected optics (de- noted by the ^ symbol), and most lenses are optimally corrected for a number 1.5 coverslip, nominally 170-pm thick. Both of these parameters are important, but the objective lens will still function adequately for many ap- plications with other tube lengths and coverslip thick- nesses. However, because the manufacturers perform chromatic corrections in different ways, multi-color ex- periments for instance, co-localization of two different colored immunofluorescent probes should be per- formed using only sets of optics that were designed to work together. This applies not only to mixing lenses of different manufacturers, but also to mixing older and newer lines of optics. The working distance of the objec- tive (the depth into the sample to which the lens can focus before it runs into the sample) is also a very important parameter, especially for confocal microscopy in thick biological samples. Despite the critical nature of this spec- D. W. PISTON Figure 1. An objective lens with typical iiKiikmgs. Although this is a Zeiss lens, most manufacturers use similar markings. itication, the working distance is not marked on most lenses. Nikon has now started writing the working dis- tance on their CFI60 optics, and it is hoped that this trend will be followed by the other manufacturers. This short article describes the relative importance of magnification and numerical aperture for digital optical mi- croscopy. Traditionally, observations made with optical mi- croscopes were detected by eye. and in this case, the size of the detector pixels given by physiological factors in the human eye is not optimal, so the magnification was increased so the sample could be "seen" better. In digital imaging, however, the magnification can be determined by the combination of resolution and detector pixel size. To understand the relative importance of NA and magnifica- tion, we must consider the basics of image formation and the effect of lens parameters on the resolution and informa- tion content in optical microscopy. Because the resolving power of an optical microscope is dependent only on the numerical aperture, magnification should be thought of as a secondary parameter whose optimal value can be deter- mined by the NA, detector pixel size, and other instrument- independent imaging parameters. Thus, NA is a more im- portant parameter than magnification in digital imaging. The practical implications of this conclusion are described tor two commonly used modes of fluorescence imasiii": widefield epi-fluorescence microscopy with a CCD camera as the detector, and laser scanning confocal microscopy with photomultiplier tube detectors. Basics of Image Formation As might be guessed by looking at the markings on any objective lens, the magnification and numerical aper- ture are important for the image-formation properties of an optical microscope. Magnification for an optical instru- ment is defined as the relative enlargement of the image over the object. Although at first glance it would seem best to use the highest magnification possible, the maxi- mal useful magnification is limited by the resolution of the imaging instrument (as described in the next para- graph). The definition of numerical aperture is more com- plicated. NA is defined by the half-angle of the objective's collection cone (a) and the index of refraction of the immersion medium (n), and is expressed by NA = n-sin(o) (Inoue and Spring. 1997, p. 32). The larger the cone of collected light, the higher the NA, and the more light that will be collected. Thus in practice. NA can be thought of as the amount of light that is collected by the objective lens; a high-NA lens collects more light than a lovv-NA lens. An analogy is with telescopes: a larger telescope collects more light just as a lens with a larger NA collects more light. Most optical microscopes also otter the option of secondary magnification between the objective lens and the detector. Use of such extra magni- fication may sometimes be required (see Table I). "but should be avoided if possible since extra light loss is introduced. As suggested above, resolution (as determined by the basic diffraction principles of light) limits the useful mag- nification in an optical microscope. Resolution (R) is de- fined as the smallest distance that two objects can be apart and still be discerned as two separate objects. There are many mathematical definitions for resolution, but a simple and reasonable approximation is R = X/(2-NA). where \ is the wavelength of the light (Inoue and Spring, 1997, p. 3 1 ). This relationship indicates that when using a high- NA lens and 500-nm (blue-green) light, the smallest re- solvable distance is -200 nm, or 0.2 pm. which agrees well with experimental values. One frequent point of con- tusion for microscope users is the difference between spatial resolution (the ability to distinguish multiple ob- jects) and spatial precision (the ability to localize a single object). Many image-processing enhancements can be used to increase the precision of localization. For exam- ple, the path of a single microtubule can be determined to --lOnm precision by pixel-fitting (e.g.. Ghosh and Webb, 1994) or deconvolution methods (e.g., Agard et ul.. 1989; Carrington et a/.. 1990; Holmes et ui. 1995). However, this is not 10-nm resolution: 10-nm resolution means that two microtubules that are 10-nm apart can be recogni/ed as two separate tubules. If multiple objects are small and close together (that is, close enough that they cannot he resolved), then no amount of image pro- cessing can differentiate between several individual ob- jects and a single object. Since there is a minimum resolvable distance for every microscope, continuing to increase the magnification past a certain point will no longer increase the information CHOOSING OBJECTIVE LENSES content of the image. Further magnification beyond this point is sometimes referred to as "empty" or meaningless magnification. This is analogous to any digital image on a computer, where pixelation is observed when an image is magnified on the screen (this can be seen, for example, by repeatedly using the "zoom in" command in Adobe Photoshop). So the question obviously arises, how should the correct magnification be chosen? A good rule is to use the Nyquist criterion, which basically says that one should collect two points per resolution size (I none and Spring, 1997, p. 513). Collecting images in this manner maximizes the information content. The use of \A2-NA) for the resolution criterion, and of R/2 for the sampling rate are both arbitrary. Many microscopists select other resolution criteria, but all of these choices are only mathematical approximations of the same physical properties. Use of any other resolution criteria would not affect the arguments presented here, although the numbers (e.g.. those shown in Table I ) would change slightly. Some attention should also be given to special consid- erations for fluorescence microscopy (Rost, 1992). Since fluorescence is subject to fluorophore saturation and pho- tobleaching effects that do not affect other optical meth- ods, the highest possible light collection efficiency is de- sirable. This consideration dictates that the highest possi- ble NA should be used. However, the highest NA lenses (NA = 1.40) are usually of a "plan-Apochromat" design; this type of lens consists of up to 14 elements and has a lower transmittance than a "Fluor" design. Also, if aque- ous samples are used, the actual NA is limited to ~ 1 .2 because of total internal reflection for higher collection angles at the interface between water and coverslip (Inoue and Spring, 1997, pp. 53-55). For these reasons, fluores- cence from an aqueous sample appears brighter through a 100X/1.30 NA FLUAR (as shown in Fig. 1) than through a 100X/1.40 plan-Apochromat objective lens. Finally, it should be noted that improvements in almost every aspect of lens design and construction (e.g.. com- puter design of complex lens combination, automated grinding of arbitrary lens shapes, new optical materials for both lenses and coatings, and computer-controlled thin film deposition for precise optical coatings) make modern objective lenses superior to and more reliable than older lenses. Although many older lenses are superb, variables during their construction made finding a good one some- what challenging, and many researchers just took what came. Today's lenses are consistently of high quality, and also offer higher transmission efficiency and lower autofluorescence than did older lenses. Numerical aperture, magnification, and resolution in widefield microscopy In a digital widefield (conventional fluorescence) mi- croscope, the image is projected onto an imaging detector (usually a CCD camera) that takes the place of the eye. Thus, to optimize the information content of the resulting digital image, the pixels on the detector must be matched to the desired image resolution. As described above, the radius of a diffraction-limited spot, R dllT ~ X/(2 NA). is a good quantity to use for the definition of resolution. In the image plane, this spot will still give the smallest resolvable object, but the width of the spot will now be M R. Based on the Nyquist criterion, the desired sam- pling rate should be twice the resolution, so we want a pixel size in the object of R vm , n ~ R d ,n/2. In practice, the pixel size in widefield microscopy is fixed by the imaging camera used, so the magnification is the only variable that can be adjusted. For the purposes of these calculations, we can assume \ = 0.5 pm (a good approximation for fluorescein (FITC) imaging). We can determine the opti- mal M to be used for a given pixel size by matching the sampling resolution in the image plane to the pixel size (P) by P = M-R s ., mp . Table I shows the results of this calculation for two typical pixel sizes: 24 //m (an older SITe 512D CCD chip) and 6.8 /jm (the more modern Kodak KAF1400 CCD chip). As can be seen from the table, the older chips (with their larger pixel sizes) require higher magnification. For these larger pixels, an extra intermediate magnification of 2.5 would be required to maximize the information content of an image collected with a lOOx/1.3 NA objective lens, and in fact cameras that use the older SITe chip usually have some extra magnification built into them. As micro-fabrication tech- nology continues to advance, however, the need for high magnification lenses will decrease. Obviously, it is not possible to purchase a 72X/1.30 NA lens (although given the popularity of the KAF1400 CCD chip, perhaps it should be), so these optimal magnifications can only serve as a guide for selection of the best objective lens. Further calculations, such as those presented in the table, reveal that a camera with a pixel size of 5.4 //m would be ideal for use with many existing lenses, such as 60X/1.4 NA. 40X/0.90 NA. and 25X/0.60 NA. It should be noted, however, that as pixel sizes get smaller, the dynamic range of the detector may be reduced. For instance, the 5.4-/jm pixels would likely be filled by fewer than 20.000 counts, which would limit the detector to 14-bit dynamic range. This is in contrast to larger pixel sizes (i.e., the 24/L/m in the SITe 512D CCD chip), which can easily deliver > 16-bit dynamic range. For applications that require high precision, such as deconvolution methods. smaller pixels may be unworkable. Numerical aperture, magnification, anil resolution in laser scanning microscopy Much has been made of the improvement in resolution provided by confocal microscopy. But this improvement is at best minimal, and is only attained for extivmcK 4 D. W. PISTON Table I Optimal magnification for detectors with different pixel sizes calculated for five numerical apertures Numerical aperture (NAl .40 * Represents the SITe512D CCD chip. t Represents the Kodak KAF1400 CCD chip. 0.90 0.60 Calculated resolutions {//m) R-diff 0.18 0.19 0.28 0.42 0.83 samp 0.090 0.095 0.140 0.210 0.415 Magnification (x) 24-^m pixels* 267 253 171 114 58 6.8-/jm pixelst 76 72 49 32 16 small pinholes. In practical fluorescence microscopy, the pinhole must be opened somewhat to increase the effi- ciency of fluorescence collection. In fact, the pinhole is almost always opened enough to negate any resolution enhancement (Sandison et ui, 1995). In this practical case, there is an improvement in rejection of out-of-focus background, but the resolution is still given by R d , IT ~ \J (2 NA), so the ideal sampling resolution remains as shown in Table I. A key point in laser scanning microscopy is that there is no longer a fixed pixel size. Because the field over which the laser is scanned can be varied (this variation is usually called "zoom," or more appropriately "elec- tronic zoom," and is analogous to using an optical zoom lens), the sampling resolution can be easily changed. For this reason, users often have a lot of trouble choosing lenses when they switch to laser scanning confocal mi- croscopy. For instance, a 40X lens with a zoom factor of 2.5 is basically equivalent to a lOOx lens with no extra zoom. Thus, a 40X/1.3 NA lens should be chosen over an equivalent lOOx/1 .3 NA lens, because it offers a poten- tially larger field of view with no fall-off in light collec- tion or resolution. In laser scanning confocal microscopy, two other pa- rameters must be considered. First is the size of the detec- tor pinhole, which depends on the magnification. Most confocal microscopes have an adjustable pinhole that is easily set to match the magnification (e.g., for equiva- lence, a 60X lens needs a pinhole 1.5-fold larger than a 40x lens). Secondly, the lens design for confocal micros- copy may, in some cases, be more important than either M or NA. This is especially true for co-localization exper- iments, in which the chromatic corrections of a plan- Apochromat make it preferable despite its lower light- collection efficiency (the same trade-off must be consid- ered for any three-dimensional microscopies based on widefield and deconvolution methods, as well). Regard- less of the lens design, however, a lower magnification lens (of equivalent NA) is almost always preferable, be- cause it offers a larger field-of-view, and delivers equiva- lent resolution. Acknowledgments This work was supported by an Arnold and Mabel Beckman Foundation Young Investigator Award, NIH grant DK53434. and the Vanderbilt Cell Imaging Re- source (underwritten by CA68485 and DK20593). Literature Cited Agard, D. A., Y. Hiraoka. P. Shaw, and J. W. Sedat. 1989. Fluores- cence microscopy in three dimensions. Methods Cell Bio/. 30: 353- 377. Carrington, W. A., K. E. Fogarty. and F. S. Fay. 1990. 3D fluores- cence imaging of single cells using image restoration. Pp. 53-72 in Non-invasive Techniques in Cell Biology, i. K. Foskett and S. Grinstein, eds. Wiley-Liss, New York. Ghosh, R. N., and W. W. Webb. 1994. Automated detection and tracking of individual and clustered cell surface low density lipopro- tein receptor molecules. Biophys. J. 66: 1301-1318. Holmes, T. J., S. Bhatlacharyya, J. A. Cooper, D. Hanzel, V. Krish- 11:1111111 1 hi. W. Lin, B. Roysam, D. H. Szarowski, and J. N. Turner. 1995. Light microscopic images reconstructed by maxi- mum likelihood deconvolution. Pp. 389-402 in The Handbook of Biological Confocal Microscopy. 2nd Edition. J. Pawley, ed. Plenum, New York. Inoue, S., and K. R. Spring. 1997. Video Microscopy: the Fundamen- tals, 2nd Edition. Plenum, New York. Keller, H. E. 1995. Objective lenses for confocal microscopy. Pp. 1 1 1 - 1 26 in The Handbook of Biological Confocal Microscopy. 2nd Edition. J. Pawley. ed. Plenum. New York. Rost, F. W. D. 1992. Fluorescence Microscopy. Cambridge Univer- sity Press, Cambridge, UK. Sandison, D. R., D. W. Piston, R. M. Williams, and W. W. Webb. 1995. Resolution, background rejection, and signal-to-noise in widefield and confocal microscopy. App. Optics 34: 3576-3588. Reference: Biol. Bull. 195: 5-11. (August, 1998) Sickling of Anoxic Red Blood Cells in Fish FERENC I. HAROSI 1 *, IONE HUNT VON HERBING 2 , AND JEFFREY R. VAN KEUREN 3 ' Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, and Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118; 'School of Marine Sciences, University of Maine, Orono, Maine 04469; and 3 Biology Department, Woods Hole Oceanographic Institution. Woods Hole, Massachusetts 02543 The occurrence of the mutant hemoglobin Hb S in hu- man red blood cells results in sickle cell anemia. This disease, including its genetic and molecular bases, has been extensively investigated and is well understood (1.2). The presence of deoxy-induced sickling of animal erythro- cytes is largely unknown, however. We examined red blood cells (RBCs)from several fish species in vitro under aerated and anoxic conditions. Our polarized light micro- scopic techniques were aimed at establishing correlations bet\\'een erythrocyte morphology, state of oxygenation. spectral absorbance, linear dichroism, and linear bire- fringence. We found no fish with intracellular HbO 2 poly- merization; but there were intraerythrocytic aggregations of deoxy Hb with a high degree of either molecular order or disorder. The ordered aggregates in the RBCs of Atlan- tic cod, haddock, and toadfish were remarkably similar in dichroic ratio magnitudes and birefringence to those in human RBCs that contain HbS. Therefore, fish hemo- globins appear to be good models of sickling disorders and polymerization-related phenomena. The conse- quences of sickling on animal health and fish aquaculture remain to be studied. Aggregates of hemoglobin in fish erythrocytes have been reported since 1865 (cited in ref. 3). Nevertheless, former studies have failed to establish causal relationships between Hb aggregation, the state of oxygenation, and the attendant optical properties of erythrocytes (4-6). As a chance micro- spectrophotometric observation, we discovered linearly di- chroic absorption of light by Hb in larval fish erythrocytes. This linear dichroism i.e., the dependence of the absorp- Received 25 February 1998; accepted 5 June 1998. * To whom correspondence should be addressed. E-mail: fharosi@ mbl.edu tion coefficient on polarization direction was associated with abnormal RBC shape, or "sickling," the common term for this condition. The cells with the distorted morphol- ogy contained intracellular "bodies" with high concentra- tions of deoxyhemoglobin; for the sake of simplicity we call these bodies "erythrosomes." Erythrosomes are therefore intraerythrocytic. aniso- tropic aggregates of polymerized deoxy Hb that become visible in the light microscope because the refractive in- dex of Hb is higher than that of the surrounding medium. Erythrosomes exhibit linear dichroism as well as linear birefringence and occur in varied forms. In the RBCs of larval haddock and cod, for example, they may appear as V. U. or "banana" -shaped structures; but at other stages, or in different species, they may be shaped like needles or rods. The sickled fish erythrocytes encountered to date have always been associated with deoxy Hb, and not the oxygenated form. Indeed, the sickling that appeared was readily reversible by aeration; that is, the erythrosomes disappear and the affected cells resume their normal shape upon oxygenation. Moreover, repeated cycles of anoxia and normoxia cause concomitant cycles of intracellular hemoglobin aggregation and dispersion. In these charac- teristics, sickling in fish mimics sickling in man. Our initial observations of sickling were made on 8- and 60-day-old larval haddock (Melanogrammus aeglefinus, family Gadidae) as well as on 118- and 125-day-old Atlantic cod (Gadus morhua, family Ga- didae), all of which derived from stocks originating in the Bay of Fundy, Canada. Because we did not know whether sickling in fish is restricted to the developmental stages of particular species or is a more widespread phe- nomenon, we extended our investigation to wild-caught and cultured specimens of several species from four other geographical locations. F. I. HAROSI ET AL Typical spectral behavior of larval Atlantic cod RBC is illustrated in Figure 1. Erythrocytes with undistorted shape yielded the expected absorption spectrum for HbO : as shown in panel a: the Soret (or y ) band peaks at 414 nm and is not dichroic (dichroic ratio of 1 ). This is consistent with the presence of a random ensemble of HbO : mole- cules (a- and /3-band peaks are at 574 and 540 nm, respec- tively). In marked contrast, the Soret-band in panel b peaks at 430 nm and shows significant linear dichroism with a peak ratio of about 2. The latter is indicative of an orderly arrangement of the chromophores in situ (no a-band and shifted /? peak to 554 nm); thus the cod Hb behaves like the human Hb S. RBCs of juvenile Atlantic cod (age, 227 and 272 days) obtained from the Narra- gansett Laboratory of the National Marine Fisheries Ser- vice (Rhode Island), also sickled extensively (92%-95% of all cells) under anoxic conditions. Therefore, RBC sickling in cod and haddock is limited neither to an iso- lated stock nor to a specific age group, but is present in both larval and juvenile stages. Of the 12 fish species tested, however, only 4 species exhibited sickling under our experimental conditions. These were the two gadids (Atlantic cod and haddock), the toadfish (Opsunus l O ro .a 01 -Q 00 Normal, oxyHb 300 350 400 450 500 550 600 650 Wavelength [nm] Sickled, deoxyHb 300 350 400 450 500 550 600 650 Wavelength [nm] Figure 1. Larval Atlantic cod erythrocyte spectra under oxygenated (a) and deoxygenated (b) conditions: absorbance. A; linear dichroism. LD: and dichroic ratio, R. Absorbance was defined as A = log I/,//,), where /, and /, are transmitted fluxes through sample and reference volumes of the preparation. Linear dichroism was proportional to trans- mission modulation by sample anisotropy. which was calibrated with an external polarizer to yield 1.0 full-scale value. Polarized absorbance components were determined (26, 27) parallel with erythrosome length for Ay. and perpendicular to it for Aj_. The dichroic ratio was computed as R = A i /A,. HEMOGLOBIN POLYMERIZATION IN FISH Figure 2. Photomicrographs of adult toadtish erythrocytes (nucle- ated, as all non-mammalian erythrocytes [9]) obtained with a Zeiss Axiophot 2 microscope in differential interference contrast mode (objec- tive: Plan Apochromat, 63/1.4, oil immersion), (a) Normal cells con- taining HbO 2 with spectra like those of Fig. la. (b-d). Sickled cells under anoxic condition, each with spectra similar to those depicted in Fig. Ib. Scale burs, 10/jni. the erythrocytes, usually forming elliptical and closed structures. The transverse-to-tangential dichroic ratio around the cells were nearly uniform, with R ~ 1 .5. These results imply a flexible fibrous aggregation of Hb in which a degree of parallelism prevails. Unlike the toadfish case, tautog Hb polymerization seems to be too feeble to distort cells into "sickles"; rather it occurs along existing cellu- lar features, such as hooplike microtubules (9). Hemoglobin gelation in the summer flounder (Para- lichthys dentatus, family Bothidae) is markedly different from that in either the toadfish or the tautog. Anoxic erythrocytes in the flounder may contain intraerythrocytic clumps without sickling. Aggregates of deoxy Hb in flounder remained isotropic and did not gel into regular structures. They exhibited no linear dichroism (R 1). To distinguish isotropic from anisotropic aggregation, we regarded a cell to be sickled only when the Soret-band's linear dichroism was at least 10% above isotropic (i.e.. R a 1.1; see Table I). Because a linearly dichroic sample should also have anisotropic polarizability, sickle cells would be expected to exhibit linear birefringence as well. Indeed, this has been observed (10) and measured (3) for human sickle cells. We found fish erythrosomes to be also birefringent. Figure 3a illustrates how toadfish erythrosomes may "light up" between crossed polarizers in various color- ations depending on their orientation with respect to the polarizer's passing direction; they are the darkest at and 90. and brightest near 45 and 1 35. Retardance and absorbance spectra obtained from a typical toadfish erythrosome are depicted in Figure 3b. The retardance spectrum shows two peaks, one at 445- 450 nm and the other at 585-590 nm; these peaks corre- spond to the longwave half-maxima of the y and /3-bands (with peaks at 430 and 556 nm, respectively). Although the latter behavior is consistent with anomalous disper- sion (\ 1,12), we currently lack a quantitative interpreta- tion. The theory of anomalous dispersion describes varia- tions in the relationship between refractive index and wavelength through regions of strong absorption: the in- dex first declines with increasing wavelength, and then sharply rises before declining again to a higher plateau on the longwave side of an absorption band. The presence of the erythrosome's linear dichroism complicates mat- ters. Here, the stronger absorption coincides with the slow direction of retardance. and the weaker absorption with the fast direction. Therefore, the two principal refractive indices undergo unequal dispersion and thus yield an "anomalous retardance." Whereas we expected erythro- somes to have t\vo intrinsic birefringent components, one due to the heme groups and the other to the globin chains, their further delineation does not appear possible at pres- ent. Based on multiple determinations of retardance shown in Figure 3b (and assuming that the width of the erythrosome equals its thickness), the average specific retardance (n = 16) was in the range of 8.4 1.4 nm/pm (450 nm) to 2.7 0.6 nm//im (540 nm). Thus, the average difference in the principal refractive indices, n^.ii,, \ . was variable in the visible spectrum between 2.7-8.4 X 10 \ This range of values is to be compared with that of crystal- line quartz, where the difference between the principal refractive indices is 9 X 10"'. The explanation for eryth- rosome hues also follows from Figure 3b. Since light transmission of an erythrosome between crossed polariz- ers is proportional to retardance at each wavelength, and because peak retardance occurs around 450 nm, the trans- mitted flux should cause blue visual sensation at white illumination. If, on the other hand, retardance is also ele- vated for longer wavelengths (expected for thicker eryth- rosomes), increased transmission would result throughout the spectrum, yielding desaturated shades of blue. The principal axes of birefringence were directly ob- servable in polarized light by sample rotation. For toadfish erythrosomes. these correspond to their short and long dimensions, as can be seen in Figure 3a. However, a compensator is also needed if the slow direction is to be distinguished from the fast. To accomplish this, we used the Pol-Scope ( 13, 14), which is equipped with an auto- matic compensator. Figure 3c depicts brightness-encoded retardance images of several anoxic toadfish erythrocytes. F. I. HAROSI ET AL Table I Fish erythrocyte properties: absorbance maxima and sickling OxyHb \ mav [nm] DeoxyHb \ mjx [nm] Species Stage Sickling Sickling Atlantic cod (Gadus morhua) Larval 414 540 574 430 554 + (-100) Juven. 414 540 574 430 554 + (-95) Haddock (Melanogrammus aeglefinus) Larval T t t t 430 556 + (-100) Juven. 414 540 575 430 556 + (95) Adult 414 542 575 430 556 + (91) Toadfish (Opsanus tun) Adult 414 540 576 430 556 + (99) Tautog (Taittoga onitis) Larval 414 540 575 430 556 + (-90) Juven. 414 542 576 430 556 + (-1) Adult 414 542 576 430 556 Summer flounder ( Paralichthys di'inanix ) Juven. 414 542 576 430 560 Killitish (Fundulus heleroclinis) Juven. 414 544 575 430 556 Sheepshead minnow ( Cvprinodon variegatus ) Juven. 415 540 578 428 560 Blueback herring (Alosa aestevalis) Juven. 414 539 575 430 555 Atlantic silverside (Menidia menidia) Juven. 415 540 576 430 560 Smooth dogfish (Musteltis canis) Adult 414 544 575 430 556 Little skate (Raja erinacea) Adult 413 538 576 429 555 Common white sucker ( Catostomus commersoni) Adult 414 540 576 430 555 * In this column. + indicates the occurrence of optically anisotropic intraerythrocytic aggregates; percentage was obtained by visual inspection of cell morphology; other fractions were based on measurable Soret-band linear dichroism (R =: 1.1). The numeric values we regard only as approximate indicators of sickling, because polymerization is reversibly dependent on subtle variations in environmental conditions. Factors promoting sickling were anoxic conditions, low pH (6.9) and elevated temperature (29C). analogous to those reported previously for Hb S. t Unavailable data. revealing bundles of linear molecular aggregation. An- other cell from the same preparation is shown in Figure 3d, where an erythrosome retardance image with superim- posed black lines indicates local slow axis direction: this direction is approximately transverse to the long dimen- sion of the erythrosome. The line scans of erythrosome retardance are consistent with a model of fibrous molecu- lar order which, taken together with the slow direction of retardation and coincident strong absorption, is in har- mony with the presence of bundles of polymerized hemo- globin molecules with their planar porphyrin groups ori- ented nearly perpendicular to the length of the aggrega- tion. These features have also been found in normal human reduced-hemoglobin crystals and in sickle cells from anemia patients (3). Our polarized light microscopic observations are com- plementary to the structural information provided by elec- tron microscopy. Thomas (15) used the latter technique on red cells of cod (Gadus cullarias) and reported a para- crystalline organization for hemoglobin in erythrocytes. For atomic-scale comparison of fish hemoglobins with Hb S, it will be necessary to combine data from several techniques, as has been done for the various forms of human hemoglobin, including refined methods in electron microscopy (16), X-ray crystallography (17), and analy- ses of globin mutations based on amino acid sequence information ( 18). The current investigation notwithstanding, we can only speculate how widespread sickling is among the nearly 20,000 extant fish species. But given the richly varied environments that fishes inhabit, a great variability in fish hemoglobins is probable. Also probable is that the evolution of fish hemoglobins has responded to various environmental stresses in many species. Thus, we antici- pate fish hemoglobins to emerge as a rich resource for genetic, evolutionary, molecular, and pathophysiological studies. The variety of sickling hemoglobins may lead to new model systems, not only for a human disease, but also for studies of fundamental importance such as poly- merization, force generation (19), and the identification of molecular contacts that make paracrystal formation possible. The phenomenon of sickling may also have practical relevance to marine fish aquaculture. Fish in culture are often under heightened stress (e.g., insufficient oxygen- ation, high bacterial load, and low pH), and they are consequently prone to infections and high mortality rates. Because a propensity for sickling could increase mortali- HEMOGLOBIN POLYMERIZATION IN FISH s 400 500 600 Wavelength [nm] 700 CD O c CO Distance [ [im] 20 CD O CO CO o5 K T Distance [|j,m] 19 Figure 3. Toadfish erythrocytes. (a| Unstained, anoxic preparation between crossed polarizers at white light illumination (photographed in a Zeiss Axiophot 2 microscope on Kodak Ektachrome 160T film), (b) Retardance (solid) and absorbance (dashed) spectra of an isolated erythrosome (3.6 x 9 fjm). (c) Retardance images from sickled and ghost cells measured at 546 nm with the Pol-Scope ( 1 3, 1 4). The retardance for each picture element (pixel) was encoded in gray between (black) and 16 nm (white), (d) Another Pol-Scope image of retardance with two line scans (inserts): longitudinal (L) and transverse (T) to the erythrosome. Scale bars, 1 ^m. ties, the molecular characterization of sickling hemoglo- bins in fishes and their attendant physiological manifesta- tions are of paramount importance. The phenomenon is also relevant to restocking programs aimed at reestablish- ing dwindling groundfish populations. Summary Sickling in fish red blood cells is described, and the phenomenon is shown to occur in several species. By correlating the morphology of RBC with spectral absorp- tion of Hb in situ, sickling is associated with the presence of deoxyhemoglobin. The polymerization of deoxy Hb is inferred from optical anisotropies measured in single cells. The magnitude of linear dichroism and the nature of linear birefringence detected in fish "erythrosomes" show close kinship to the corresponding properties deter- mined previously from human sickle cells and hemoglo- bin crystals. The range of oxygen tension for sickling has not been determined and. therefore, we do not know whether it is within physiological limits. Also unknown is whether sickling in fish is pathological or, perhaps, reflective of a physiological condition with as yet obscure significance. In either case, however, further inw 10 F. I. HAROSI ET AL. lions promise to provide new insights into the requisite conditions that produce intracellular hemoglobin poly- merization. Methods Sample preparation. Experiments were conducted at room temperature (20-23C). Our primary buffer (ma- rine teleost Ringer's solution) contained (in mM): NaCl 140, KC1 2.5, CaCl 2 1.5, MgSO 4 1, NaH,PO 4 0.5, NaHCO, 0.5. and HEPES 10, adjusted to pH 7.3 with NaOH. This was used (air-saturated) in about 10-fold excess to dilute whole blood for oxygenated preparations. Samples for microscopy were prepared in between two No. 1 'A cover glasses sealed around the edge with a mol- ten mixture of paraffin and Vaseline. The usual procedure was to place 3 /J\ of blood on a thoroughly cleaned (20. 2 1 ) microscope cover glass, either with or without a poly- L-lysine (P-1274. Sigma) coating (22. 23). add and mix 15-30 /jl of desired buffer, cover, blot, and seal. Anoxic preparations were obtained in one of four ways: ( 1 ) By letting metabolically active blood cells deplete O : . The effectiveness of this method is variable: for extensive sickling to occur, it may take from a few hours to 2- 3 days. (2) By mounting erythrocytes and metabolically active retinal cells together in a sealed preparation, where complete deoxygenation may take only 30-120 min. This was the condition under which we first observed sickling. (3) By using N ; -purged dithionite solution (from 100 mM Na : S:O 4 stock) (24) and N : -purged buffer ( 1:1 ) in prepar- ing blood samples. (4) By the use of a calcium-free buffer (marine teleost Ringer without added calcium, containing 1 mM EGTA. at pH 6.9. and purged with N : for 1 h) and blood stored under light mineral oil (Paraffin, Fisher Scientific). The purpose of the oil is to keep the blood airtight, as previously reported (10). Additionally, we found that the oil prevents clotting for long periods (tested up to 8 weeks for toadfish blood); the latter method was found most effective in producing large, well-aligned erythrosomes. Determination of uhsorhancc and linetir ilichro- /.v;/i. We used the dichroic microspectrophotometer (25- 27) with a measuring beam cross section of about 0.6 X 2 fj,m on cells in optical isolation. This instrument is a single-beam, phase-modulated, polarized light micro- scope equipped with automatic, wavelength-scanning and recording photometry. Glycerin immersion-type micro- scope objectives were used for both condenser (32X/0.4 Ultrafluar. Zeiss) and objective ( lOOx/1.3 UV-F100, Ni- kon). Visualization was implemented with infrared light and a video camera system with tape recording. Lineur birefringence determination. The laboratory in- strument was built around a modified Axiovert 10 (Zeiss) microscope utilizing quartz components and fiberoptic links between a 150-W xenon light source (Oriel Corp.) and a spectrograph-detector combination. Its adjustable cross section microbeam (about 1.5 X 4 /vin) was passed through a rotatable polarizer (Glan-Thompson type); the condensor (32X/0.4 Ultrafluar, Zeiss); the preparation af- fixed to a rotatable, sliding stage; the objective (lOOx/ 1.2 Ultrafluar, Zeiss); and a slidable analyzer (HN38S, Meadowlark Optics). The transmitted light was collected and focused on the entrance slit of a spectrograph (MonoSpec 18, Jarrell Ash), which in turn dispersed it over an intensified diode array detector (Model 1455B, EG&G PARC). Electronic scanning of the latter provided records of spectral responses in the range of 350-650 nm. This instrument was also capable of determining ab- sorbance and linear dichroism from static transmission measurements taken at appropriate sample orientations and polarizer settings. For the determination of linear birefringence, the preparation was positioned between crossed polarizers with the sample's principal ("slow" and "fast") axes oriented at 45 to the direction of polarization. The basis for signal processing was provided by the formula /(\) = /,,(\){siir[F(\)/2] + 1/EF). where /(\) is the transmitted flux at sample retardance of F(\) radians. /,,(\) the flux through the system for polarizer and analyzer axes set parallel [with F(\) = 0]. and EF is the extinction factor defined as EF = / r (\)//,(\) with /, (\) being the transmitted flux for crossed polarizers (28). It followed from the foregoing that sin : [F(\)/2] = |/(\) - /, (\)]//,,(\). where from the retardance (in nm) at wave- length \ (nm) could be obtained as F(\) = ([/(\) - /, (\)]/ / ; ,(\)} l/: X/TT (assuming equality between the sine of small angles and their values in radians). The latter relationship was implemented on data sets from spectral scans of 7(\). /,(\) and /,,(\) by software operations using ORIGIN (Mi- crocal Software). Birefringence is linked to retardance by the optical path difference in the sample between the fast and slow waves propagating through thickness \i>lic cuiiipiiriments of gastropod hepatopancreas * Enzyme activity (/jmol/min/g wet tissue weight )$ Genus Fractiont CCO CS DL-BHBDH D-BHBDH L-BHBDH LDH Stylommatophora Archachatina hepatopancreas tissue n.ni n.ni- n.m. 0.13 0.06 0.77 0.09 n.m. heart tissue n.m n m. n.m. 35.74 8.60 n.d. n.m. kidney tissue n.ni n m. n.m. 0.54 0.10 0.58 0.08 n.m Brad\baena mito 1.25 0.10 0.83 0.1 1 0.14 0.08 0.21 0.12 0.14 0.08 0.33 0.1 cytosol 0.16 0.06 0.22 0.06 0.85 0.06 0.23 0.08 0.98 0.14 4.35 0.8 Arion mito 1.10 0.11 1.90 0.10 n.d. n.d. n.d. n.m cytosol 0.03 0.01 0.21 0.04 2.44 0.09 0.07 0.04 2.60 0.11 n.m Basommatophora Physa mito 0.87 0.08 4.71 0.50 1 .02 0.28 3.07 0.73 n.d. n.m cytosol 0.17 0.08 1.37 0.34 0.53 0.18 0.99 0.19 0.22 0.04 n.m Helisoma mito 4.85 0.65 3.58 0.29 0.52 0.15 1.94 0.44 n.d. n.ni cytosol 0.04 0.04 0.39 0.08 0.12 0.05 0.13 0.04 0.09 0.01 n.m Stagnicola hepatopancreas mito 2.81 0.21 1.40 0.20 2.97 0.14 7.00 0.67 n.d. 0.38 0.2 cytosol 0.76 0.08 0. 1 8 0.06 0.67 0.10 1.01 0.19 n.d. 1.25 0.2 heart mito 9.48 0.85 5.72 3.66 n.ni. 13.20 1.96 n.d. 6.00 3.3 cytosol 5.01 2.96 1.95 0.55 n.m. 1 1.61 1.85 n.d. 34.57 1.7 Neritopsina Helicina mito 6.54 1.24 1.49 i* O.I 1 0.45 0.09 0.53 0.32 n.d. 0.17 0.1 cytosol 0.31 0.11 0.37 0.25 0.25 0.18 0.21 0.10 n.d. 1.72 0.8 Architaenioglossa Campeloma mito 2.12 0.12 1.82 0. 1 1 0.46 0.27 0.65 0.04 n.d. n.d. cytosol 0.01 0.01 0.06 0.02 0.07 0.02 0.15 0.06 n.d. n.d. Pomacea hepatopancreas mito 1.82 0.62 0.89 0.45 1 .68 0.63 1.53 0.70 n.d. 0.44 0.2 cytosol 0.03 0.01 0.25 0.06 0.19 0.1 1 0.33 0.13 n.d. 0.48 0.01 heart mito 3.25 1.35 17.05 1.87 n.m. 0.55 0.18 n.d. 0.32 0.1 cytosol 0.81 0.37 14.18 2.73 n.m. 0.09 0.05 n.d. 7.73 2.3 kidney mito 2.97 1.83 4.09 0.56 n.m. 0.47 0.09 n.d. 0.44 0.3 cytosol 0.15 0.10 0.31 1 0.05 n.m. 0.14 0.10 n.d. 0.92 0.4 * Most gastropods were collected from fields and ponds near the University of Guelph. in Guelph, Ontario, Canada, or purchased (Pomacea, Campeloma) from local aquarium stores. The terrestrial prosobranchs. Helicina orbiculata. were collected near Jacksonville. Florida. The giant African snails, Archachatina ventricosa, were from our laboratory population established with animals provided by the Toronto Metro Zoo. All snails and slugs were kept in terraria or aquaria at room temperature (22 2C) and fed lettuce. We observed no effect of duration of time spent under these conditions on BHBDH compartmentation or enantiomeric specificity. Taxonomic classification of gastropods was as in Ponder and Lindberg (101 (Table II). A. ventricosa tissues were not fractionated. They were prepared as in Stuart and Ballantyne. (4) For enzyme measurements in all other gastropods, excised tissues were placed in approximately 20 volumes of sucrose buffer ( 100 mM sucrose. 20 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesul- fonic acid] (HEPESl. pH 7.51. Cells were disrupted by five passes of a Potter-Elvejhem homogenizer operated at low speed (<100 rpm). This homogenate was centrifuged at KXOOOx g for 10 min to separate it into mitochondnal (pellet) and cytosolic (supernatant) fractions. The mitochondrial pellet was resuspended in a volume of buffer equal to the original buffer addition, thus maintaining equal the dilution of both fractions. Both fractions were sonicated with three 5-s bursts of a Vibra-Cell sonicator (Sonics & Materials Inc., Danbury, CT) set to 80% output, 50 watts. These preparations were used directly in the measurement of enzymes. We used CCO to mark the mitochondrial membrane, CS to mark the leakage of matrix enzymes from mitochondria damaged in the fractionation process, and LDH to evaluate the contamination of the mitochondrial fraction with cytosolic enzymes. CCO, CS, and LDH were measured as in Stuart and Ballantyne. (4) The assays for D-, L- and DL-BHBDH contained 2 mM NAD and either 200 mM D- or L-BHB or 400 mM DL-BHB. respectively (BHB omitted for control) in 50 mM imidazole. pH 8.0. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). t "tissue" = whole tissue homogenate: "mito" = mitochondrial fraction; "cytosol" = cytosolic fraction. t CCO = cytochrome C oxidase; CS = citrate synthase; LDH = lactate dehydrogenase. DL-. D- and L-BHBDH = activity measured with racemic mixture of DL-/3-hydroxybutyrate, high-purity D-/3-hydroxybutyrate, and high-purity L-/3-hydroxybutyrate. respectively, n.d. = not detected; n.m. = not measured. Values are means standard error; n = 5. 14 J. A. STUART ET AL low L-BHBDH activity observed in the mitochondrial fraction thus can be attributed to cytosolic contamination. Low D-BHBDH activities in Bradvbaena, Arion, and Archachatina hepatopancreas were likely due to contami- nating i -BHB in the commercial D-BHB preparation (L-BHB contamination was up to 2%). As we used a [o-BHB] of 20 mM in the assays, as much as 0.4mA/ L-BHB could have been present. Heart and kidney were fractionated in the same way as the hepatopancreas. Fractionation of heart tissue re- sulted in a lower recovery of mitochondria and greater leakage into the cytosol of mitochondrial matrix enzymes (Table I), perhaps because of the small size of these tis- sues (Stagnicola hearts averaged 0.8 mg; Pomacea hearts averaged 10 mg). Nonetheless, the distribution of BHBDH activity approximated those of CCO and CS in these tissues, indicating that BHBDH activity is localized to the mitochondria in basommatophoran heart and archi- taenioglossan heart and kidney. In all of these tissues, BHBDH was specific for D-BHB, with no oxidation of L-BHB observed. The pattern of D- and L-BHBDH distribution in Archti- chatimi (Table I) parallels that seen in Cepaea. In C. nemoralis tissues, no mitochondrial form of BHBDH was found (5). Instead, a cytosolic L-BHBDH was present in hepatopancreas and kidney, and a cytosolic D-BHBDH was found in heart and kidney. Similarly, D-BHB was oxidized by Archaclmtina heart and kidney homogenates and L-BHB was oxidi/ed by kidney and hepatopancreas. This configuration of BHBDH organization appears to be characteristic of the Stylommatophora. We investigated the evolution of cytosolic BHBDH isoforms by mapping the occurrence of the mitochondrial D-BHBDH and cytosolic L-BHBDH of gastropod hepato- pancreas onto a gastropod phylogeny (8) using the Mac- Clade (9) software program (Fig. 1 ). Mollusc phylogenies based upon morphological (10) and molecular (8, 11-13) data support the monophyly of Gastropoda, within which pulmonates are monophyletic. Within Pulmonata, the Sty- lommatophora and Basommatophora are each also mono- phyletic. For this analysis, we have considered the presence of mitochondrial D-BHBDH to be the ancestral condition. Though BHBDH activity is undetectable in marine gas- tropods, it is found in all freshwater and terrestrial gastro- pods that we have studied. Thus, the enzyme appears to have first arisen in a gastropod ancestral to these groups. Our phylogenetic analysis suggests that the cytosolic L- BHBDH isoform evolved a single time in an ancestral stylommatophoran. Gastropods of this order are almost exclusively terrestrial and compose the vast majority of terrestrial snails and slugs (14). To test whether the pres- ence of L-BHBDH correlates with terrestriality, we in- cluded the distantly related terrestrial gilled gastropod Helicina orbiculata Pomacea bridges* Campeloma decisum Bradybaena similans Cepaea nemoralis Anon subfuscus Archachatina ventncosa Physa gynna Biomphalaria glabrata O Stagnicola elodes Heltsoma tnvolvis Figure 1. Phylogenetic relationships among freshwater and terres- trial gastropod taxa used in this study. Unfilled lines represent the pres- ence of mitochondrial D-/3-hydroxybutyrate dehydrogenase (and absence of L-/3-hydroxybutyrate dehydrogenase). Filled lines denote the presence of cytosolic L-/3-hydroxybutyrate dehydrogenase (and absence of D-/3- hydroxybutyrate dehydrogenase). Hatched line = equivocal occurrence of L-/3-hydroxybutyrate dehydrogenase, denoting that the enzyme arose at an undetermined point along this lineage. Cepucti iirmoralis data are from Stuart and Ballantyne (5). Biomphalaria xlahmhi data are from Meyer ct al. (15). Helicinci (Neritomorpha) in our analysis. The absence of i -BHBDH in Hclichui. however, suggests that the occur- rence of this isoform correlates exclusively with phyloge- netic position. A general upregulation of BHBDH activities appears to have occurred in tissues of pulmonate gastropods. In pulmonate hearts, exceptionally high activities of D- BHBDH (approximately two orders of magnitude greater than in Pomacea heart) (Table I) suggest that D-BHB is particularly important as a metabolic substrate in these tissues. High activities of all enzymes of ketone body metabolism in Sttignicolu cloiles. C. lu'iuomlis, and A. vcntricosa indicate a substantial flux through this pathway in all pulmonates. D-BHB levels in hemolymph are as high as those of glucose in the basommatophoran pulmo- nate Biomplnihirici i>lcihrcitii (15). These snails, and tissues isolated from them, actively oxidize ketone bodies. BHB K.ETONE BODY METABOLISM IN GASTROPODS 15 Table II Higher classification f 10) of gastropod species used for measurements of BHBDH activity' Gastropoda Orthogastropoda Neritimorpha Helicinoidea Helicina orhiculata I terrestrial) Apogastropoda Architaenioglossa Viviparoidea Campeloma decisum (freshwater) Pomacea bridges: (freshwater) Heterobranchia Euthyneura Pulmonata Basommatophora Physa gyriiui (freshwater) Helisoma trivolvis (freshwater) Stagnicola elodes (freshwater) Stylommatophora Arcliacluirinu ventricosa (terrestrial I Brad\haena similaris (terrestrial) Arion subfuscits (terrestrial) appears, therefore, to be an important metabolic substrate in pulmonate snails. Unlike mammals, these snails show a decrease in D-BHB levels in the hemolymph during starvation, suggesting that pulmonates may routinely use ketone bodies as energy substrates, whereas mammals confine their use to times of starvation. This difference may be related to a decreased emphasis on amino acid metabolism and a low capacity for extrahepatic oxidation of fatty acids in pulmonates. The metabolic organization of tissues in these organisms suggests that they use ketone bodies which, unlike fatty acids, are freely soluble, as a means of transporting lipid carbon from central stores to peripheral tissues for oxidation. In stylommatophoran pulmonates, upregulation of BHBDH activity has been followed by the elaboration of new isoforms of the enzyme. This may have occurred through an initial loss of the transmembrane amino acid sequence from the membrane-bound mitochondria! BHBDH. to allow the enzyme to function in the cytosol. In stylommatophoran hepatopancreas and kidney, this en- zyme may have been further modified to act upon L- BHB rather than D-BHB. However, this scenario for the occurrence of cytosolic BHBDH isoforms assumes diver- gence from the ancestral mitochondria! D-BHBDH. Alter- natively, the cytosolic enzymes could derive from other proteins, unrelated to the mitochondria] o-BHBDH, and have achieved functional similarity through evolutionary convergence. This convergence appears to have occurred in the evolution of D- and L-LDHs in bacteria (16). On the other hand, divergence of cytosolic L-BHBDH from cytosolic D-BHBDH is suggested by the difficulty of sepa- rating these isoforms when they are electrophoresed to- gether on a gel that separates proteins on the bases of size and charge (5). Analyses of primary structures are necessary to determine the relatedness of the three BHBDH isoforms. The use of both enantiomers of a single metabolic sub- strate in routine energy metabolism is unusual in the ani- mal kingdom, though other examples of this phenomenon exist among molluscs. Both D- and L-alanine are found in tissues of some marine bivalves. The occurrence of D- alanine appears to be related to a role in osmoregulation (17). Both D- and L-specific isoforms of LDH also occur within individual cephalopods (18). although the physio- logical significance of these has not been established. The advantages of the stylommatophoran cytosolic BHBDH isoforms are also not immediately obvious. The existence of both D- and L-BHBDH may allow the metabolic parti- tioning of BHB between specific tissues. Enzyme activi- ties indicate that ketone bodies could be synthesized in the kidney from fatty acids under normal conditions. Both D- and L-BHBDH are present in stylommatophoran kid- neys, which are thus able to produce both forms of BHB. Each of these enantiomers of BHB may be specifically targeted to a tissue, with D-BHB being oxidized by heart and L-BHB by hepatopancreas. This adaptation could be related to the apparently greater role for ketone bodies in the intermediary metabolism of pulmonate gastropods i.e.. a refining of a much-used pathway. The phylogenetic pattern of hepatopancreas BHBDH stereospecificity and subcellular distribution in gastropods suggests that L-BHBDH. and perhaps also D-BHBDH. could be valuable characters for assessing phylogenetic relationships within the Gastropoda. Both enzymes can be rapidly and inexpensively assayed. The presence of hepatopancreas L-BHBDH may be a useful defining char- acteristic of the Stylommatophora. As such, it will be especially interesting to identify which isoforms of BHBDH are present in tissues of the Archiopulmonata, a group of much-debated phylogenetic position. Cer- tainly, the presence of L-BHBDH in stylommatophoran gastropods should be noted by population geneticists, as staining of electrophoretic gels of gastropod tissues with racemic DL-BHB mixtures will give results that are a function, in part, of the phylogenetic position and tissue of the snail examined. Acknowledgments We thank John Colbourne for help with phylogenetic analyses. Also, thanks to Drs. Fred G. Thompson and Harry G. Lee. and Tom Mason, Invertebrate Curator at the Toronto Metro Zoo, for their assistance with tindi and identifying gastropod species. 16 J. A. STUART ET AL. Literature Cited I . Nevvsholme, E. A., and A. R. Leech. 1983. Bioehemixtry for the Medical Sciences. John Wiley. New York. 2 Robinson, A. M., and D. H. Williamson. 1980. Physiological roles of ketone bodies as substrates and signals in mammalian tis- sues. Phyxinl. Rev. 60: 143-187. 3. Beis, A., V. A. Zamniit, and E. A. Newsholme. 1980. Activities of 3-hydroxybutyrate dehydrogenase, 3-oxoacid CoA-transferase and acetoacetyl-CoA thiolase in relation to ketone-body utilisation in muscles from vertebrates and invertebrates. Eur. J. Biochcni. 104: 209-215. 4. Stuart, J. A., and J. S. Ballantyne. 1996. Correlation of environ- ment and phytogeny with (he expression of /3-hydroxybutyrate de- hydrogenase in the mollusca. Camp. Biochem. Phyxiol. 114B: 153- 160. 5. Stuart, J. A., and J. S. Ballantyne. 1997. Tissue specific forms of /3-hydroxybutyrate dehydrogenase oxidize the D- or L-anantiom- ers of /3-hydroxybutyrate in the terrestrial gastropod Cepaea neino- rulix. J. Exp. Zool. 278: 115-1 IS. 6. Stuart, J. A., and J. S. Ballantyne. 1997. Importance of ketone bodies in the intermediary metabolism of the terrestrial snail Archu- clnirinii vi'ntrieoxu: evidence from enzyme activities. Camp. Bio- cliem. Phyxiol. 117B: 197-201. 7. Lehninger, A. L.. H. C. Sudduth, and J. B. Wise. 1960. D-beta- hydroxybutyric dehydrogenase of mitochondria. J. Bid. Chein. 235: 2450-2455. 8. Harasewych, M. G., S. L. Adamkevvicz, J. A. Blake, D. Saudek. T. Spriggs, and C. J. Bull. 1997. Phytogeny and relationships of pleurotomariid gastropods (Mollusca: Gastropoda): an assessment based on partial 18S rDNA and cytochrome c oxidase I sequences. Mol. Mar. Bin/. Biotech. 6: 1 -20. 9. Maddison, W. P., and D. R. Maddison. 1992. MucCltu/e: Analy- sis nt Pli\lg. Ser. 3: 317-328. 24 Forsythe. J. W., and W. F. Van Heukelem. 1987. Growth. Pp. 135-156 in Cephalopod Life Cycles. Vol. 2. Comparative Reviews. P. R. Boyle, ed. Academic Press, London. 25. Rosenberg, A. A., K. F. Wiborg, and I. M. Beck. 1980. Growth of Todarodes sagittatus (Lamarck) (Cephalopoda: Ommastrephi- dae) from the Northeast Atlantic, based on counts of statolith growth rings. Sarsiu 66: 53-57. 26. Forsythe, J. W., and R. T. Hanlon. 1989. Growth of the Eastern Atlantic squid, Loligo forbesi Steenstrup (Mollusca: Cephalopoda). Aquacult. Fish. Manage. 20: 1-14. 27. Forsythe, J. W. 1993. A working hypothesis on how seasonal temperature change may impact the growth of young cephalopods. Pp. 1 33- 143 in Recent Advances in Cephalopod Fisheries Biologv. T. Okutani. R. K. O'Dor. and T. Kubodera. eds. Tokai University Press. Tokyo. 28. Grist, E. P. M., and S. des Clers. 1998. How seasonal tempera- ture variations may influence the structure of annual squid popula- tions. IMA J. Math. Appl. Med. Biol. 15: I -23. 29. Arkhipkin, A., and A. Mikheev. 1992. Age and growth of the squid Sthenoteuthis pteropus (Oegopsida: Ommastrephidae) from the Central East Atlantic. J. E\p. Mar. Biol. Ecol. 163: 261-276. 30. Arkhipkin, A., and V. Laptikhovsky. 1994. Seasonal and in- terannual variability in growth and maturation of winter-spawning llle.f argentinus (Cephalopoda, Ommastrephidae) in the Southwest Atlantic. At/uat. Living Resour. 7: 221-232. 31. Gould, S. J. 1977. Ontogeny and Phvlogeny. Harvard University Press, Cambridge. 501 pp. 32. Jackson, G. D., and J. H. Choat. 1992. Growth in tropical cepha- lopods: an analysis based on statolith microstructure. Can. J. Fish. Ai/uat. Sci. 49: 218-228. 33. Alford, R. A., and G. D. Jackson. 1993. Do cephalopods and larvae of other taxa grow asymptotically? Am. Nat. 141: 7 17-728. 34. Pauly, D. In press. Why squids though not fish may be better understood by pretending they are. S. Afr. J. Mar. Sci. Reference: Bioi Bull. 195: 21-29. (August. 1998) Metamorphic-Signal Transduction in Hydroides elegans (Polychaeta: Serpulidae) Is Not Mediated by a G Protein ERIC R. HOLM, BRIAN T. NEDVED, EUGENIC CARPIZO-ITUARTE, AND MICHAEL G. HADFIELD* Kewalo Marine Laboratory, University of Hawaii, 41 Ahui St., Honolulu, Hawaii 96813 Abstract. Evidence from larvae of hydrozoans, gastro- pods, and barnacles suggests that G protein-coupled re- ceptors mediate induction of settlement and metamorpho- sis in response to environmental cues. We examined re- sponses of larvae of the serpulid polychaete Hydroides elegans to neuropharmacological agents to determine if G protein-coupled receptors or their associated signal- transduction pathways regulated induction of metamor- phosis by bacterial cues. Larvae of Hydroides elegans metamorphose rapidly and in high proportions when ex- posed to bacterial biofilms. Neither the G-protein activa- tor Gpp[NH]p nor the inhibitor GDP-/3-S affected meta- morphosis. Although the nonspecific phosphodiesterase inhibitors IBMX, theophylline, and papaverine induced larvae to metamorphose, RO-20-1724 (an inhibitor selec- tive for cAMP-specific phosphodiesterase IV) and the cy- clic nucleotide analogs db-cAMP and db-cGMP had no effect on metamorphosis. The adenylate cyclase activator forskolin inhibited responses of larvae to inductive bacte- rial biofilms. These apparently conflicting results may be due to side effects of IBMX, theophylline, papaverine, and forskolin on ion transport. The phorbol ester TPA, an activator of protein kinase C, also had no effect on larval metamorphosis. These experiments indicate that G protein-coupled receptors and signal transduction by the Received 23 December 1997; accepted 27 May 1998. * To whom correspondence should be addressed. E-mail: hadfield hawaii.edu Abbreviations: AC/cAMP. adenylate cyclase/cyclic AMP; db-cAMP. dibutyryl-cyclic AMP; db-cGMP, dibutyryl-cyclic GMP; DMSO, di- methyl sulfoxide; FSW, filtered seawater; GDP-/3-S, guanosine S'-O- (2-thiodiphosphate); Gpp[NH]p. 5'-guanylylimidodiphosphate; IBMX. 3-isobutyl-l-methylxanthine; PI/DAG/PKC, phosphatidylinositol/di- acylglycerol/protein kinase C; TPA, phorbol-12-myristate-13-acetate. adenylate cyclase/cyclic AMP or phosphatidyl-inositol/ diacylglycerol/protein kinase C pathways are not compo- nents of the morphogenetic pathway that is directly re- sponsible for processing metamorphic cues in H. elegans. Introduction Several decades of research have demonstrated the im- portance of biochemical and physical cues in determining, on small spatial scales, where larvae of marine inverte- brates settle and metamorphose (see Scheltema. 1974: Crisp, 1984; Svane and Young, 1989, for reviews). Identi- fication of the exact nature and ecological role of exoge- nous cues remains an active field of study. Over the last 20 years, however, increased attention has focused on the physiological mechanisms underlying detection of these cues and the initiation of metamorphosis. The approach generally taken in this work consists of exposing larvae to solutions of potentially neuroactive compounds, in the presence or absence of a cue, and noting the responses. Although such experiments may be difficult to interpret (Pawlik, 1990; Leitz, 1997), a common theme is devel- oping from the results. In the marine invertebrate larvae that have been most extensively studied, it appears that G protein-coupled re- ceptors and their associated signal-transduction pathways play an important role in regulating metamorphosis. In the hydrozoans Hydractinia echinata (Miiller, 1985; Leitz and Miiller, 1987; Leitz and Klingmann, 1990; Schneider and Leitz, 1994) and Mitrocomella polydiademata (Free- man and Ridgway, 1990), the phosphatidylinositol/diacyl- glycerol/protein kinase C (PI/DAG/PKC) pathway trans- duces the metamorphic signal provided by bacterial cues. In addition, Leitz and Wirth ( 1991 ) found that H. echinata 21 22 E. R. HOLM ET AL metamorphosed in response to the G-protein activators ortho- and metavanadate, suggesting that G protein-cou- pled receptors are involved in the process. Schneider and Leitz ( 1994) proposed that the bacterial inducer of meta- morphosis acted by first binding to a G protein-coupled receptor. In the barnacle Balanns ainphi trite, two signal - transduction systems, the adenylate cyclase/cyclic AMP (AC/cAMP) pathway (Rittschof et ui. 1986; Clare et ui, 1995) and the PI/DAG/PKC pathway (Yamamoto et al, 1995; Holm et al., unpubl. data), appear to regulate meta- morphosis. Clare (1996) proposed a model linking the two pathways; in this model the exogenous metamorphic cue was bound to a G protein-coupled receptor. Finally, in the abalone Ha/iotis rnfescens, the AC/cAMP pathway transduces the metamorphic signal provided by com- pounds found on the surface of crustose coralline algae, while the PI/DAG/PKC pathway facilitates the response to these compounds (Morse et al., 1980; Trapido-Rosen- thal and Morse, 1985, 1986; Baxter and Morse, 1987; Morse, 1990, 1991 ). The receptor of the metamorphic cue did not appear to be associated with a G protein, but evidence suggested that the separate, facilitating pathway was activated by binding of amino acids to a G protein- coupled receptor (Baxter and Morse, 1987. 1992; Wo- dicka and Morse, 1991). G protein-coupled receptors and their associated signal- transduction pathways present a compelling general model for detection of a variety of stimuli in an exception- ally broad range of organisms (Carr, 1992). It is not clear, however, whether the regulation of metamorphosis by G proteins is a general feature of marine invertebrate larvae (Hadfield. 1998). For larvae of Huliotis rnfescens, at least, a G protein-coupled receptor does not bind the metamor- phic signal (Baxter and Morse, 1987). There are several other classes of receptors (Feger et ai, 1994) that could conceivably function in marine larvae to detect exogenous cues for settlement and metamorphosis. These include lectins, which have been implicated in the metamorphosis of larvae of the spirorbid polychaete ./anna hmsiliensis (Kirchman et al., 1982; Maki and Mitchell, 1985), and ligand-gated ion channels. To test the hypothesis that G protein-coupled receptors or the AC/cAMP and PI/DAG/PKC signal-transduction pathways regulate metamorphosis in the serpulid poly- chaete Hydroides elegans, we examined the response of larvae of this species to various neuropharmacological agents. Hydroides elegans (Haswell, 1883) is a common member of the shallow subtidul fouling community throughout tropical and warm temperate seas (Hadfield et al.. 1994). In the laboratory, competent larvae settle and metamorphose after as little as 15 min of exposure to bacterial biofilms ( Hadfield et ul.. 1994; Carpizo-Ituarte and Hadfield, 1998). Experiments with larvae of other polychaetes, including Capilelln <idcs elegans. Except for Gpp[NH]p and RO-20-1724, the concentra- tion ranges we tested for these compounds spanned effec- tive concentrations previously determined to induce meta- morphosis in larvae of other marine invertebrates. We could find no comparable data for Gpp[NH]p and RO- 20-1724. RO-20-1724 was obtained from Calbiochem, San Diego, California; all other compounds were pur- chased from Sigma, St. Louis, Missouri. Statistical analysis The results of the experiments were analyzed using either two-sample /-tests or Wilcoxon rank-sum tests. The percentage of larvae metamorphosing in each replicate petri dish was subjected to the angular transformation, and the means and variances for each treatment were calculated. Variances for the two treatments to be com- pared were then tested for homogeneity using the F-max test (Sokal and Rohlf, 1981). If variances were homoge- neous, the transformed data were compared by two-sam- ple /-test. Otherwise the untransformed data were ana- lyzed by the nonparametric Wilcoxon rank-sum test. All /-tests were calculated by hand; the nonparametric tests were conducted using the NPAR 1 WAY procedure in SAS (SAS Institute Inc.. 1989). Significant results were identi- fied using the sequentially rejective Bonferroni procedure (Holm, 1979; Rice, 1989) to correct for multiple tests. Results Compounds affecting the activity of G proteins Neither the G-protein activator Gpp[NH]p nor the in- hibitor GDP-/?-S affected metamorphosis of the larvae of Hydroides elegans (Fig. 1A. B). In the absence of a stimulatory bacterial biofilm, Gpp[NH]p did not induce metamorphosis (Fig. 1A), and GDP-/2-S failed to inhibit metamorphosis in response to a biofilmed surface (Fig. IB). Compounds affecting the AC/cAMP transduction pathway In two separate trials, the phosphodiesterase inhibitors IBMX, theophylline, and papaverine all induced meta- morphosis in the absence of a biofilm (Fig. 2A, B). Con- centrations at which we observed maximal responses were the same in both trials; 10 4 M for IBMX (Mest. P 0.001. both trials), 10 - M for theophylline (/-test. P < 24 E. R. HOLM ET AL Table I Compounds tested for effects on metamorphosis of larvae o/Hydroides elegans Activity Predicted effect on metamorphosis Compounds affecting G proteins Gpp [NH] p GDP-/3-S Compounds affecting the AC/cAMP signal-transduction pathway IBMX Theophylline Papaverine RO-20-1724 db-cAMP db-cGMP Forskolin Compounds affecting the PI/DAG/PKC signal-transduction pathway TPA Activates G proteins Induction Inhibits activation of G proteins Inhibition Inhibits phosphodiesterases* Induction Inhibits phosphodiesterases* Induction Inhibits phosphodiesterases* Induction Inhibits cAMP-specific phosphodiesterase IV* Induction Increases intracellular cAMP Induction Increases intracellular cGMP Induction Activates adenylate cyclase* Induction Activates protein kinase C Induction A brief description of the activity of each compound is provided (Activity). Predicted Effect describes the result one would expect if G proteins. or the affected signal-transduction pathway, mediated responses of larvae to metamorphic cues. See text for details. * Activity increases intracellular cAMP or cGMP. 0.001, both trials), and 10 5 M for papaverine (/-test, P < 0.001, trial 1; Mest, P < 0.005, trial 2). IBMX at \0~ 3 M caused a large number of larvae to metamorphose without constructing a primary or secondary tube. Figure 2 shows only the percentage of larvae that had undergone normal metamorphosis (characterized by permanent at- tachment to the substratum and construction of either a primary or secondary tube, see Materials and Methods) when exposed to IBMX at IQ~* M. Papaverine was a much less effective inducer of metamorphosis than either IBMX or theophylline, with a maximum mean percentage of metamorphosis < 20%, as compared to > 80% for both IBMX and theophylline (Fig. 2A, B). Exposure of larvae to theophylline at 10~ 2 M was fatal. In the second trial (Fig. 2B) both IBMX (rank-sum test, P < 0.05) and papaverine (rank-sum test, P < 0.05) inhibited normal metamorphosis at their highest concentrations, although the effects were not strong. These compounds may have been toxic at the highest concentrations we tested. The selective phosphodiesterase inhibitor RO-20-1724 exhib- ited no inductive effects (Fig. 3). Neither the adenylate cyclase activator forskolin, the cAMP analog db-cAMP. nor the cGMP analog db-cGMP induced metamorphosis in the absence of biofilm (Fig. 4). In the presence of a stimulatory biofilm, however, 10~ 4 M forskolin strongly inhibited metamorphosis (Fig. 5A, /-test, forskolin vs. DMSO control, P < 0.001). Inhi- bition did not appear to be the result of toxicity, as larvae exposed to 10~ 4 A/ forskolin in this experiment and in the absence of biofilm swam and behaved normally. We observed no mortality in these trials. Neither db-cAMP nor db-cGMP inhibited responses of larvae to biofilms (Fig. 5B). Compounds affecting the PI/DAG/PKC transduction pathway Exposure of larvae to the phorbol ester TPA caused no significant induction of metamorphosis as compared to the appropriate DMSO controls (Fig. 6). Table II lists the compounds tested, their predicted ef- fect on metamorphosis of larvae of Hydroides elegans if G protein-coupled receptors or the AC/cAMP or PI/DAG/ PKC signal-transduction pathways mediated responses of larvae to metamorphic cues, and the effect we observed in our experiments. Discussion A growing body of research on the effects of neuro- pharmacological agents on marine invertebrate larvae suggests that G protein-coupled receptors and their associ- ated signal-transduction pathways are important regula- tors of larval settlement and metamorphosis, either di- rectly through sensation and processing of exogenous metamorphic cues, or indirectly by modifying responses to these cues. It is not clear, however, whether this role for G proteins in metamorphosis is a characteristic of all marine invertebrate larvae (Hadfield, 1998). Our experi- ments indicate that G protein-coupled receptors or signal transduction by the AC/cAMP or PI/DAG/PKC pathways METAMORPHOSIS OF HYDROIDES ELEGANS 25 A. 100 en "en o "I- o .2 "CD CD CD CL B. en 'en O 9- o -2 "55 CD O CD CL 90 10 Gpp[NH]p Biofilm Filtered Seawater Controls 10' 7 M 10' 6 M 10' 5 M Concentration 100 90 80 70 60 50 40 30 20 10 GDP-p-S + Biofilm Biofilm Filtered Seawater Controls 10' 7 M 10' 6 M 10' 5 M 10' 4 M Concentration Figure 1. Metamorphosis of larvae of Hydroides elegans exposed to activators and inhibitors of G proteins. (A) Response to the G-protein activator Gpp [NH]p. (B) Effect of the G-protein inhibitor GDP-/3-S on metamorphosis in response to bacterial biofilms. Points represent means of 4 replicates; error bars are standard deviations. AC/cAMP or PI/DAG/PKC pathways. Baxter and Morse (1987) found that Gpp[NH]p and GDP-/3-S did not di- rectly affect metamorphosis of larvae of Halintis rufes- cens, but larvae were induced to metamorphose by appli- cation of forskolin, IBMX, and theophylline, suggesting that the metamorphic signal was likely to be transduced by the AC/cAMP pathway, although not by a G protein- coupled receptor (Baxter and Morse, 1987; Morse, 1990). If signal transduction by the AC/cAMP pathway were a necessary event in the metamorphic sequence in Hydro- ides elegans, then competent larvae should have meta- morphosed when exposed to the phosphodiesterase inhib- itors, db-cAMP, and the adenylate cyclase activator for- skolin. The nonspecific phosphodiesterase inhibitors IBMX. theophylline, and papaverine induced significant levels of metamorphosis (Fig. 2); however, we observed no effect of the selective inhibitor RO-20-1724 (Fig. 3). Additionally, neither forskolin nor db-cAMP caused lar- vae to metamorphose (Fig. 4). Forskolin had the opposite A. en 'tn O _c Q. O CD o CD CL 100 -i 90 80 70 60 50 - 40 30 - 20 10 -I IBMX Theophylline Papaverine Biofilm Filtered Seawater Controls 10' 8 M 10' 7 M 10' 6 M 10' 5 M 10 J M 10' 3 M 10' 2 M Concentration do not mediate responses of larvae of the serpulid poly- chaete Hydroides elegans to metamorphic cues. If cues for settlement and metamorphosis of larvae of H. elegans were bound by a G protein-coupled receptor, we would have expected competent larvae to ( 1 ) meta- morphose when exposed to the GTP analog Gpp[NH]p. and (2) fail to respond to inductive bacterial biofilms when those biofilms were presented in combination with GDP-/J-S, a competitive inhibitor of G protein activation by GTP. We observed no increase in metamorphosis over FSW control treatments in the presence of Gpp|NH]p (Fig. 1 A), and no inhibition of response of larvae to bio- filmed surfaces when exposed to GDP-/3-S (Fig. IB). These results imply that the receptors responsible for de- tecting the metamorphic cue in H. elegans are not associ- ated with G proteins. The absence of evidence for the involvement of G pro- tein-coupled receptors does not exclude the possibility that the metamorphic signal is transduced by either the B. en 'en O "- O CD CD CL 100 - 90 - 80 - 70 - 60 50 40 30 20 10 Controls 10' 6 M 10' 5 M 10'" M Concentration 10" J M Figure 2. Metamorphosis of larvae of Hydroides elegans exposed to the phosphodiesterase inhibitors IBMX, theophylline, and papaverine. (A) and (B) present results from two separate trials. Note that the con- centration ranges (x axes) differ between trials. Points represent means of 5 replicates in (A), 4 replicates in (B); error bars are standard devia- tions. * - significantly different from the FSW control treatment at P == 0.05. 26 E. R. HOLM ET AL. 100 -, C/5 90 -- RO-20-1724 'c/5 e DMSO Control O t 80 - ^ Biofilm Q. 70 - v Filtered Seawater O E 60 - -2 50 - lr CD 40 - 30 - CD O 20 - CD CL 10 - - Controls 10' 7 M 10' 6 M 10' 5 M 10' 4 M 10' 3 M Concentration Figure 3. Metamorphosis of larvae of Hydroides elegans exposed to RO-20-1724, a selective inhibitor of the mammalian cAMP-specific phosphodiesterase IV. Exposure of larvae to DMSO served as the control for the RO-20-1724 treatment. Points represent means of 4 replicates; error bars are standard deviations. effect, inhibiting metamorphosis of//, elegans in response to bacterial biofilms (Fig. 5). Alternatively, the metamorphic signal may be trans- duced by cGMP. IBMX. theophylline, and papaverine are not selective for any of the cyclic nucleotide phosphodies- terase isozymes (Thompson, 1993); they inhibit phospho- diesterases that hydrolyze cGMP as well as those that hydrolyze cAMP. Exposure of larvae to db-cGMP. how- ever, neither induced (Fig. 4) nor inhibited (Fig. 5B) meta- morphosis. Although the results presented here appear to be contra- 100 CD C/5 O f 90 - 80 - B db-cAMP db-cGMP *- Q- 70 e DMSO Control O 60 - Forskolin 50 - r Biofilm "CD 40 - v Filtered Seawater - 30 o 20 CD Q_ 10 - - V Controls 1Q- 8 M 10' 7 M 10" 6 M 10' 5 M 10"" M Concentration Figure 4. Metamorphosis of larvae ot Hydroitles elegans exposed to the cyclic nucleotide analogs db-cAMP and db-cGMP, and the adenylate cyclase activator forskolin. Exposure of larvae to DMSO served as the control for the forskolin treatment. Points represent means of 4 repli- cates; error bars are standard deviations. A. 100 n 90 - '8 80 e- o CD 'CD CD Q_ 70 - 60 - 50 - 40 - 30 - 20 - 10 Forskolin + Biofilm DMSO + Biofilm Biofilm Filtered Seawater Controls 10' 8 M 10' 7 M 10' 6 M 10' 5 M 10' 4 M Concentration B. "e- o E ro "CD CD CL 100 -, 90 80 70 - 60 50 40 30 - 20 10 db-cAMP * Biofilm db-cGMP + Biofilm Biofilm Filtered Seawater Controls 10' 8 M 10 7 M 10' 6 M 10' 5 M 10 4 M Concentration Figure 5. Metamorphosis of larvae of Hydroides elegans exposed to bacterial biofilms in combination with forskolin. db-cAMP, and db- cGMP. (A) Response to biofilmed surfaces in the presence of forskolin. The DMSO + Biofilm treatment served as the control for larvae exposed to forskolin. (B) Response to biofilmed surfaces in the presence of db- cAMP and dh-cGMP. Points represent means of 4 replicates; error bars are standard deviations. * - significantly different from the control treat- ment at P s 0.05. dictory, they may be explained by the effects on ion trans- port of some of the compounds used. IBMX (Kopf ct ai, 1983; Simasko and Van, 1993; Usachev and Verkhratsky, 1995), theophylline (Kopf et cil., 1983), and papaverine (Fujioka, 1984; Iguchi et ai, 1992) all have effects on calcium transport that can be either independent of or stronger than their effects on intracellular cAMP levels. Forskolin can block both potassium (Watanabe and Gola, 1987; Coombs and Thompson. 1987; Hoshi ct til., 1988; Garber et at., 1990) and calcium (Park and Kim. 1996) channels, and desensitize acetylcholine receptors (Wag- oner and Pallottu. 1988; White. 1988). independently of its effects on cAMP. Calcium ions are important second messengers in the metamorphic pathway of some hydro- /oan larvae (Freeman and Ridgway, 1987, 1990), and may serve the same function in larvae of polychaetes (Ilan el a!., 1993) and barnacles (Rittschof ct tit., 1986; Clare, METAMORPHOSIS OF HYDROIDES ELEGANS 27 1996). Metamorphosis of larvae of several phyla of ma- rine invertebrates can be induced or inhibited by elevated concentrations of potassium (reviewed in Woollacott and Hadtield, 1996) or by application of potassium-channel blockers (reviewed in Pawlik, 1990). The exact mecha- nism by which potassium affects metamorphosis has yet to be determined. Ongoing research in our laboratory indicates that cal- cium and potassium play important roles in the metamor- phic morphogenesis of H. elegans. Addition of calcium ions to FSW enhances metamorphosis in the presence of biofilms, and pulse application of potassium induces metamorphosis in the absence of other cues (unpubl. data; Carpizo-Ituarte and Hadfield. 1996, 1998). Calcium and potassium channel blockers inhibit metamorphosis in re- sponse to cesium pulses and bacterial films, respectively (unpubl. data: Carpizo-Ituarte and Hadfield, 1998). It seems likely that the results we observed after application of the nonspecific phosphodiesterase inhibitors and for- skolin are due to their effects on ion transport, rather than on cAMP or cGMP levels. This conclusion is supported by the lack of inhibitory or stimulatory effects from db- cAMP, db-cGMP, or the selective phosphodiesterase in- hibitor RO-20-1724. On the basis of this evidence, we conclude that the AC/cAMP pathway does not transduce the metamorphic signal in larvae of H. elegans. Our data also indicate that metamorphosis does not require signal transduction by the PI/DAG/PKC pathway. We attempted to induce metamorphosis with the protein kinase C activator TPA, the phorbol ester found to be the most effective stimulator of metamorphosis in two species of hydrozoans (Miiller, 1985; Freeman and Ridgway, 1990). No effect was observed, implying that the PI/DAG/ PKC pathway does not directly participate in the meta- 100 -i TPA C/5 'c/5 O f 90 - 80 > DMSO Control r Biofilm I Q- 70 v Filtered Seawater O 60 - v CT3 50 CD 40 "c 30 CD O 20 CD Q_ 10 - - I _-_ _&= Controls 10' 9 M 10' 8 M 10 7 M Concentration 10' b M 10' 5 M Induction Induction Induction Induction Induction Induction Induction None Induction None Induction None Induction Inhibition Figure 6. Metamorphosis of larvae of Hvdroidex elegans exposed to the protein kinase C activator TPA. Exposure of larvae to DMSO served as the control for the TPA treatment. Points represent means of 4 replicates; error bars are standard deviations. Table II 1'iti/icled and ohscrvctl effects of compounds tested on lnn'ae of Hydroides elegans Effect on metamorphosis Predicted Observed Compounds affecting G proteins Gpp [NH] p Induction None GDP-^-S Inhibition None Compounds affecting the AC/cAMP signal-transduction pathway IBMX Theophylline Papaverine RO-20-1724 db-cAMP db-cGMP Forskolin Compounds affecting the PI/DAG/PKC signal-transduction pathway TPA Induction None See Table I and the text for descriptions of the activity of each compound. Predicted Effect describes the result one would expect if G proteins, or the affected signal-transduction pathway, mediated re- sponses of larvae to metamorphic cues. morphic process. The possibility that G proteins and the PI/DAG/PKC pathway transduce signals that modify re- sponses of larvae to the metamorphic cue, as in Haliotis rufescens (Trapido-Rosenthal and Morse, 1985; Baxter and Morse, 1987), cannot be eliminated, however. Our results indicate that neither G protein-coupled re- ceptors nor signal transduction by the AC/cAMP or PI/ D AG/PKC pathways are components of the morphogene- tic pathway (in the sense of Baxter and Morse, 1987) directly responsible for sensation and processing of bacte- rial settlement cues in larvae of Hydroides elegans. We are investigating the possibility that the receptors that detect these cues are either ligand-gated ion channels sim- ilar to amino acid taste receptors in channel catfish (Teeter ct a/.. 1990; Brand el a/., 1991), or lectins such as those suggested to mediate responses to bacterial biofilms in larvae of the spirorbid polychaete Janna brasiliensis (Kirchman et /., 1982; Maki and Mitchell, 1985). Acknowledgments This research was funded by grants from the Office of Naval Research to C. M. Smith and MGH (No. N00014- 95-1-0196), and MGH (N00014-95-1-1015). EC-I was supported in part by a scholarship for graduate studies from CONACYT/UABC, Mexico. We thank D. Rittschof and two anonymous reviewers for valuable comments on preliminary drafts of the manuscript. 28 E. R. HOLM ET AL. Literature Cited Baxter. G., and D. E. Morse. 1987. G protein and diacylglycerol regulate metamorphosis of planktonic molluscan larvae. Proc. Natl. Acad. Sci. USA 84: 1867-1870. Baxter, G. T., and D. E. Morse. 1992. Cilia from abalone larvae contain a receptor-dependent G protein transduction system similar to that in mammals. Biol. Bull, 183: 147-154. Biggers. VV. J., and H. Laufer. 1992. Chemical induction of settle- ment and metamorphosis of Capitella capitata Sp. I (Polychaeta) larvae by juvenile hormone-active compounds. Invenebr. Reprod. Dev. 22: 39-46. Brand, J. G., J. H. Teeter, T. Kumazawa, T. Huque, and D. L. Bayley. 1991. Transduction mechanisms for the taste of amino acids. Physio!. Behav. 49: 899-904. Bryan, P. J., P.-Y. Qian, J. L. Kreider, and F.-S. Chia. 1997. Induc- tion of larval settlement and metamorphosis by pharmacological and conspecific associated compounds in the serpulid polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 146: 81-90. Carpizo-Ituarte, E., and M. G. Hadfield. 1996. Biofilm and ionic stimulation of metamorphosis in the polychaete Hydroides elegans. Am. Zoo/. 36: 10A. Carpizo-Ituarte. E.. and M. G. Hadh'eld. 1998. Stimulation of meta- morphosis in the polychaete Hydroides elegans Haswell (Serpuli- dae). Biol. Bull. 194: 14-24. Carr, W. E. S. 1992. Recurring themes and variations: an overview and introduction. Bint Bull. 183: 143-146. Cheung, W. Y., and Y. M. Lin. 1974. Purification and characteriza- tion of cyclic 3', 5'-nucleotide phosphodiesterase from bovine brain. Methods En-ymol. 38: 223-239. Clare, A. S. 1996. Signal transduction in barnacle settlement: calcium re-visited. Biofmtling 10: 141-159. Clare, A. S., R. F. Thomas, and D. Rittschof. 1995. Evidence for the involvement of cyclic AMP in the pheromonal modulation of barnacle settlement. J. E.\p. Biol. 198: 655-664. Coombs, J., and S. Thompson. 1987. Forskolin's effect on transient K current in nudibranch neurons is not reproduced by cAMP. J. Neurosci. 7: 443-452. Crisp, D. J. 1984. Overview of research on marine invertebrate larvae. 1 940- 1 980. Pp. 1 03 - 1 26 in Marine Biodeterioration: an Interdisci- plinary Study, J. D. Costlow and R. C. Tipper, eds. Naval Institute Press. Annapolis. Maryland. Feger, J., S. Gil-Faloon, and C. Lamaze. 1994. Cell receptors: defi- nition, mechanisms and regulation of receptor-mediated endocytosis. Cell Mot Biol. 40: 1(139-1061. Freeman, G., and E. B. Ridgvtay. 1987. Endogenous photoproteins, calcium channels and calcium transients during metamorphosis in hydrozoans. Roux's Arch. Dev. Biol. 196: 30-50. Freeman, G., and E. B. Ridgway. 1990. Cellular and intracellular pathways mediating the metamorphic stimulus in hydrozoan planu- lae. Roux's Arch. Dev. Biol. 199: 63-79. Fujioka. M. 1984. Lack of a causal relationship between the vasodila- tor effect of papaverine and cyclic AMP production in the dog basilar artery. Br. J. Pharnuicot 83: 113-124. Garber, S. S., T. Hoshi, and R. W. Aldrich. 1990. Interaction of forskolin with voltage-gated K* channels in PC 12 cells. J. Neurosci. 10: 3361-3368. Gilman, A. G. 1987. G proteins: transducers of receptor-generated signals. Anini. Rev. Biochem. 56: 615-649. Hadfield, M. G. 1998. Research on settlement and metamorphosis of marine invertebrate larvae: past, present and future. Biofoitlint; 12: 9-29. Hadfield. M. G., C. C. Unabia, C. M. Smith, and T. M. Michael. 1994. Settlement preferences of (he ubiquitous fouler Hydroides elegans. Pp. 65-74 in Recent developments in Biofoii/ing Control. M. F. Thompson. R. Nagabhushanam, R. Sarojini. and M. Fin- german, eds. Oxford and IBH Pub. Co., New Delhi. Holm. S. 1979. A simple sequentially rejective multiple test proce- dure. Scu/ul. J. Statist. 6: 65-70. Hoshi, T., S. S. Garber. and R. W. Aldrich. 1988. Effect of forskolin on voltage-gated K* channels is independent of adenylate cyclase activation. Science 240: 1652-1655. Iguchi. M., T. Nakajima, T. Hisada, T. Sugimoto, and Y. Kurachi. 1992. On the mechanism of papaverine inhibition of the voltage- dependent Ca + * current in isolated smooth muscle cells from the guinea pig trachea. J. Pharmacot E\p. Ther. 263: 194-200. Ilan, M., R. A. Jensen, and D. E. Morse. 1993. Calcium control of metamorphosis in polychaete larvae. /. Exp. Zoot 267: 423-430. Jensen, R. A., and D. E. Morse. 1990. Chemically induced metamor- phosis of polychaete larvae in both the laboratory and ocean environ- ment. J. Chem. Ecol. 16: 911-930. Kirchman, D., S. Graham, D. Reish. and R. Mitchell. 1982. Lectins may mediate in the settlement and metamorphosis of Jainui fDexi- ospira) brasiliensis Grube (Polychaeta: Spirorbidae). Mar. Biol. Lett. 3: 131-142. Kopf, G. S., C. A. Lewis, and V. D. Vacquier. 1983. Methylxan- thines stimulate calcium transport and inhibit cyclic nucleotide phos- phodiesterases in abalone sperm. Dev. Biol. 99: 115-120. Leitz, T. 1997. Induction of settlement and metamorphosis of cni- darian larvae: signals and signal transduction. Invertehr. Reprod. Dev. 31: KW-122. Leitz, T., and G. Klingmann. 1990. Metamorphosis in Hvdractinia: studies with activators and inhibitors aiming at protein kinase C and potassium channels. Roux's Arch. Dev. Biol. 199: 107-113. Leitz, T., and W. A. Miiller. 1987. Evidence for the involvement of Pi-signaling and diacylglycerol second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata Fleming. Dev. Biol. 121: 82-89. Leitz, T., and A. Wirth. 1991. Vanadate, known to interfere with signal transduction. induces metamorphosis in Hydractinia (Coelen- terata; Hydrozoa) and causes profound alterations of the larval and postmetamorphic body pattern. Differentiation 47: 1 19-127. Maki, J. S., and R. Mitchell. 1985. Involvement of lectins in the settlement and metamorphosis of marine invertebrate larvae. Bull. Mar. Sci. 37: 675-683. Morse, A. N. C. 1991. How do planktonic larvae know where to set- tle? Am. Sci. 79: 154-167. Morse, D. E. 1990. Recent progress in larval settlement and metamor- phosis: closing the gaps between molecular biology and ecology. Hull Mar. Sci. 46: 465-483. Morse, D. E.. H. Duncan, N. Hooker, A. Baloun, and G. Young. 1980. GABA induces behavioral and developmental metamorpho- sis in planktonic molluscan larvae. Fed. Proc. 39: 3237-3241. Miiller, VV. A. 1985. Tumor-promoting phorbol esters induce meta- morphosis and multiple head formation in the hydroid Hvdractinia. Differentiation 29: 216-222. Park, T.-J., and K.-T. Kim. 1996. Cyclic AMP-independent inhibi- tion of voltage-sensitive calcium channels by forskolin in PC 1 2 cells. J. Neurochem. 66: 83-88. Pawlik, J. R. 1990. Natural and artificial induction of metamorphosis of Phragmatopoma lapidosa californica (Polychaeta: Sabellariidae), with a critical look at the effects of bioactive compounds on marine invertebrate larvae. Bull. Mar. Sci. 46: 512-536. Posternak, T., and G. VVeimann. 1974. The preparation of acylated derivatives of cyclic nucleotides. Methods Enzwno/. 38: 399-409. Rice, W. R. 1989. Analyzing tables of statistical tests. Evolution 43: 223-225. Rittschof, I)., J. Maki, R. Mitchell, and J. D. Costlow. 1986. Ion and METAMORPHOSIS OF HYDROIDES ELEGANS 29 neuropharmacological studies of barnacle settlement. Netherlands J. Sea Res. 20: 269-275. SAS Institute Inc. 1989. SAS/STAT User's Guide. Version f>. 4th <'. North Carolina. 846 pp. Scheltema, R. S. 1974. Biological interactions determining larval set- tlement of marine invertebrates. Thalassia Jugosl. 10: 263-296. Schneider, T., and T. Leitz. 1994. Protein kinase C in hydrozoans: involvement in metamorphosis of Hydractinia and in pattern forma- tion of Hydra. Roux's Arch. Dev. Biol. 203: 422-428. Seanion, K. B., W. Padgett, and J. W. Daly. 1981. Forskolin: unique diterpene activator of adenylate cyclase in membranes and intact cells. Proc. Ntitl. Acad. Sci. USA 78: 3363-3367. Simasku, S. M., and S. Van. 1993. 3-Isobutyl-l-methylxanthine in- hibits sustained calcium current independently of cyclic AMP in neuronal and endocrine cells. Mol. Pharmacol. 44: 622-627. Sokal, R. R., and F. J. Rohlf. 1981. Biometry. W. H. Freeman. San Francisco. 859 pp. Svane, I., and C. M. Young. 1989. The ecology and behaviour of ascidian larvae. Oceanogr. Mar. Biol. Annu. Re\'. 27: 45-90. Teeter, J. H., J. G. Brand, and T. Kumazawa. 1990. A stimulus- activated conductance in isolated taste epithelial membranes. Bio- /)/iv.v. J. 58: 253-259. Thompson, W. J. 1993. Cyclic nucleotide phosphodiesterases: phar- macology, biochemistry and function. Pp. 287-313 in Intracellnlar Messengers. C. W. Taylor, ed. Pergamon Press, Oxford. Trapido-Rosenthal, H. G., and D. E. Morse. 1985. i.-a, u'-diamino acids facilitate GABA induction of larval metamorphosis in a gastro- pod mollusc (Haliotis rufescens). J. Comp. Physio/. B. 155: 403- 414. Trapido-Rosenthal, H. G., and D. K. Morse. 1986. Availability of chemosensory receptors is down-regulated by habituation of larvae to a morphogenetic signal. Pmc. Null. Acad. Sci. USA 83: 7658- 7662. Usachev, Y., and A. Verkhratsky. 1995. IBMX induces calcium release from intracellular stores in rat sensory neurones. Cell Cal- cium 17: 197-206. Wagoner, P. K., and B. S. Pallotta. 1988. Modulation of acetylcho- line receptor desensitization by forskolin is independent of cAMP. Science 240: 1655-1657. Watanahe, K., and M. Gola. 1987. Forskolin interaction with volt- age-dependent K channels in Helix is not mediated by cyclic nucleo- tides. Neurosci. ten. 78: 21 1-216. White, M. M. 1988. Forskolin alters acetylcholine receptor gating by a mechanism independent of adenylate cyclase activation. Mol. Pharmacol. 34: 427-430. Wisely, B. 1958. The development and settling of a serpulid worm. Hvdroides nonegica Gunnerus (Polychaeta). Ansr. J. Mar. Freshwa- ter Res. 9: 351-361. Wodicka, L. M., and D. E. Morse. 1991. cDNA sequences reveal mRNAs for two G signal transducing proteins from larval cilia. Biol. Bull. 180: 318-327. Woollacott, R. M., and M. G. Hadfield. 1996. Induction of metamor- phosis in larvae of a sponge. Invertehr. Kiol. 115: 257-262. Yamamoto, H., A. Tachihana, K. Matsumura, and N. Fusetani. 1995. Protein kinase C (PKC) signal transduction system involved in larval metamorphosis of the barnacle, Balanus amphitrite. Zool. Sci. 12: 391-396. Reference: Biol. Bull 195: 30-42. (August. 1998) Transport Pathways in the Neotropical Sponge Aplysina SALLY P. LEYS 11 AND HENRY M. REISWIG 2 1 Department of Biology, University of Victoria, Victoria. British Columbia, Canada, V8W 3N5; 2 Redpath Museum and Biology Department, McGill University, 859 Sherbrooke St., Montreal, Quebec, Canada: and ' Bellairs Research Institute of McGill University; St. James. Barbados Abstract. Strands of cells distinct from the rest of the tissue were found running lengthwise through the endo- some of four species of the sponge Aplysina. Although the strands were more highly pigmented than the adjacent tissue and could be removed intact with forceps, ultra- structural studies revealed no obvious barrier separating the cells in the strands from the rest of the tissue. The strands consist of stretches of elongate cells tightly aligned along densely bundled collagen fibrils, and areas of other elongate cells that possess numerous filopodia. When sponges were fed fluorescent latex beads in situ. beads were taken up and transported specifically into the strands; eventually they were found at the tip of the sponge and further down the stalk, away from the site of feeding. Beads injected into endosomal strands were also transported upwards in the strands to the tip of the sponge. Video microscopy of cells in strands that had been ex- posed along a portion of their length showed no bulk movement of cells; but individual cells were seen mov- ing in both directions along the strands at 0.025- 0.04 pm-s '. The endosomal cell strands are suggestive of a primitive nutrient transport pathway in sponges. Introduction Sponges were the first multicellular animals to arise from unicellular protists during early metazoan evolution, at least 600 million years ago (Steiner etal., 1993; Reitner and Mehl, 1995), and their tissue organization, accord- ingly, is quite simple. Sponges are diploblastic metazoans that possess no organ systems; their tissues consist of a limited number of cell types that surround and penetrate a mesohyl of collagen. The entire animal is organi/ed Received 30 March 1998; accepted I June 1998. around canals and chambers through which water is chan- neled for feeding and respiration. Within the Porifera. however, some groups differ mark- edly from this fundamental plan. The Hexactinellida, for example, are mostly syncytial (Reiswig, 1979; Mackie and Singla. 1983), a condition that allows symplastic transport of food (Perez, 1996; Wyeth et a/.. 1996) and rapid conduction of action potentials that control the feed- ing current (Leys and Mackie, 1997). The Cladorhizidae, on the other hand, are deep-sea demosponges that are effectively carnivorous and lack flagellated chambers, which are otherwise a key poriferan character (Vacelet and Boury-Esnault, 1995). In a group as old as the Pori- fera, other exceptions to the basic sponge plan probably exist. One intriguing possibility lies within demosponges of the genus Aplvsimi. These sponges, which are known as bacteriosponges for the vast number of bacteria living within their mesohyl, belong to the Verongida, an order that contains numerous pharmaceutically interesting chemicals (Bergquist el ai, 1991; Ciminiello et a/., 1994, 1997). An unusual feature of some sponges in this genus is the presence of longitudinal strands of elongate cells in the endosome. The strands, up to lOO/um in diameter and particularly densely populated by bacteria, constitute a quasi-tissue that runs vertically through sponges of tube, chimney, and rope-form alike. Despite their prominence in these sponges and their marked absence in other members of the phylum studied to date, the strands have received only passing mention in the literature. Tsurumi and Reiswig (1997) suggested that the "endosomal" tissue strands in Aplysinu cauli- I'onnis (a rope-form sponge) serve as migration routes for rapid transport of cells for tip growth. Similar elongate cells, in the endosome and around the water canals in Verongia (synonymous with Aplysina). were suggested 30 SPONGE TRANSPORT PATHWAYS 31 to he contractile cells that act together to control water flow through the canal system (Vacelet, 1966). To have tested either of these hypotheses would have required cell labeling studies and, if possible, video-enhanced-contrast microscopy of live tissue, techniques that were not readily available until recently. We have now undertaken both approaches and present here a detailed description of the endosomal tissue strands and their role in transport of materials. Materials and Methods Description of the sponges and collection site Aplysina caulifonnis (probably synonymous to A. fiilva). a neotropical rope-form sponge, grows at the tip (Wulff, 1990), reaching about 1 cm in diameter, but up to several meters in length. It is pink or purple on the outside and yellow inside. At depths of 7- 10 m, A. cauli- fonnis is typically branched and grows only 20-30 cm in height. Aplvsiiw lacunosa is a mustard-yellow tube- form sponge. The tubes are 10-50 cm in length and can occur singly, but more often several of them bud from the same base. The tubes are 5-10 cm in diameter with an atrial diameter of 2-5 cm. Aplysina rigida, a branching stick-form sponge, is pink outside and yellow on the in- side, but is both thicker (up to 5 cm in diameter) and taller (up to 1 m in height in 7-m-deep waters) than A. caulifonnis. Aplysina fistularis is a robust, branching, stick-form sponge in Barbados, but it may be tube-form in other habitats in the Caribbean. A. fistularis is yellow outside and has an irregular outer surface, but it lacks the lacunae characterizing A. lacunosa', it is 3-5 cm in diameter and reaches 20-30 cm in height at depths of 7- 10m. Species of Aplysina were manipulated in situ and col- lected by scuba from coral rubble at a depth of 7- 10 m off the Bellairs Research Institute, St. James, Barbados (1310'N, 5940'W). Field experiments Latex bead feeding. Plastic bags (4x7 cm) open at both ends were slipped over the branches of A. caulifonnis in situ and, about 5- 10 cm from the top, were gently secured at both ends with plastic twist ties (Figs. 1 and 2a). A plastic syringe and needle were used to inject the bag with 5 ml of 1-^rn fluorescent latex beads (Molecular Probes, Eugene, OR) diluted 1 :5 with seawater. The beads were not treated with serum to prevent clumping, because trial experiments showed that clumping did not interfere with bead uptake. The bead-filled bags were left on the sponges for 4 h and then removed; the beads had been cleared from the bags during this period. Two branches were collected for fixation at 4 and 24 h, and 2, 3, 7, 1 1, and 1 3 days after feeding. The sponges were cut off at a bag with beads Tip Middle 5cm Lower i/WVM/ Figure 1. Diagram of a branch of Aplysinu caulifonnis showing the attachment of a plastic bag that is used to feed latex beads to the sponge. Also shown are the three segments (Tip. Middle, and Lower) that the branch was cut into; the segments were used in determining the fate of the beads. point 15 cm from the top, placed in a plastic bag, and taken to the laboratory, where each piece was cut into three segments: the top 5 cm (Tip), the middle 5 cm where the bag had been secured (Middle), and the bottom 5 cm (Lower) (Fig. 1). The segments were fixed in 2% para- formaldehyde in seawater and placed in the refrigerator at about 4C. After 4 hours the fixative was poured off and replaced with fresh, cold 2% paraformaldehyde. Insertion of latex beads. Whole branches of A. cauli- fonnis attached to loosely buried coral rubble were col- lected in plastic bags (with the coral) and brought into the laboratory; the sponges were never removed from seawater. The sponge branches were cut 7 cm from the tip, but not quite in two; thus a hinge of tissue still con- nected the tip segment to the main stem. The sponge was placed in a large bucket of seawater and, with the aid of a dissecting microscope poised over the bucket, l-um latex beads were injected with a plastic syringe and needle onto a severed cell strand and its immediate area. The wound was sewn together with dental floss and the sponges were returned to the field no more than 4 h after collection and reattached with plastic cable ties to other pieces of coral rubble. Preliminary experiments showed that, despite wave action, the wound healed and new tis- sue grew over the dental floss within 1 -2 days. Sponges treated in this way were recollected 1, 2, 3, 7. 11, and 13 days after wounding, cut into three segments as described above, and fixed in 2% paraformaldehyde in seawater. Laboratory analysis of field experiments Sponge pieces were transported while still in fixative to the University of Victoria. British Columbia, Canada, where 32 S. P. LEYS AND H. M. REISWIG they were rinsed in phosphate-buffered saline (PBS) for 4 h, infiltrated overnight in 1:1 PBS and Tissue Tek (O.C.T. Compound 4583, Sakura Finetechnical, Tokyo), and embed- ded in 100% Tissue Tek by freezing in liquid nitrogen. Blocks of embedded tissue were stored at 20C. Longitudi- nal sections (30 /um) from each segment (Tip, Middle, and Lower) of one of the two branches from each collection period were cut on a cryomicrotome. Sections were mounted on slides coated with poly-L-lysine and stored at 20C. Sections were viewed with a Leitz Aristoplan epifluores- cence microscope equipped with the F1TC filter (450-490- nm excitation) through which the tissue appeared green and orange and the latex beads bright yellow. Latex beads were counted in 10 sections chosen at random from each segment of the sponge for every time interval of collection. The location of the beads was scored as follows: i) in the tissue (i.e., in or near the flagellated chambers) of the sponge; ii) at the edges of water canals; and iii) in cell strands. Where beads were too numerous to count (for example in the pieces collected within the first 24 h after treatment), 100 beads were first counted, and estimates of every hundred beads visible thereafter were made. Selected sections were later stained in hematoxylin and eosin but not cleared in xylene, and then mounted in PBS-glycerol. This procedure allowed us to see the location of beads within the strands. Video microscopy Pieces of A. cunlifonnis were transferred to flow- through seawater tanks at the Bellairs Research Institute without removal from seawater. A 4-cm piece of a sponge branch was cut in two lengthwise with a shaip disposable scalpel. Where a strand was exposed without damage along a portion of its length, the piece of branch was tied with dental floss to a glass microscope slide with the cut surface against the slide. With the exposed strand facing upwards, the slide was rested on supports at either end in a How-through pertusion chamber so that the sponge did not touch the bottom of the dish. The pertusion cham- ber was set on the stage of a Zeiss compound microscope and the strands were filmed with a lOx objective lens for up to 5 h using a Panasonic CCD color video camera; the image was digitally enhanced with an Omnex real-time digital image processor (Imagen Corp.) and recorded on Fuji video tapes in real time with a Panasonic VCR. Pho- tographs were taken from the monitor of a Technitron television with a Nikon FG camera on TMAX 400 ASA Him. Bright field microscopy Pieces of A. cauliformis were cut in half lengthwise to expose the tissue strands and placed strand-side up, in a 5-cm diameter dish of seawater. on the stage of a Leitz Orthoplan microscope. The exposed surface was illumi- nated with two adjustable microscope lamps, and photo- graphs were taken on 100 ASA Fujicolor print film. Electron iiiu'roscop\ Tissue strands were dissected, with a scalpel and fine forceps, out of freshly collected sponge branches. Strands and whole pieces of sponge with exposed strands were fixed in a cocktail of cold 1% osmium tetroxide and 2% glutaraldehyde in 0.45 M sodium acetate buffer, pH 6.4, and 10% sucrose in the final volume. Pieces were kept in the refrigerator at 4C for 4 h, and the fixative was then changed. After 1 6 to 20 h, the fixative was poured off, and the tissue was rinsed in 0.45-/um Millipore-filtered seawater and stored in 70% ethanol for transport to the University of Victoria, British Columbia. Tissue strands and pieces of sponge were then further dehydrated to 100% ethanol, stained en Hoc in 0.5% uranyl acetate overnight at the 80% ethanol stage, rinsed three times in propylene oxide, infiltrated overnight in 1:1 Epon (Taab 812) and propylene oxide, and embedded in 100% Epon at 60C. Silver sections were cut on a Reichert ultramicrotome with a diamond knife, stained with lead citrate, and viewed in a Hitachi XOOO transmission electron microscope. H};iire 2. (a) Aplysina cauliformis attached to coral rubble, with plastic bags (arrowhead) tilled wilh latex beads attached to each branch. Latex beads were "fed" to the sponge branches in the plastic bags, and the tale of ingested materials was determined, (b-e) Live tissue strands of A. cauliformis. Bar: 1 mm. (f-h) Latex beads in longitudinal sections of preserved sponge branches. Bar: 50 //m. (b) A strand in a longitudinal section of a sponge. The strand (arrow) is darker than the adjacent tissue and divides to go around the spongin skeleton. The arrowhead indicates a piece of the broken spongin skeleton, (c) Two exposed strands (arrows) in a longitudinal section are similar in color to the tissue at the outer layer of the sponge, (d) A cross section of a sponge branch showing three strands (arrows), which are easily identified by their pink color and by their slight separation from the neighboring tissue. Polychaete worms (*) are an abundant macrosymbiont in these sponges and either use water canals or fashion their own pathways through the sponge, (e) A tissue strand can be lifted up with a dissecting needle, indicating the integrity and slight elasticity of the strands. ( f) Four hours after feeding, latex beads (arrowheads) were well distributed through- out the tissue but were not in the strands (s). (g) Two days after feeding, the beads (arrowheads) were predominantly in the tissues around the water canals (*): strands (s). (h) Eleven days after feeding, the beads (arrowheads) were only found in the tissue strands (s) of the tip segment of the branches. Very few were found in other tissues of the sponge at this time. SPONGE TRANSPORT PATHWAYS 33 Results Description of the strands The tissue of A. caitliformis contained longitudinal strands running from the base to the tip of the sponge (Fig. 2b). The strands were darker than the adjacent tissue. and were flecked here and there by white cells that ap- peared to be highly retractile. The strands were most similar in color to the tissue at the cortex (Fig. 2c) and the tissue at the tip of the sponge. Up to 12 strands could be identified in any one cross section of a sponge branch (Fig. 2d). Strands ranged in diameter from 20 to 100 pm. 34 S. P. LEYS AND H. M. REISW1G hut the diameter of any one strand was rather uniform for its entire length. Strands could be traced for at least 3 cm and often for more than 6 cm along a sponge branch. The strands tended to be straight, with only slight bends and turns where they coursed around the spongin skeleton (e.g.. Fig. 2b). and there were no cross-connections be- tween strands. Typically, strands branched or forked at the tip of the sponge, and if a strand was located along the cortex or edge of the sponge, a branch was often directed towards the cortex. The strands were sufficiently separate from the rest of the tissue in the sponge that they could be lifted by one end with forceps and gently pulled free from the sponge. The strands were slightly elastic, as can be seen by the ability of a dissecting needle to pull a portion of a strand away from the sponge tissue (Fig. 2e). However, more than a moderate pulling resulted in the breakage and slow curling of the excised ends. Occasionally, when they were pulled out of the sponge, the strands frayed at the edges and pieces at the edges were left behind. Other parts of the sponge lacked this integrity and could not be removed intact from the sponge. Examination of the live tissues of A. lacunosa, A. fistu- Uiris, and A. rigidu revealed tissue strands in all three species. Strands were always distinctly more pink than the surrounding tissue, up to 100/ym in diameter, and traceable for at least 3 cm. and often for the full length of the sponge. Sections 1-cm thick cut with a scalpel from top to bottom of these sponges revealed fewer strands at the base than at the middle or tip of the sponge, and none of the strands were especially close to the cortex. In one section of A. lacnnosu. 24 different strands were identi- fied. Most of the strands could be followed for 4 cm or more along the length of the sponge. Electron microscopy Ultrastructural examination revealed no clear barrier separating the tissue strands from the rest of the sponge tissue. Only in one instance was a string of connected cells 60-//m long found at the edge of the strand. More commonly the edges of strands were lined simply by collagen. Strands were identifiable in sections of the sponge tissue by the presence of an increased number of aligned elongate cells, vast numbers of bacteria, and a much denser collagenous mesohyl in which the collagen tibrils were often aligned in a single direction (Fig. 3A. B). Elongate cells (40-50-yum long and 2.5 I-/JITI wide) with numerous filopodia were separated laterally by about 10 fjm (Fig. 3A). These cells typically lacked conspicuous nucleoli and contained many small vesicles. Other cells in the strands (possibly spherulous cells) were stretched around large vesicles containing an electron-dense mate- rial (Fig. 3A). Along particularly dense bands of aligned collagen there were highly elongate cells with a well- developed cytoskeleton (Figs. 3B, 4A). Cells in the strand were often found in the process of phagocytosing bacteria (e.g.. Fig. 4B). The tissue outside of the strands consisted mostly of a comparatively loose collagenous mesohyl containing numerous bacteria, although fewer than in the strands; scattered amoeboid-like cells: and flagellated chambers. The sponge cortex consisted of many spherulous cells containing large inclusions, and some elongate cells em- bedded in collagen that was not aligned in any particular direction (Fig. 4C). Strands from other species of Aplysina were identical in ultrastructure to those in A. ccudiformis. Cells in strands that were preserved after they had been forced to recoil at the edges by cutting and tweezing appeared no different than those in strands that had been fixed while still within the sponge. In both cases, cells were elongate when lying beside aligned collagen fibrils and irregularly ovoid when not. Feeding and insertion of lute.x heads In the feeding experiment, the beads were first seen in the flagellated chambers and pinacoderm (Fig. 5a) and later in cells in the mesohyl (Figs. 5B and 20. Four hours after feeding, beads were found in all the sponge tissues of the middle segment except the cell strands (Fig. 2f). By 2 to 3 days after feeding the beads were concentrated in the tissues around the water canals (Fig. 2g). At this time only a few beads were found in the cell strands. However. 7 to 11 days after feeding, many beads were found in the tip segment, where they were specifically lodged in the cell strands (Fig. 2h); very few were found in adjacent tissues. Closer examination of selected stained sections showed that the beads were within the cells in these strands. At this level of resolution it appeared that the beads were in the elongate cells rather than in the spherulous cells, but the strands with beads were not ex- amined by electron microscopy to confirm this observa- tion. The relative proportion of beads in the tissue, at the edges of canals, and in the tissue strands over the course of 13 days after latex bead feeding and insertion is shown in Figures 6 and 7. In the feeding experiment (Figs. 6A and 7A) most latex beads in the middle segment were first found in the sponge tissues (triangles) and later at the edges of water canals (squares) during the first week after feeding. The number of beads in the middle segment declined during the second week after feeding. Although many beads were counted in the strands in the middle segment at day 2 (see Table I), these were far outnum- bered by those beads that were counted around the edges of the canals and in the tissue at this time. In the second week, 227c of the beads counted in all segments were in the strands in the tip segment 1 1 days after feeding (Fig. SPONGE TRANSPORT PATHWAYS 35 Figure 3. Longitudinal sections of tissue strands from ,4/>/vwmi um/i/c/n (transmission electron microscopy). (A) A section showing several elongate cells with numerous filopodia (arrows). These cells lie lengthwise throughout the strand within a bacteria-filled (b) collagenous mesohyl (co). Other cells in the strands contain large inclusions of electron-dense material (*). Bar: 10 pm. (B) Some cells in the tissue strands were highly elongate and were tightly aligned within dense bundles of collagen fibrils (arrows). Bar: 10 /jm. 6A: tip segment). However, 7 to 11 days after feeding, 95% of all the beads counted in the tip segment alone were in the strands (Fig. 7 A). Many beads were found around the water canals in the lower segment in the feed- ing experiment. A week after feeding, 63% of the beads counted in the lower segment were found in the strands (Fig. 6A: lower segment). Latex beads that were inserted into the sponge branches about 7 cm from the tip were transported predominantly upward to the tip 2 weeks after insertion (Fig. 6B: tip segment); very few beads reached the segment of the sponge below the insertion point. One day after their insertion, the beads were abundant in the general tissue of the sponge in the middle segment, much as was found in the feeding experiment. After 3 days a large proportion of the beads were found at the edges of the water canals, and a few (5%) were in the tissue strands in the middle segment. Whereas the number of beads counted in the 36 S. P. LEYS AND H. M. REISWIG ^. Figure -I. Aspects of the cell strands and the cortex of Aplyxinu cinilifonnix (transmission electron microscopy). (A) A higher magnification of cells among densely bundled collagen fibrils (co) in strands, showing bundles of electron-dense fibrils, which may be microtilaments ( arrowheads!, and of microtubules (arrows), in the cells. The niicrotubules were identified as such from their diameter in high magnification electron micrographs. Bar: 2 /jm. (B) An example of a cell within a strand in the process of engulfing a bacterium (b) Bar: 1 pm. (C) The cortex of the sponge. Just inside the dermal layer or pinacoderm (p) are numerous spherulous cells (sp) with large inclusions, and individual elongate cells (arrowheads) lying in a collagenous mesohyl (co). These elongate cells are far shorter than those in the strands and arc not in large tracts of aligned collagen fibrils. Bar: ? /jm. middle segments declined toward the end of the second week, their number in the tip segment increased at this time. After 7 days. 42% of the beads counted in the tip segment were in the strands: 13 days after feeding, how- ever, most beads in the tip segment were in the tissue (Fig. 7B). SPONGE TRANSPORT PATHWAYS 37 Figure 5. Latex bead uptake by ,4/j/v.w'm; fuiiliforniis (transmission electron microscopy). (A) Four hours after feeding, latex beads (Ib) were found in flagellated chambers (fc) and (B) in ameboid cells in the mesohyl. Bar (A): 5 pm: (B): 2 pm. Attempts to record movement of cells in the tissue strands of Aplysina were hampered by the opacity of the tissue strands; indeed, only cells at the edges of strands could be monitored by transmitted light microscopy. Con- sequently, strands that were exposed by cutting the sponge branch longitudinally were illuminated from above, and viewed at a low magnification with a 10X objective lens. The image was magnified by both the video camera and the digital image processor. With this system, highly refractile cells, which may correspond to the spherulous cells shown in Figure 3A, were the most conspicuous element in the strands; the movements of the smaller cells could not be monitored. The refractile cells showed no obvious movement as a group in either direc- tion along the strand. Nevertheless, more than 2 h after filming began on the sponge, several individual cells definitely moved along the strand in both directions at rates of 0.025 ^rn-s" 1 to0.04^m-s ' (/; = 8) (Fig. 8). Thus, the movement was probably not caused by contrac- tion of the strand after wounding. Discussion Endosomal tissue strands have not been described in sponges other than the genus Aplysina. nor is there any record that a specialized structure is involved in the distri- bution of particulates in sponges. We have shown here, however, that when latex beads are fed to A, cauliformis, they are taken up and transported into the tissue strands; eventually they end up at the tip of the sponge or further down the stalk. Sponges are well-known to have highly motile cells, whose rate of movement (2 to 21 mm -day"'; Bond. 1992) although slower than that of crawling by Amoeba is comparable to. and even faster than crawling by fibroblasts and neutrophils (Bray. 1992). Furthermore, at the growing edge of sponges, these cells (particularly those of a similar type) often become aligned in tracts (Bond and Harris, 1988). Similar tracts of cells, some- times referred to as cell "cords," have been described in various demosponges where their suggested role has been in growth (Brien, 1976), regeneration (Levi, 1960), and remodeling (Diaz, 1979). In each of these cases, the pri- mary role of the cell cords was thought to be in skeleto- genesis (Simpson, 1984), as demonstrated by Teragawa ( 1986) for the keratose sponge Dysidea etheria. However, none of these tracts or cords of cells is so permanent a structure within the sponge, nor so widespread and mor- phologically uniform within a genus, as the endosomal tissue strands in Aplvsina. Nor can any of those tracts be so readily extracted from the rest of the tissue as can Aplvsina' s tissue strands. The bead uptake experiments here show that the strands are involved in transport of materials taken in during feeding. Most of the beads that were fed or inserted into the sponges were excreted within a week; relatively few ended up in the strands, which suggests that most of the food taken in by flagellated chambers and the adjacent pinacoderm is probably distributed to cells locally, and wastes are probably expelled from the same area. How- ever, a role for the strands as transport pathways is sup- ported by the observation that a substantial proportion of beads were in the strands in the tip segment 2 weeks after 38 S. P. LEYS AND H. M. REISWIG 100 Tip 80 * Tissue 60 o Canal edge 40 * Strand 20 X^ -^- c9. . -S - -(. - - - I 2 3 7 11 Time after feeding (Days) 100 80 60 40 20 Tip 1 2 3 7 11 Time after bead insertion (Days) ro c 4hrs 0) a. 100 80 60 40 20 'U 8 0) CL 100 80 60 40 20 100 80 60 40 20 Middle Figure 6. Latex head experiments. Percent of the total number of beads counted in all sections of all three segments (Tip. Middle, and Lower) at each time interval, in the tissue (triangles), at the canal edges (squares), and in the strands (crosses): (A) feeding; (B) insertion. See text for explanation. the middle portion of the sponge was ted. The beads found in the strands 2 days after the sponges were fed were far outnumbered by the beads counted around the B T3 0) ro 0) .o O t: o I 100 80 60 40 20 100 80 60 40 20 *- Tissue o Canal edge "Strand 1 2 3 7 11 13 Time after feeding (Days) 1 2 3 7 11 13 Time after bead insertion (Days) Figure 7. Latex bead experiments. Percent of all the beads counted in the tip segments only: (A) feeding: (B) insertion. See text for explanation. canal edges or in the tissue (see Table I). The delayed appearance of the beads in the tip segment 7 days after feeding is best explained by the time required for the cells transporting the beads to crawl first to the strands and then along the strands to the tip, where they accumulated. That downward transport also occurs is suggested by the number of beads found in strands in the lower segments a week after feeding, and later around the water canals. The beads found around the canal edges in the lower segment may have been transported down and then moved to the canals for excretion, or they may have been rein- gested after excretion from the middle section. Aplysiiui fiilvu has been shown to grow rapidly, at an average rate of 2 cm month ' (minimum 0.2 cm month"', maximum 15 cm month '; calculated from Wulff, 1990). For this rate of growth, rapid translocation of materials to the tip would be necessary despite the ability of flagellated chambers at the tip to take in and distribute nutrients locally. Downward transport might supply regions of the sponge that possibly feed less and function primarily as a stalk. Video microscopy showed that individual cells moved within strands for distances of 30 pm or more at a rate of 2.2 to 3.5 mm day '. At this rate of movement, material could be transported 5 cm in 2 weeks, if trans- ported more or less in a straight line. The large numbers of bacteria in the strand might support the energy require- SPONGE TRANSPORT PATHWAYS 39 Table I The number of latex beads counted in the tissues, around the water canals, and in the strands of each segment of fed branches of Aplysina cauliformis Number of beads Collection period Segmenf 1 In each segment region h Total per segment Total per day (all segments) Percent of beads in each segment/day Tissue Canal edge Strand day 1 , 4 h Middle 20.000 9,400 7 29.407 29.407 day 1, 24 h Lower 572 3,765 20 4,357 9.792 44.5 Middle 3,348 1,060 6 4,414 45.1 Tip 876 144 1 1 .02 1 10.4 day 2 Lower 5,116 19.400 65 24,581 106.472 23.1 Middle 23.400 49.950 548 73.898 69.4 Tip 1.554 6.366 73 7.993 7.5 day 3 Lower 2.890 5.219 48 8.157 1 1 .536 70.7 Middle 319 3.042 (1 3.361 29.1 Tip 14 3 1 18 0.2 day 7 Lower 78 355 766 1.199 18.554 6.5 Middle 3,806 12.753 346 16,905 91.1 Tip 12 7 431 450 2.4 day 1 1 Lower 1,090 12,779 290 14,159 30.698 46.1 Middle 2,843 3,914 2.212 8.969 29.2 Tip 535 37 6,998 7.570 24.7 day 13 Lower 1,148 3,617 39 4.804 6.394 75.1 Middle 247 1,274 30 1 .55 1 24.3 Tip 5 5 29 39 0.6 J Lower. Middle, and Tip segments of each sponge branch fed latex beads. h Beads were counted in 10 sections from each segment of sponge (Lower. Middle. Tip) each day. and were noted as being in three regions of the segment: Tissue (flagellated chambers and associated tissues); Canal edge (cells specifically around the edges of water canals); and Strand (only within cell strands). ments of so many moving cells, and this notion is substan- tiated by the ample phagocytosis of bacteria seen in cells in the tissue strands. The presence of tissue strands in tube- and stick-forms of Aplysina suggests that the mechanism of transport is efficient for growth regardless of sponge form, and indeed has been maintained by at least four species in this genus. Whether more distantly related verongiid sponges also possess tissue strands or similar structures for nutrient transport or other functions is not known. Feeding in sponges has been well-documented, and with the exception of the two examples cited in the intro- duction, the Cladorhizidae and the Hexactinellida, particle uptake in sponges occurs at the choanocytes in the flagel- lated chambers or at the pinacoderm-lined incurrent ca- nals. Food is transferred via food vacuoles from the choa- nocytes to amebocytes. which deliver the nutrients locally to other cells, and wastes are excreted via the excurrent canals (Kilian, 1952; Weissenfels, 1976; Willenz, 1980; Imsieke, 1994). Directional translocation of cells and nu- trients occurs in gemmule formation (Rasmont and de Vos, 1974) and during oogenesis (Fell, 1983), but this transport occurs over very short distances. Hexactinellid sponges possess perhaps the longest transport pathway known in the Porifera, but translocation is intrasyncytial and occurs through a highly dynamic three-dimensional network of reticulated tissue, rather than along fixed path- ways (Wyeth et al.. 1996; Leys, 1998). Although sponges have not previously been shown to possess specific transport pathways for nutrients, all in- vertebrates have developed means of distributing nutri- ents. Many higher invertebrates such as ascidians. most echinoderms. and crustaceans have a well-developed fluid circulatory system with numerous types of hemocytes which, in addition to their many other functions, transport nutrients. Others invertebrates, however, move food around in a much slower manner: branching corals trans- locate nutrients to the tip of branches for tip growth (Buchsbaum Pearse and Muscatine, 1971); gorgonians have cells that travel through the stem canals and solenia. a collagenous filled mesohyl, to deliver nutrients to the other tissues of the animal (Murdoch, 1978); fixed t 40 S. P. LEYS AND H. M. REISWIG Figure 8. Cell movement in tissue strands (video microscopy). A highly retractile cell (arrowheads) was traced as it moved for 3(1 ^m along a tissue strand at 0.033 /ynvs' 1 . Time of frames: (A) (I min: (B) 5 min; (C) 15 min. Bar: 100 ^m. chymal cells in turbellarians are thought to act as a sort of stationary intraccllular circulatory system (Pedersen, 1961); and acid phosphatase staining has shown that amehocytes in the hemal lacuna of crinoids (again, a col- lagen-filled pathway) are involved in digestion and trans- port of nutrients (Hein/eller and Welsch, 1997). We cannot rule out a role for the tissue strands in contracting the ostia and water canals to control water flow as suggested for the elongate cells in Verongici ( Va- celet, 1966): nonetheless, there is little evidence to sup- port this hypothesis. Although some cells in the strands have what appears to be a highly developed actin cy- toskeleton, and the strands recoiled gently after being cut, none of the strands were wrapped around the water canals or ostia sufficiently to be able to reduce water flow if contracted. Furthermore, localized patches of elongate cells were present in valve- or sphincter-like structures around the water canal system, and these quite possibly function in controlling water flow through the sponge as suggested by Vacelet (1966). and by Reiswig (1971) for those sponges in which flow is not controlled by flagellar activity. The facts that video microscopy did not reveal all cells in the strands to be moving, and that not all cells in the strands are elongate, could also be taken as evidence against the transport hypothesis. But the thickness ( 100 jjm} of the strands allowed only a few cells at the edges of strands to be observed at high magnification by video microscopy. Despite the low magnification and use of epi-illumination. a few of the highly refractile cells could be clearly seen to travel for significant distances along the length of the strand. It could also be true, however, that not all the cells in the strand transport material. Thin sections of the strands showed that only some cells in the strands had a highly developed actin cytoskeleton and were aligned along densely bundled collagen fibrils. Other cells were clearly elongate with filopodia at either end, but were not associ- ated with the densely bundled collagen fibrils. Fibroblasts in culture orient themselves along grooves in culture dishes (Carter. 1967; Clark ct uL, 1980). and on collagen substrates they take on a bipolar spindle morphology with filopodia at either end (Elsdale and Bard. 1972). Further- more, Harris ( 1987) has demonstrated that the forces ex- erted by fibroblasts in culture are far greater than is neces- sary for cell crawling, and that the prime function of these cells is to bundle and align collagen to prepare the path for migratory cells (Stopak and Harris, 1982). An analogous situation could exist in Aplysina strands. We suggest that the role of the highly elongate cells with a well-developed actin cytoskeleton is primarily to bundle and align the collagen fibrils, creating a pathway that cells transporting nutrients can recognize and follow, thereby allowing the rapid transport of materials either to the tip or to the base of the sponge (Fig. 9). In this fashion, apoplastic nutrient transport by ameboid cells has become specialized in the genus Aplysina to the extent that these sponges have a differentiated structure that parallels fluid transport systems in other animals, but is a typically novel poriferan solution. Acknowledgments We thank the director and staff at the Bellairs Research Institute (BRI) of McGill University, in Barbados, for the use of facilities while we were conducting the field work SPONGE TRANSPORT PATHWAYS 41 Figure 9. Diagram of the proposed route taken by latex beads (Ib) that were fed to Aplysina caii/iformis. Beads are taken in via the incurrent canals (ic) to the flagellated chambers (fc), where they are ingested (1) by choanocytes. The beads are then transferred to wandering amoebocytic cells (2), which encounter strands of elongate cells (ec) and highly bundled collagen fibrils (co). The amoebocytic cells follow the direction of the collagen, thereby transporting the latex beads up and down the strand (3) as shown by the arrows. for this study. We also thank L. Verhegge. M. Tsurumi, and G. O. Mackie for comments on the manuscript. This research was supported by a Commander C. Bellairs Post- doctoral Fellowship from BRI and McGill University to SPL, and by a research grant from the Natural Science and Research Council of Canada (OGPOO 1427) to G.O.M. Literature Cited Bergquist, P. R., P. Karuso, R. C. Cambie, and D. J. Smith. 1991. Sterol composition and classification of the Porifera. Biocliem. Sysl. Ei-1000 Da. At the end of dialysis, concentrations of total Ca and free Ca were measured on samples withdrawn from inside the dialysis bags (dialyz- able + nondialyzable Ca) and from the external solution (dialyzable Ca). Nondialyzable Ca concentration was cal- culated by subtraction. Calcium speciation modeling Results from dialysis experiments were compared to theoretical estimates of Ca speciation by using the equilib- rium speciation model MINTEQA2/PRODEFA2 (Allison et al.. 1991). Values for the molar concentration of the major inorganic ions (Na + , K + , Ca 2+ , Mg 2+ , Cl. SO 4 2 , and total CO : ) in blood and EPF of M. mercenaria were obtained from Crenshaw ( 1972a). Since blood SO 4 : ~ and CO 2 concentrations were not available, the corresponding EPF values (Crenshaw, 1972a) were substituted as an approximation. The total CO 2 molarity provided by Cren- shaw (1972a) was entered in the model as CO 3 2 . MINTEQA2 was run using various permutations of the given concentrations to understand more clearly changes in Ca speciation with changing blood and EPF composi- tion. This included adding a hypothetical Ca-binding pro- tein to examine organic Ca speciation. The MINTEQA2 model provided percentage distributions of free, inor- ganic, and organic Ca species formed under the given conditions. Electrophoresis Plasma and EPF were subjected to discontinuous SDS- polyacrylamide gel electrophoresis (SDS-PAGE) using the method of Laemmli (1970), with a 3% stacking and 7.5% resolving gel. Protein concentrations in the samples [plasma = 1.34 0.15 ing/ml, n = 5; EPF = 0.97 0.17 mg/ml, n = 5; determined by Bradford's (1976) dye binding method] were adjusted by dilution such that the amount of protein applied per lane was 40-60 ^g. Stack- ing (100 V d.c.; 2 h) and resolving (200 V d.c.; 4 h) were carried out at 15C. Gels were stained either with 0.1% Coomassie brilliant blue R-250 (Hames and Rickwood, 1981) or with a periodic acid-silver stain for glycopro- teins, as described by Dubray and Bezard (1982). Protein molecular weights were determined by comparison with a standard mixture containing myosin (205 kDa). /3-galac- tosidase (116 kDa), phosphorylase B (97.4 kDa), BSA (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). Results Quahogs injected with 45 Ca :+ and 'H-tyrosine showed a progressive decrease in radioactive counts in the right shell EPF, the site of injection (Fig. la, b). Concentrations of both materials decreased 3 orders of magnitude over a period of 4 to 4.5 h. Samples of blood from the anterior and posterior adductor muscles and of EPF from the left shell exhibited a corresponding increase in '"Ca 2 ^ and 'H- tyrosine counts. Counts from the four sampling points converged at approximately 3 h, indicating complete mix- ing of the introduced radiolabel with the blood. The high molecular weight BSA, on the other hand, showed less tendency to redistribute into the circulatory system (Fig. Ic). Although a decline in BSA concentration was ob- served in the right EPF, a difference of 3 orders of magni- tude remained between counts at the point of injection and at the three other sampling sites at 4 h post-injection. 46 P. S. NAIR AND W. E. ROBINSON l&'+Vt = 1000000 | \ a re O .A 100000 i ~~ ' .--_ Tt a 10000 i 1000 ; ^ 100 i 1 n ( 112345 1UUUUU : >, b 0) 10000 i \ '3> ^"-~-. , 1000 i ' ~"T\^1^ =r-5 ^~ ro 100 i 10 i ? 1 ( 112345 1UUOUU 5 ^ C < 10000 '~' s m 1000 a 100 10 ._... A. I2H" y "v time (h) Figure 1. Radioactivity (cpm) in Mercenaria mercenaria extrapal- lial fluid (EPF) and blood following injection of (a) 45 Ca (MW = 40), (b) 3 H-tyrosine (MW = 181), and (c) I4 C-BSA (MW = 66 kDa) into the right shell EPF. Extrapallial fluid samples were drawn from the central zone of the right ( O ) and left ( ) shells; blood samples were drawn from the anterior (. . .A. . .) and posterior (----) adductor muscles. Lines were drawn by a logarithmic-fit function using SlideWrite Plus* V4.0. Furthermore, I4 C-BSA concentration in the left shell EPF appeared to remain constant over the 4-h period. The time required for the introduced BSA to become homogeneous with the blood was estimated to be at least 22 h. A confirmatory experiment using nonlabeled BSA was performed to determine whether the BSA remained solu- bilized in EPF or became adsorbed to the mantle tissue during the course of the experiment. SDS-polyacrylamide gels of EPF and blood plasma showed that the 66-kDa band of BSA was present only in the right shell EPF (the point of introduction) in samples taken 3 h and 6 h post- injection (results not shown). Staining intensities of the bands at 3 and 6 h were similar, indicating that the BSA was not bound to tissues. If BSA was present in the left shell EPF and in the plasma samples at 3 and 6 h. it was below the detection limits of SDS-PAGE. The shapes of the semilog plots in Figure 1 indicate a rapid decrease in injected counts during the first 30 min, followed by a longer phase of gradual decline and attain- ment of an apparent steady state over the next 4-4.5 h. This suggests that the release of radiolabel from the EPF may be biphasic. Apparent depuration constants (kjs) were calculated from the slopes of tangents to the initial and final portions of the plots. Depuration constants for the rapid phase (k d ,) were estimated as 9.7, 12.0, and 2.4 h~' for 45 Ca, 3 H-tyrosine. and I4 C-BSA. respectively, whereas constants for the slow phase (kj : ) were 0.1, 0.08, and 0.04 h"'. Biological half-lives calculated from the kj : values were 6.9 h for 45 Ca, 8.7 h for 'H-tyrosine. and 17.3 h for I4 C-BSA. The mean concentration of total Ca measured in the blood plasma of 10 quahogs was 10.9 1.3 mM. of which 0.4 0.1 mM was free Ca :+ . Bound Ca was estimated as 10.5 1.2 mM. Equilibrium dialysis of the plasma sam- ples revealed that the majority of bound Ca (7.2 mM; Table I) was nondialyzable (i.e., bound to ligands >1000 Da), but 1.1 mM was dialyzable. and 0.2 mM was free Ca. These concentrations reflect a 78% recovery of total Ca following dialysis; the remainder was presumably bound to the walls of the dialysis bags. Expressed as percentage of total Ca recovered, bound Ca (nondialyz- able and dialyzable) and free Ca :+ amounted to 84.7, 12.9, and 2.4%, respectively (Table I). Inorganic Ca speciation obtained by MINTEQA2 pre- dicted that a high proportion of plasma Ca should be present as free Ca 2+ (78.9%; Table II, permutation 1), and the remainder as bound Ca (primarily CaSO 4 , 20.2%). However, this prediction is inconsistent with the values of 2.4% free Ca 2+ and 12.9% bound-dialyzable Ca ob- tained by the dialysis experiments (Table I). If the SO 4 : concentration used in model permutation 1 (46.1 mM, Crenshaw, 1972a) was replaced by the seawater SO 4 : concentration (28.2 mM, Mantoura et 50 kDa) that were not detected in the plasma, indicating that the outer mantle epithelium contains an effective barrier to at least some high molecular weight proteins. These ob- servations are consistent with those of Bielefeld et al. (1993), who found the outer mantle epithelium of the Table II Theoretical speciation of calcium in Mercenaria mercenaria blood plasma and extrapallial fluid Model permutation* Sample CaSO 4 CaHCO, CaOIT Ca-Protein 1 Plasma 78.9 20.2 <0.9 trace EPF 85.9 19.3 <0.9 trace 2 Plasma 85.9 13.1 <0.9 trace EPF 86.5 12.5 <0.9 trace 3 Plasma 2.3 1.0 96.7 EPF 2.2 0.4 96.9 Note: Values expressed as percentage of total calcium. Results were obtained using the equilibrium speciation model MINTEQA2 (Allison et al.. 1991). * Premutations: (1) concentrations of inorganic ions taken from Crenshaw (1972a): (2) sulfate concentration decreased from 46.1 to 28.2 mM', (3) 60 mM of hypothetical Ca-binding protein (15:1 Caiprotein) with log,,, K a of 2.1 AT' added. 48 P. S. NAIR AND W. E. ROBINSON 100 100 mM protein Figure 2. Percentage distribution of free Ca (ionic Ca :+ ) in the blood plasma of Mercenaria mercenaria in the presence of a hypotheti- cal Ca-binding protein with 15 Ca-binding sites. The lines represent a range of binding affinity constants tested (log,,, K a = 2-6 A/~'). Data were generated using the equilibrium speciation model MINTEQA2 (Allison et a/.. 1991). snail Biomphalaria gluhrutii impermeable to injected horseradish peroxidase (MW = 40 kDa). An important factor governing the direction of Ca flux across the outer mantle epithelium is anaerobiosis. An- aerobiosis induced upon shell closure is accompanied by redissolution of CaCO, from the shell to maintain a more uniform plasma pH through the bicarbonate buffering sys- tem (Crenshaw and Neff, 1969; Hudson, 1992; Littlewood and Young, 1994). This leads to a net Ca flux away from the central zone EPF. On the other hand, injury to the shell causes remobilization of Ca into the EPF. Calcium for shell repair is derived predominantly from the calcium cells of the mantle and foot (see Watabe, 1983), by disso- lution of CaCOj and Ca,(PO 4 ) : spherules. Because the quahogs in our experiments had damaged shells and were kept out of water for as long as 4.5 h, the 3-h timeframe for Ca exchange in this study probably reflected opposing Ca fluxes resulting from anaerobiosis and attempts at shell repair. Other studies have found similar timeframes for Ca exchange in bivalves. 4S Ca introduced in the seawater took 2 h to reach a steady state with the mantle in Argopecten irmdians (Wheeler el inica. Mm: Biol. 114: 423-428. Dubray, G., and G. Bezard. 1982. A highly sensitive periodic acid- silver slam for 1 ,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal. Blot-hem. 199: 325-329. Falini, G., S. Albeck, S. Weiner, and L. Addadi. 1996. Control of aragonite orcalcite polymorphism by mollusc shell macromolecules. Science 271: 67-69. Feher, J. J., C. S. Fullmer, and R. H. Wasserman. 1992. Role of facilitated diffusion of calcium by culhindin in intestinal calcium absorption. Am. ./. Physioi. 262: C517-C526. Graf, F., and J. C. Meyran. 1983. Prcmolt calcium secretion in mid- gut posterior ceaca of the crustacean Orchestin: ultrastructure of the epithelium. J. Morphol. 177: 1-23. Greenaway, P. 1971. Calcium regulation in the freshwater mollusc Limmieii staginilis (L.) (Gastropoda: Pulmonata). II. Calcium move- ments between internal calcium compartments. J. Exp. Biol. 54: 609-620. Hames, B. I)., and I). Rickwood. 1981. Gel Electrophoresis of Pro- teins: A Pniclictil Approach. IRL Press, Oxford. 290 pp. Hudson, R. I,. 1992. Ion transport by the isolated mantle epithelium ol i he freshwater clam. Unio coiiiplimtiis. Am. ./. Physioi 263: R76- R83. Jones, R. G., and W. I,. Davis. 1982. Calcium-containing lysosomes in the outer mantle epithelial cells of Ainbleimi. a fresh-water mol- lusc. Anal. Rec. 203: 337-343. Karbach, U. 1992. Paracellular calcium transport across the small intestine. J. Nun: 122: 672-677. Kawaguchi, T., and N. Watabe. 1993. The organic matrices of the shell of the American oyster Crassostrea vi>\t>inicti Gmelin. J. Exp. Mar. Biol. Ecol. 170: 11-28. Keith, J., S. Stockwell, D. Ball, K. Remillard, D. Kaplan, T. Thann- hauser, and R. Sherwood. 1993. Comparative analysis of macro- molecules in mollusc shells. Comp. Biochem. Phvsiol. 105B: 487- 496. Kessler, G., and M. Wolfman. 1964. An automated procedure for the simultaneous determination of calcium and phosphorous. Clin. Chem. 10: 686-687. Laemmli, U. K. 1970. Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature 227: 680-685. IJttlewood, D. T. J., and R. E. Young. 1994. The effect of air-gaping behaviour on extrapallial fluid pH in the tropical oyster Crassostrea rhizophorae. Comp. Biochem. Physioi. 107A: 1-6. Lucu, C. 1994. Calcium transport across isolated gill epithelium of Ciin-iiuis. J. Exp. Zoo/. 268: 339-346. Mantoura, R. F. C., A. Dickson, and J. P. Riley. 1978. The com- plexation ot metals with humic materials in natural waters. Estuarine Coastal Mar. Sci. 6: 387-408. Marsh, M. E., and R. L. Sass. 1983. Calcium-binding phosphopro- tem particles in the extrapallial fluid and innermost lamella of clams. 7. Exp. Zoo/. 226: 193-203. May, P. M., P. W. Linder, and D. R. Williams. 1977. Computer simulation of metal-ion equilibria in biofluids: models for the low- molecular-weight complex distribution of calcium (II), magnesium (II), manganese (II), iron (II), copper (II), zinc (II), and lead (II) ions in human blood plasma. ./. Chem. Soc: Da/ton Trans. 6: 588- 595. Meenakshi, V. R., P. L. Blackwelder, P. E. Hare, K. M. Wilbur, and N. Watabe. 1975. Studies on shell regeneration I. Matrix and mineral composition ot the normal and regenerated shell of Pomacea pahtdosa. Comp. Biochem. Phvsiol. SOA: 347-351. Misogianes, M. J., and N. D. Chasteen. 1979. Extrapallial fluid: a chemical and spectral characterization of the extrapallial fluid of Mytilus eilulis. Anal. Biochem. 100: 324-334. Miyamoto, H., T. Miyashita, M. Okushima, S. Nakano, T. Morita, and A. Matsushiro. 1996. A carbonic anhydrase from the nacre- ous layer in oyster pearls. Proc. Natl. Acad. Sci. USA 93: 9657- 9660. Neff, J. M. 1972. Ultrastructure of the outer epithelium of the mantle in the clam Mercenaria mercenaria in relation to calcification of the shell. Tissue Cell 4: 591-600. Putnam, F. W. 1975. The Plasma Proteins: Structure. Function, and Genetic Control. Vol. 2. Academic Press, New York. 423 pp. Robinson, W. E., and D. K. Ryan. 1988. Transport of cadmium and other metals in the blood of the bivalve mollusc Mercenaria merce- naria. Mar. Binl. 97: 101 - 109. Saha, A., T. K. Jana, and A. Choudhury. 1988. The extrapallial fluid of Macoma hirminica: an environment for calcium carbonate deposition. Philipp. ./. Sci. 117: 395-399. Saleuddin, A. S. M., and S. C. Kunigelis. 1984. Neuroendocrine con- trol mechanisms in shell formation. Am. Zoo/. 24: 91 1-916. Samata, T. 1990. Ca-binding glycoproteins in molluscan shells with different types of ultrastructure. Veliger 33: 190-201. Scott, B. J., and A. R. Bradwell. 1983. Identification of the serum binding proteins for iron, zinc, cadmium, nickel and calcium. Clin. Chem. 29: 629-633. Sorenson, A. K., D. S. Wood, and L. B. Kirschner. 1980. Electro- physiological properties of resting secretory membranes of lamelli- branch mantles. ./. Gen. I'hvsiol. 75: 21-37. Timmermans, L. P. 1973. Mantle activity following shell injury to the pond snail, Lymnaea stagnulis L. Malaco/ogia 14: 53-61. CALCIUM IN QUAHOG BLOOD AND EPF 51 Tripp, M. R., L. A. Bisignani, and M. T. Kenny. 1966. Oyster amoebocytes in vitro. J. Invert. Path. 8: 137-140. Wada, K., and T. Fujinuki. 1976. Biomineralization in bivalve mol- luscs with emphasis on the chemical composition of the extrapallial fluid. Pp. 175-190 in The Mechanisms of Mineralization in the Invertebrates and Plants. N. Watabe and K. M. Wilbur, eds. Univer- sity of South Carolina Press, Columbia. Watabe, N. 1983. Shell repair. Pp. 289-316 in The Mollusca. vol. 4, A. S. M. Saleuddin and K. M. Wilbur, eds. Academic Press, New York. Weiner, S. 1984. Organization of organic matrix components in min- eralized tissues. Am. Zoo/. 24: 945-951. Wheeler, A. P., and E. W. Harrison. 1982. Subcellular localization and characterization of HCOr-ATPase from the mantle of the fresh- water clam. Anodonta cataracta. Comp. Biochem. Physio/. 71B: 629-636. Wheeler, A. P., and C. S. Sikes. 1984. Regulation of carbonate calci- fication by organic matrix. Am. Zoo/. 24: 933-944. Wheeler, A. P., P. L. Blackwelder, and K. M. Wilbur. 1975. Shell growth in the scallop Argopecteii irnulians. I: Isotope incorporation with reference to diurnal growth. Biol. Hi///. 148: 472-482. Wheeler, A. P., K. W. Rusenko, D. M. Swift, and C. S. Sikes. 1988. Regulation of in vitm and in vivo CaCO 1 crystallization by fractions of oyster shell organic matrix. Mar. Biol. 98: 71-80. Wilbur, K. M. 1972. Shell formation in molluscs. Pp. 103-145 in Chemical Zoologv, vol. VII, M. Florkin and B. T. Scheer, eds. Aca- demic Press. New York. Wilbur, K. M., and A. M. Bernhardt. 1984. Effects of amino acids, magnesium, and molluscan extrapallial fluid on crystallization of calcium carbonate: i;i vitro experiments. Biol. Bull. 166: 251-259. Wilbur, K. M., and A. S. M. Saleuddin. 1983. Shell formation. Pp. 235-287 in The Molliixca. vol. 4, A. S. M. Saleuddin and K. M. Wilbur, eds. Academic Press, New York. Ziegler, A. 1996. Ultrastructural evidence for transepithelial calcium transport in the anterior sternal epithelium of the terrestrial isopod Porcellio scaber (Crustacea) during the formation and resorption of CaCO, deposits. Cell Tissue Res. 284: 459-466. Reference: Biol. Bull. 195: 52-59. (August. 1998) Induction of Extra Claws on the Chelipeds of a Crayfish, Procambarm clarkii ISAMU NAKATANI 1 *. YOSHINORI OKADA 2 . AND TAKUJI KITAHARA 1 1 Department of Biology. Faculty of Science. Yamagata University. Yainagata 990-8560, Japan; ~ Department of Biology, Faculty of Science. Okayama University, Okayama 700-8530, Japan Abstract. In a crayfish, Procambarus clarkii, growth of an extra claw was induced by making a V-shaped wound in the proximal end of the propodus of the second and third chelipeds. Two nerve bundles were damaged by the wounding. Some type of extra growth developed on the propodi of 13 of the damaged chelipeds: a pair of extra claws (2 chelipeds). a pair of extra dactyls (1 cheliped), a single extra dactyl (2 chelipeds), and a single slight projection (8 chelipeds). The extra claws and dactyls de- veloped from the peripheral side of the propodus away from the wound site. One of a pair of extra dactyls and the single extra dactyls could be moved only slightly, either manually or by the crayfish. The other extra dactyls could be moved by the crayfish. Muscles were associated with each of the extra and primary claws. The muscles attached to the double extra claw or dactyl were inner- vated by nerve bundles that were branches from the pri- mary thick nerve bundles. One possible explanation for these findings is that the severed nerve fibers in the thick nerve bundles regenerate, elongate into aberrant roots, and form extra claws or dactyls. Introduction The occurrence of double claws has been observed in the past. They have been reported to occur naturally on the cheliped of the American lobster, Homants ameri- canus, from the primary propodus near the dactyl (Faxon, 1881 ) and from the base of the primary propodus (Cole, 1910). In the crayfish. Procciinhunts clarkii, extra claws have been reported to occur naturally on the first and third chelipeds (Nakatani et a!., 1997). Both of these extra Received 1 August 1997; accepted 7 May 1998. * To whom correspondence should be addressed. E-mail: nakatani@ sci.kj.yamagata-u.ac.jp claws developed from the base of the primary propodus. These crayfish were not able to move the dactyls of the extra claws, although they could be moved manually. Nerve bundles were observed to branch from the primary thick nerve bundles into the extra claw on the third cheli- ped (Nakatani et ai, 1997). Lateral outgrowths on the first cheliped of crayfish can be induced by wounding. In one study (Murayama et al., 1994), outgrowths developed in about \0 c / f of wounded chelipeds. This percentage was increased to 61.9% by increasing the width and depth of the wound and remov- ing the tissue in the wounded area (Nakatani. 1996). Many of these outgrowths have serrations and hooks. However, there is no articulation between the primary propodus and the new structure. These structures, hereafter called "lateral outgrowths." may be distinguished from an extra claw or extra dactyl by the absence of an articulation. The objectives of the present study were to induce the growth of extra claws and to investigate the degree of movement and innervation of the extra growth. Materials and Methods The experimental crayfish Crayfish (Procambarus clarkii) of both sexes were col- lected, without regard to molting stage, from ponds in the suburbs of Yamagata City, Yamagata, Japan. The length of the carapace varied from 15.2 to 36.6 mm. We used both intact and eyestalk-ectomized specimens because eyestalk removal promotes growth and decreases molt interval (Fingerman and Fingerman, 1974; Nakatani and Otsu, 1979; 1981; Suzuki, 1980). Wounds The dorsal side of the base of the propodus of the second and third chelipeds was wounded using small scis- 52 EXTRA CLAW ON CHELIPED OF CRAYFISH 53 sors. The V-shaped incision was made to a depth of about half the width of the base of the propodus (Fig. 1 ). The ventro-median tissues were destroyed by inserting a nee- dle ( 1 mm in diameter) into the wound. In 34 crayfish, a total of 134 chelipeds were wounded. Both eyestalks of five of the crayfish were removed from the base 10 to 20 days post-operation. Rearing wounded crayfish From September 1996 to March 1997, the wounded crayfish were reared until they had molted two or three times. The animals were kept separately in individual containers (300 X 240 X 105 mm) under a natural photo- period at room temperature (18-26C). Prawn pellets (Super B; Nihon Nosan Industry, Japan) and dry persim- mon leaves were placed in the rearing containers so that the crayfish could feed on them ad libitum. Sensory neuron responses A cheliped with extra dactyls was cut off at the base of the merus and held on a glass slide by rubber bands. The exoskeleton at the base of the merus was removed and the nerve bundles were exposed. To detect responses from the sensory neurons, the point of a needle was used to push the extra dactyls in a closing direction. Action potentials from the exposed nerve bundles were recorded extracellularly, using suction electrodes. Mechanical force exerted by closure of dactyls Measurements were taken on the force exerted by clos- ing the dactyls. The cheliped with double extra dactyls was cut off at the base of the merus and prepared as described above. Nerve bundles were stimulated using a pair of tungsten hook electrodes. Repetitive pulses (single pulse, 10 ms in duration; 20 Hz) were applied for 0.5-2 s to hook electrodes connected to an electronic stimulator (SEN1 101; Nihon Kohden, Japan). The mechanical force exerted by closure of each of the two dactyls was recorded V-shaped wound Dactyl Carpus Propodus Pollex Figure 1. Diagram of the second cheliped of the crayfish showing V-shaped wound at the base of the propodus. simultaneously with a thermal array recorder (TRA1 100; Nihon Kohden) via a high-gain force-displacement trans- ducer (SB-1T-H; Nihon Kohden) coupled mechanically to the tip of the dactyl by a silk thread filament. The sensory nerve responses and mechanical force were recorded at room temperature (23-28C). Obsen>ations on nen'e bundles and muscles Observations were made on the nerve bundles and mus- cles of the propodus. The exoskeletons of the propodus and carpus of the above-mentioned chelipeds were re- moved, and nerve bundles and muscles were exposed and placed in physiological solution for crustaceans (van Harreveld, 1936). The nerve bundles were stained with a 0.005% solution of methylene blue dissolved in physio- logical solution and were observed under a dissecting microscope. The stained chelipeds were then fixed over- night at 0C in a 20% formalin solution acidified to pH 3.8 with an acetate buffer. The muscles were observed with a dissecting microscope and a polarizing light micro- scope (BHSP. Olympus, Japan). Results Development of extra structures Data on extra structure development are shown in Table I. Most of the chelipeds healed normally, but 13 (9.7%) formed extra structures. A pair of claws, a pair of dactyls, and single dactyls developed from the side away from the wound, and single, slight projections developed at the wound. These projections ranged in size from 0.3 to 1.8 mm at the second or third post-operation molt. Be- cause extra structures developed at such a low frequency, we cannot draw any conclusions about the effect of eye- stalk removal on their development. Morphology of extra claws A pair of extra claws that developed on one of the damaged chelipeds is shown in Figure 2. Here, the dactyl and pollex were torn off at the first post-operation molt. However, a pair of projections 0.6-mm long developed on the remaining untorn part (arrowheads in Fig. 2A). Two extra claws appeared on the propodus near the pri- mary dactyl, and the primary dactyl and pollex regener- ated at the second molt. The lengths of the three dactyls (Fig. 2B, a, b, c) and the single contralateral dactyl were 0.5. 1.9, 1.7, and 3.3 mm, respectively. The three claws elongated at the third molt, and the shape of each claw was normal. There was no boundary among the propodi of these claws. The lengths of these three dactyls (Fig. 2C, a, b, c) and the single contralateral dactyl were 2.9, 3.7, 3.7. and 4.4 mm. respectively. 54 I. NAKATANI ET AL Table I Summary of wounding experiment in the crayfish Procambarus clarkii (values represent number of chelipeds) Developed extra structures 1 Double Single Wounded Autotomized Claws Dactyls Dactyl Projection Healed normally 4 Eyestalks With 1 14 10 1118 93 Without 20 1 1010 17 1 A total of 134 chelipeds from 34 crayfish received a V-shaped incision. After 10 to 20 days, both eyestalks were removed from 5 of the animals (20 chelipeds). : Some animals autotomized chelipeds post-operatively. 3 Extra structures developed in 13 chelipeds. 4 Most of the chelipeds healed normally, with no outgrowths. Figure 2. Development of extra claws on the third chehped of the crayfish after a wound was made in the propodus. Both eyestalks were removed at the base on day 1 1 after wounding. (A) Two projections 0.6-mm long (arrowheads) developed on the propodus, but the dactyl and pollex were torn off at the first post-operation molt. (B) The projec- tions developed into two poor claws at the second molt, and the untoni remaining part regenerated the dactyl and pollex. (C) The claws devel- oped fully at the third molt. A and B. the castoff exuviae after the second and third molts, respectively, a, primary claw; b and c, extra claws. Bar = 5.0 mm. Morphology of a pair of extra dactyls Two dactyls appeared on one of the chelipeds of a crayfish with eyestalks. The V-shaped wound seemed to have healed normally at the first molt. However, extra dactyls appeared on the right second cheliped at the sec- ond molt (Fig. 3, a, b). The extra dactyls developed on the primary propodus near the primary dactyl, although the wound was made at the base of the propodus. The primary claw was angled about 45 to the left of the propodus near the primary dactyl. The angle of the extra dactyls and the primary claw was about 90. The extra dactyls a and b of Figure 3 are hereafter called "extra dactyl a" and "extra dactyl b," respectively. The lengths of the carapace, extra dactyls a and b, and primary and contralateral dactyls were 39.5, 2.9, 3.1, 5.1, and 3.1 mm, respectively. Morphology of single extra dactyl A single extra dactyl that developed on the propodus of the second right cheliped at the second molt is shown in Figure 4. The extra dactyl was located between the base of the primary propodus and the base of the primary dactyl. The extra and primary dactyls were 2.0- and 3.7- mm long, respectively. Movement of the extra and primary dactyls In the example shown in Figure 2, the crayfish could move the primary dactyl but not the two extra dactyls at the second molt. Extra claw b was usually in an open position, but opened readily when it was closed manually. After the third molt, the crayfish could move the extra and primary dactyls. It always moved these three dactyls asynchronously and often spontaneously moved the pri- mary dactyl synchronously with extra dactyl b. These EXTRA CLAW ON CHELIPED OF CRAYFISH 55 Figure 3. Two extra dactyls on the right second cheliped produced after the propodus was wounded. (A) Extra dactyls in the open state. (B) Extra dactyls in the closed state, a and b, extra dactyls; p, primary dactyl. Bar = 5.0 mm. three dactyls would move synchronously when one of them was touched. In the diagram shown in Figure 3, the crayfish could move the extra dactyls and the primary dactyl. When it was picked up. it moved the extra dactyl a more frequently than the primary dactyl. However, it moved these dactyls simultaneously if one of them was touched. The single extra dactyl in Figure 4 and extra dactyl b in Figure 3 could be moved only slightly, either manually or by the crayfish. Muscles and innen'iition Muscles were observed to correspond to each claw in the propodus. In view of their locations, these muscles could control the opening and closing of the three claws. The closer muscles are shown in Figure 5A. Four muscles in the propodus with two extra dactyls (Fig. 3) were dissected out as shown in Figure 6B. In this figure, muscles m 1 , m2, m3, and m4 are the closer muscle of the primary dactyl, the closer muscles of extra dactyls b and a, and the opener muscle of extra dactyl a, respec- tively. The mean ( standard error of the mean) lengths of 10 sarcomeres of each of these muscles after being fixed with formalin solution were 6.4 0.3, 6.2 0.3, 7.7 0.4, and 5.8 0.5 pm. There were two thick nerve bundles in the intact propo- dus of the second cheliped (Fig. 6A): one ran toward its dactyl along the boundary between the opener and closer muscles; the other ran to its pollex along the closer mus- cle. The extra dactyls shown in Figure 3 were innervated with nerve bundles from the primary nerve trunks. A thick nerve bundle ran toward the primary dactyl between the two closer muscles (Fig. 6B, m2 and m3) of the extra dactyls. At the proximal part of the propodus. the nerve bundle ran between the closer muscle of the primary dac- tyl (ml ) and the opener muscle of the extra dactyl a (m4) (Fig. 6B. C). In a propodus with two extra claws, the nerve bundles branched to each claw from the primary nerve trunk (Fig. 5B). Extracellular recording An extracellular recording was made from the nerve bundle at the merus of the cheliped with two extra dactyls. The neuron in the nerve bundle responded to the push toward closing of extra dactyl b (Fig. 7). However, the neuron did not generate action potentials when the dactyl moved in the opposite direction. Therefore this response might be the action potential from proprioceptors. Mechanical force generated by dactyl movement The mechanical force generated by the movement of the primary and extra dactyls in response to electrical Figure 4. A single extra dactyl that developed on the right second cheliped. Both eyestalks were removed on day 12 after the cheliped was damaged. The arrowhead shows an articulation. Bar = 5.0 mm. 56 I. NAKATANI ET AL stimulation of the nerve bundle in the merus was mea- sured. The force generated by the primary dactyl was about 8- and 3-fold greater than that generated by extra dactyls a and b, respectively (Fig. 8). The primary dactyl moved spontaneously and asynchronously with the extra dactyls just before the electrical stimuli were applied to the nerve bundles (Fig. 8A. A'). Discussion There have been previous reports of double extra claws developing from the carpus (Bateson, 1894) and coxa (Przibram, 1921) of the crayfish (Astacns fluvkitilis) and from the propodus of the cheliped of lobster (Homarus americanux) (Cole, 1910). Recently, two extra claws that Figure 5. Innervation and muscles in the propodus of a cheliped with two induced extra claws. The cheliped is the same as that shown in Figure 2C. (A) Muscles corresponding to each claw (dorsal view). ( B ) The nerve trunks in the primary propodus branch, and each branched nerve bundle innervates a claw (ventral view), a. primary claw; h and c. extra claws; ml, m2, and m3, closer muscles associated with (he primary claw (a) and extra claws (h. ci. respectively. Arrowhead shows (he nerve trunks. Bar = 1.0 mm. "-^^^^P" '^||- ; Figure 6. Innervation and muscles in a propodus with double extra dactyl and in a normal cheliped. The cheliped with extra dactyls is the same as that shown in Figure 3. (A) Two nerve bundles (arrowheads) in the propodus of the second cheliped. (B and C) Branched nerve bundles from the primary nerve trunks (arrowheads) innervate the extra dactvls. ml. closer muscle of the primary claw; m2 and mX closer muscles of the extra dactyls b and a, respectively; m4. opener muscle of the extra dactyl a. a, b. and p are extra dactyl a, extra dactyl b. and primary dactyl, respectively. Bar = 1.0 mm. developed naturally in P. chirkii were described (Nakatani ct al., 1997). These single claws were borne on the first and third chelipeds one on each propodus. The extra EXTRA CLAW ON CHELIPED OF CRAYFISH 57 Tjp. 0.5s Figure 7. Extracellular recording from nerve trunks in the merus of a cheliped with extra dactyls. Thick bar signifies manual closing of the extra dactyl shown in Figure 3. claws induced in the present study are essentially similar to those naturally occurring ones. The naturally occurring extra claws reported by Cole (1910) and Nakatani el at. ( 1997) developed from the base of the primary propodus. Lateral outgrowths from experimental incisions devel- oped from the wounded site on the outer surface of the propodus (Murayama el /., 1994; Nakatani, 1996). Thus, in the present study, the wound was made at the base of the propodus. However, all of the extra claws or extra dactyls developed on the distal side of the wound. This suggests that the extra claws reported by Cole (1910) and Nakatani el <;/. ( 1997) were caused by damage at a site more proximal to the extra claw for example, at the carpus. Although there were no extra propodi, the shape and location of the a pair of extra dactyls observed in this study were similar to those of the naturally occurring one reported by Faxon (1881) on the cheliped of H. ameri- canus. As in the present study, these extra claws devel- oped away from the base of the propodus. The propodi of the extra claws described by Faxon were about one- sixth as long as the extra dactyls, and each was separated from its primary propodus by an articulation. Lateral outgrowths on chelipeds have been assumed to result from the abnormal healing of a natural wound (Su- zuki and Odawara, 1971; Shelton el ai, 1981: Okamoto, 1991; Nakatani el ai, 1992). Many of those outgrowths consisted of a pair of projections without any articulation. The proximal and distal surfaces of the wound each devel- oped one projection, and they faced each other as mirror images (Nakatani, 1996). However, in the present study, the extra dactyls developed away from the damaged site. Different mechanisms may be involved in the develop- ment of a lateral outgrowth and of an extra claw or dactyl. In the propodus it was observed that one nerve trunk runs in the direction of the pollex and another to the dactyl. Primary dactyl Extra dactyl a Stimulation A' Primary dactyl Extra dactyl a Stimulation 1 s B 0.5g Primary dactyl 0.1g Extra dactyl b Stimulation Extra 0.5g dact V' b Extra 0.1g dactyl a Stimulation 1 s 0.5g 1 s MMilMI^ k^^Mk ,^iMkjtJ Uk^^jU^ "' ^WW* ^H^pj ^^R^PP 0.5g 0.1g 1 s Figure 8. Movements of the primary and extra dactyls. The movements generated by each of two of three dactyls were recorded simultaneously when repetitive pulses (single pulse, II) ms in duration; 20 Hz) were applied to the nerve trunks in the merus. which was monitored in each bottom trace. The same cheliped shown in Figure 3 was used. A', the time-scale for enclosed part with broken line in A was magnified 4-fold. 58 I. NAKATANI ET AL. Lateral outgrowths were induced by wound damage of the nerve trunk that runs toward the pollex (Nakatani. 1996). Meanwhile, in the present study, both nerve trunks may have been damaged by wounding, because of the depth of the wound and the location of the nerve trunks. The crayfish reported by Nakatani et til. (1997) could not move the dactyl of its natural extra claws, despite the presence of muscles and nerve bundles. In contrast, all of the extra dactyls in the present study could be moved by the crayfish. Further studies are needed to explain this difference, but some speculation is possible. Closer and opener muscles, and innervation of these muscles, are necessary for a crayfish to move its dactyls. The crayfish claw is moved by two muscles controlled by five efferent axons: one inhibitor and one excitor to the opener muscle, and one inhibitor and two excitors a "fast" and a "slow" to the closer muscle (van Harreveld and Wiersma, 1937: Wiens, 1976). Both the primary and extra claws can be functional if the axons are adequately branching and innervate the muscles of each claw. The primary and extra dactyls may move synchronously if the above-mentioned axons branch to each muscle. However, the crayfish observed in this study could move all three dactyls synchronously or asynehronously. Atwood ( 1973) reported that a wide variety of synaptic mechanisms and muscle fiber properties permit delicate control within a simple framework in the crayfish claw. The articulation of the dactyl also affects its mobility. An extra dactyl b could be moved only slightly by hand, even though it had well-developed closer and opener muscles, and innerva- tion (Fig. 6B, C). For an extra claw or dactyl to develop, some of the severed nerve fibers must separate from the primary nerve trunks. The present results suggest that severed nerve fibers regenerate; some of them elongate to form aberrant roots and extra claws or extra dactyls. Aberrant roots have been observed in the abdominal nerve cord of the crayfish P. simiilans by Bittner et al. (1974). They reported that neurons projected aberrant roots toward the periphery after the third abdominal ganglion was removed. Also, Goransson et al. ( 1988) showed that in crayfish P. clarkii. the regenerating neurons would orient themselves in the proper direction to make connections toward the preferred target area. Further, the neurons do not respond to posi- tional cues during the first week of regeneration. Other studies have shown that transplanted tissues can induce growth of new structures. Kao and Chang (1996) proved that dactyl, pollex, and ischium tissues of the crab claw all had claw-transforming activity if they were auto- transplanted into the autotomi/,ed stump of the fourth walking leg. Furthermore, by autotransplantation of a pol- lex into the eye sockets, a claw with complete proximal segments (ischium, merus, carpus and manus) developed (Kao and Chang, 1997). In the cockroach Blattella ger- nia/iica. graft/host junctions of the leg regenerated seg- mented structures consisting of two copies of all struc- tures distal to the point of the junction (French. 1976). French (1976) speculated that the graft and host do not heal together and interact, but rather regenerate autono- mously in mirror-image symmetry of the original graft and host levels. Further studies are needed to explore related issues in crayfish. Acknowledgments We are grateful to Dr. Toshiyuki Nishida, Professor Emeritus, University of Hawaii, for his valuable com- ments and for improving the manuscript. Literature Cited Atwood, H. L. 1973. An attempt to account for the diversity of crusta- cean muscles. Am. Zool. 13: 357-378. Bateson, \V. 1894. Materials for the Study of Variation, Treated With Especial Recant to Discontinuity in the Origin of Species: X\ l i. Mac- millan. London. New York. 598 pp. Bittner, G. D., M. L. Ballinger, and J. L. Larimer. 1974. Crayfish CNS: minimal degenerative-regenerative changes after lesioning. J. Exp. Zool. 189: 13-36. Cole, L. J. 191(1. Description of an abnormal lobster cheliped. Biol. Hull. 18: 252-268. Faxon, W. 1881. On some crustacean deformities. Bull. Mas. Comp. Zool. 8: 257-274. Fingerman, M., and S. W. Fingerman. 1974. The effects of limb removal on the rates of ecdysis of eyed and eyestalkless fiddler crabs. L'cu piixilator. Zool. Jb. Phvsiol. Bel. 78: 301-309. French, V. 1976. Leg regeneration in the cockroach, Blatel/a gcnnaii- ica II. Regeneration from a non-congruent tibial graft/host junction. J. Embiyol. Exp. Morphol. 35: 267-301. Goransson. L. G., W. P. Hunt, and S. J. Velez. 1988. Regeneration studies on a crayfish neuromuscular system. II. Effect of changing the nerve entry point into the muscle field on the gradient of innerva- tion. ./. Nciirobiol. 19: 141-152. Kao, H. VV., and E. S. Chang. 1996. Homeotic transformation of crab walking leg into claw by autotransplantation of claw tissue. Biol. Hull. 190: 313-321. Kao, H. W., and K. S. Chang. 1997. Limb regeneration in the eye sockets of crabs. Hiol. Hull. 193: 393-400. Murayama, O., I. Nakalani, and M. Nishita. 1994. Induction of lateral outgrowths on the chelae of the crayfish. Procambarus clarkii (Girard). Crust. Res. 23: 69-73. Nakatani. 1. 1996. Morphology of lateral outgrowths induced on che- lipeds of the crayfish. Procambarus clarkii (Girardl. Crust. Res. 25: 142-150. Nakatani, I., and T. Otsu. 1979. The effects of eyestalk. leg. and uropod removal on the molting and growth of young crayfish. Pro- cambarus clarkii. Biol. Bull. 157: 182-188. Nakatani. 1., and T. Otsu. 1981. Relation between the growth and the molt interval in the eyestalkless crayfish. Procambarus clarkii. Comp. Kiocliem. Physiot. 68A: 549-553. Nakatani, I., K. Vamauchi, and O. Murayama. 1992. Abnormalities found in the chela of the crayfish, Procambarus clarkii (Girard). Res. Cnist. 21: 207-209. Nakalani, I., Y. Okada, and T. Yamaguchi. 1997. An extra claw on the first and on the third cheliped of the crayfish. Procambarus clarkii (Decapoda, Cambaridae). Crit.itaccatia 70: 788-798. EXTRA CLAW ON CHELIPED OF CRAYFISH 59 Okamoto, K. 1991. Abnormality found in the cheliped of Geryim affini.i xrunuliitu.'i Sakai. Res. Crust. 20: 63-65. Przibram, H. 1921. Die Bruchdreifachbildung im Tierriche. Wilhelin Rt*i<.\ Arc/i. /inr-A/fcVi. Org. 48: 205-444. Shelton, P. M. J.. P. R. Truby, and R. G. J. Shelton. 1981. Naturally occurring abnormalities (Bruchdreitachbildungen) in the chelae of three species of Crustacea (Decapoda) and a possible explanation. J. Embtyol. Exp. Morphol. 63: 285-304. Suzuki, H., and T. Odawara. 1971. Malformation found in the cheli- peds of two edible crabs. Res. Crust. 4-5: 191-195. Suzuki, S. 1980. The effects of leg-removal to the molting of the crab. Sesarma (Holometopus) haematocheir. Res. Crust. 10: 61-68. van Harreveld, A. 1936. A physiological solution for freshwater crus- taceans. Proc. Sue. Exp. Biol. 34: 428-432. van Harreveld, A., and C. A. G. Wiersma. 1937. The triple innerva- tion of crayfish muscle and its function in contraction and inhibition. J. Exp. Biol. 14: 448-461. Wiens, T. J. 1976. Electrical and structural properties of crayfish claw motoneurons in an isolated claw-ganglion preparation. J. Comp. Phvsiol. 112: 213-233. Reference: Biol. Bull 195: 60-69. (August. 1998) Isolation and Characterization of Endostyle-Specific Genes in the Ascidian dona intestinalis MICHIO OGASAWARA* AND NORIYUKI SATOH Department of Zoology, Graduate School of Science, Kyoto University, Sakyn-ku, Kyoto 606-01, Japan Abstract. The endostyle is a special organ in the pharynx of Urochordata, Cephalochordata, and Cyclostomata. It may have arisen in the common ancestor of these taxa, along with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has a functional homology to the vertebrate thyroid gland. The endostyle is therefore one of the structures key to the understanding of the origin and evolution of chordates. In the present study, we isolated and characterized cDNA clones for four endo- style-speciric genes, CiEiulsl, CiEntls2. CiEnds3. and CiEnds4, of the ascidian Cionu intestinalis. Although the predicted amino acid sequences of the gene products CiENDSl, CJENDS2, and CJENDS3 showed no similarity to known proteins, their mean hydropathy profiles suggest that they are secretory proteins. In addition, C1ENDS3 con- tained a unique repeat of 10 amino acids [R(QPCI)- (RRPC)!]. CiEndsl and CiEnds2 were expressed in zone 6, a protein-secreting glandular element of the endostyle, and CiEnds3 was expressed in zone 2, another secretory zone. CiEnds4, a cytoplasmic actin gene, was predominantly ex- pressed in zones 3 and 5, which are supporting elements of the endostyle. The amino acid sequences of CiENDS 1 and CJENDS2 resembled each other. In addition, they resembled a zone-6-specific gene product ( HrENDS2 ) of another ascid- ian, Halocvnthia roret~i. The results suggest that these genes are conserved among ascidian species, and therefore they (as well as CiEnds3 for the protein with a unique motif) may be useful probes for further analyses of molecular mechanisms involved in endostyle development. Introduction The endostyle is a specialized organ in the pharynx of tunicates, cephalochordates, cyclostomates, and certain Received 23 February 1998; accepted 20 May 1998. * To whom correspondence should he addressed. E-mail: ogasawara @ ascidian. /ool. kyoto-u.ac.jp prosobranchiates (Orton. 1912). The ascidian endostyle forms a trough-shaped structure in the ventral wall of the pharynx which extends from the fore-part of the pharynx to the esophagus (see Figs. 1A and 2D). In 1834, Lister first investigated how food particles in the feeding current are trapped in the pharynx of appendicularians. Fol ( 1876) found that the food is trapped by a mucous substance produced from the endostyle of tunicates. Thereafter, the ascidian endostyle has been intensively investigated by histological and ultrastructural observations (Olsson. 1963. 1965: Aros and Viragh. 1969; Fujita and Nanba, 1971); by the examination of ' ::> I incorporation (Thorpe et til.. 1972; Dunn. 1974); by histochemical detection of thyroperoxidase (Fujita and Sawano, 1979); and by partial purification of thyroperoxidase and its enzyme activity (Dunn. 1980). It is commonly considered that the endostyle of lower chordates may be a homolog and primitive antecedent of the vertebrate thyroid gland, mainly because the organ incorporates iodine (Harrington. 1957. 1958; Salvatore. 1969). However, the endostyle is a mucus-secreting and food-collecting organ, and the ability to concentrate io- dine is restricted to a small region of the organ (Olsson, 1963). The general organization of the ascidian endostyle is depicted in Fig. 1 B. The cells of this organ are differen- tiated into eight or nine strips, or zones, that run parallel to one another in longitudinal orientation. The cells of each zone are highly specialized in morphology and func- tion. The cells of zones 7. 8. and 9. like the thyroid cells of higher vertebrates, have an iodine-concentrating activity. The cells of zones 2, 4, and 6 have numerous secretory granules. These cells are believed to secrete the proteins or mucoprotein related to the digestion of food. The cells of zones 1. 3. and 5 are considered supporting elements and also as elements that might play a role in catching and transporting food. We are interested in molecular developmental mecha- 60 ASC1DIAN ENDOSTYLE-SPECIFIC GENES 61 B 8 I I supporting elements I I protein-secreting elements Illlllll iodine-concentrating elements 1 2 Zone Figure 1. Diagram of the ascidian endostyle. (A) Transverse section of the adult body showing the position of the endostyle (En) in the ventral wall of the pharynx (Ph). BWM, body wall muscle; Int. intestine; PhG, pharyngeal gill; Tu. tunic. (B) Enlargement of the endostyle show- ing compositional elements or zones of the endostyle. Zones 1. 3, and ? are supporting elements; zones 2. 4, and 6 are protein-secreting glandular elements; and zones 7. 8. and 9 are iodine-concentrating elements, equivalent to the thyroid gland of vertebrates. [Based on descriptions of Barrington (1957). Thorpe et nl. (1972). Fujita and Nanba (1471). and Dunn (1974)]. nisms that permitted or accelerated the advent of chordates. The phylum Chordata consists of the subphyla Urochordata (tunicates). Cephalochordata (amphioxus). and Vertebrata. Chordates are categorized as deutero- stomes, along with two other invertebrate groups, echino- derms and hemichordates, as was supported by molecular phylogenic studies (Wada and Satoh, 1994; Turbeville et /., 1994). Chordates share several characteristic features including a notochord. a dorsal hollow nerve cord, and pharyngeal gill slits. In addition, lower chordates (includ- ing tunicates, amphioxus, and lampreys) share an endo- style. We have emphasized that these hallmarks of the chordate body plan apparently evolved with the emer- gence of creatures resembling tadpole larvae (Satoh, 1995; Satoh and Jeffery. 1995). Therefore, investigations of the organization of these structures are of salient impor- tance in attempts to understand the origin of chordates. Coincidently with this change in the mode of larval locomotion, most of the primitive chordates or chordate ancestors may have shifted their feeding system to the use of pharyngeal gill slits for extracting suspended food from the water and of an endostyle for secreting mucus to catch the food particles (e.g., Brusca and Brusca, 1990). This possibility suggests that, in addition to the notochord and nerve cord, the pharyngeal gill and endostyle are key organs that can be used to explore molecular mechanisms involved in the emergence of chordates. In other words, we believe that the origin and evolution of chordates may be approached by isolating genes specific to the pharyn- geal gill or endostyle and by analyzing how these genes are organized during evolution in various deuterostomes. In previous studies, we isolated cDNA clones for pharyn- geal gill-specific genes (HrPliGI and HrPhG2: Tanaka et ul.. 1996) and for endostyle-specific genes (HrEnd.il and HrEnds2; Ogasawara et ai. 1996) from the ascidian Halocvnthia roretzi. which belongs to the order Pleuro- gona. Both endostyle-specific genes are expressed in zone 6 and encode secreted proteins. In the present study, we attempted the isolation of cDNA clones for endostyle- specific genes from Cionu intestinalis, which belongs to the order Enterogona. If both ascidian species conserve endostyle-specific structural genes, they may serve as models for the investigation of the molecular mechanisms involved in the organization of other chordate groups. Materials and Methods Biological materials Adults of dona intestinalis. C. savignyi. and Snela clava were collected near the Marine BioSource Educa- tion Center of Tohoku University, Onagawa, Miyagi and Otsuchi Marine Research Center, Ocean Research Insti- tute. University of Tokyo. Iwate, Japan. After the dissec- tion of adult specimens, tissues and organs were quickly frozen in liquid nitrogen, and kept at -80C until use. Isolation of RNAs and construction of cDNA libraries Total RNA was extracted from the endostyle and pha- ryngeal gill of C. intestinalis by the AGPC method (Chomczynski and Sacchi, 1987). Poly(A) + RNA was pu- rified with oligotex dT30 beads (Roche Japan. Tokyo). Complementary DNA was synthesized and cDNA librar- ies were constructed as described in a previous report (Ogasawara et al, 1996). An endostyle cDNA library was constructed using a uni-ZAP-II vector (Stratagene, La Jolla. CA). Isolation and sequencing of cDNA clones for endostyle- specific genes The endostyle cDNA libraries were screened differen- tially. Duplicate filters of the library were made; one was hybridized with a [ 32 P] -labeled total cDNA probe pre- pared from 5 fj,g of poly(A) + RNA of endostyle under high-stringency conditions, and the other was hybridized with a [ 3: P]-labeled total cDNA probe of pharyngeal gill under the same conditions. Plaques that showed positive hybridization with the endostyle probe but were negative for the pharyngeal-gill probe were selected and isolated by two rounds of screening. The specificity of the clones positive for the endostyle was confirmed by a Northern blot analysis. The clones were prepared for sequencing by controlled nested deletion from either the T3 or T7 62 M. OGASAWARA AND NORIYUKI SATOH side and sequenced using the ABI PRISM 377 DNA Se- quencer (Perkin Elmer, Norwalk. CT). Northern blot analvsis The Northern blot hybridization was carried out by the standard procedure (Sambrook et ai, 1989), and the filters were washed under high-stringency conditions. DNA probes for blot hybridizations were labeled with ["Pj- dCTP using a random primed labeling kit (Boehringer Mannheim, Heidelberg, Germany). In situ hybridization Juveniles of C. intestinalis, C. savignyi, and Styela clava were fixed in 4% paraformaldehyde in 0.5 M NaCl, 0.1 M MOPS buffer at 4C for 12 h. Probes were synthe- sized by following the instructions from the supplier of the kit (DIG RNA Labeling kit; Boehringer Mannheim). The /;/ situ hybridization of whole-mount specimens was earned out basically as described previously (Ogasawara et til., 1996). For the in situ hybridization of sectioned specimens, samples were dehydrated with a graded series of alcohol, embedded in polyester wax (BDH), and sec- tioned at 6 fj.m. Results As in the case of the isolation of cDNA clones for the endostyle-specific genes of H. roretzi (Ogasawara et al., 1996), differential screenings of a C. intestinalis endostyle cDNA library with total cDNA probes for the endostyle and pharyngeal gill yielded several cDNA clones specific to or enriched in the endostyle library. The preliminary in situ hybridization analysis of sectioned specimens dem- onstrated that transcripts of four cDNA clones were spe- cific to the endostyle. We named the corresponding genes CiEndsl (Ciona intestinalis endostyle gene J_), CiEnds2, CiEndsS, and CiEnds4. During the screening procedures, we noticed that the CiEndsl transcript was abundant in the library, representing nearly 10% of the library clones. When 1 /jg of poly(A) 1 RNA of the endostyle was elec- trophoresed, we detected the transcript as a band stained with 0.5 /yg/ml ethidium bromide (data not shown). Characterization of cDNA clone for CiEndsl As shown in Figure 2C, the Northern blot analysis of the CiEndsl transcript in various tissues and organs of a C. intestinalis adult detected the transcript of about 2.3 kb only in the endostyle. Hybridization signals were not de- tected in the pharyngeal gill, body-wall muscle, intestine, or gonad. The nucleotide sequence of the cDNA clone for CiEndsl will appear under the accession number of ABO 10895 in the DDBJ, EMBL, and GenBank nucleotide sequence databases. The insert of the cDNA clone con- sisted of 2265 nucleotides, including 17 adenyl residues at the 3' end. The clone contained a single open reading frame (ORF) of 1950 nucleotides, which predicted a poly- peptide of 650 amino acids (Fig. 2A). The calculated molecular mass (Mr) of the CiEndsl -encoded protein (CiENDSl) was 75.5k. CiENDSl did not show any sequence motifs shared by transcriptional factors, a transmembrane domain, nuclear localization signals, or motifs found in growth factor pro- teins. However, as shown in Figure 2B, the mean hydrop- athy profiles of CiENDSl showed that the N-terminus was highly hydrophobic. This region had a typical signal peptide sequence that consisted of a positively charged residue (amino acid position 2; K, Lys), a hydrophobic (3-13) region of 10-15 residues, a charged residue (posi- tion 15; S, Ser), and a residue containing the short side chain (position 16: A. Ala). A predicted cleavage site of the signal peptide was evident behind the Ala (position 16). This sequence motif strongly suggests that CiENDSl is a secretory protein, with a probability of 82% deter- mined by using the PSORT Program (Online. PSORT World Wide Web Server: Available: http://psort.nibb. ac.jp). In addition, CiENDSl contained four putative N- linked glycosylation sites (Fig. 2 A). The //; situ hybridization of whole-mount specimens demonstrated that the signals were restricted to the endo- style (Fig. 2D). In addition, the in situ hybridization of sectioned specimens demonstrated that the signals were not distributed over the entire regions of the endostyle but rather were restricted to zone 6 (Fig. 2E). No signals above background level were found in the control speci- men hybridized with the sense probe (data not shown). Characterization of cDNA clone for CiEnds2 The nucleotide sequence of cDNA clone for CiEnds2 will appear under the accession number of ABO 1 0896 in the DDBJ. EMBL, and GenBank nucleotide sequence databases. The insert of the cDNA clone consisted of 2107 nucleotides, including 25 adenyl residues at the 3' end. The occurrence of a 2.3-kb-long CiEnds2 transcript only in the endostyle (Fig. 3C) suggested that the cDNA was close to full-length. The clone contained a single ORF of 1950 nucleotides, which also predicted a polypeptide of 650 amino acids (Fig. 3A). The calculated Mr of the C/m/\2-encoded protein (CJENDS2) was 77.3 k. C1ENDS2 may also be a secretory protein. As shown in Figure 3B, the mean hydropathy profiles of C1ENDS2 showed that the N-terminus was highly hydrophobic. This region had a typical signal peptide sequence that consisted of a positively charged residue (amino acid position 2; K, Lys), a hydrophobic (3-13) region of 10-15 residues, a charged residue (position 15; N, Asp), and a residue ASCIDIAN ENDOSTYLE-SPECIFIC GENES 63 B MKVLL I LLAF I AAASAFSYGNGYGYGYNKCY6SYKGYSSGCYSYGYRK CYVYPKSQVFCYN I PYKKSWCSYKYYEPVLHVYPGCDCGTEGWTEKTV ADLE I EMTNLLKEALLK I TTEMNNCKTTFVEQLKSS I EQYKLNVKNKL FNYYAYY I QSAKTDEERENL I KKRDDA I KEYNEELDKKRDDV I LKCEE DVADKLKC I ADYHTKLVENGVECLKTRLTK I VDYTTTLTAKCVOYVKN YVACHMS I LEQKKSYYRSFLHKVHGSSEWEKVTVOAV I QLYHOQEVAK I TALATEYATKLATWKLKL I MNYSCAYRCYMSNGC I RFYKKRYYSTCK RYGCWYKYKTRYCFVRYCLQPFKFCFNPTKYTGLKTCVFPAVVRDGAT I IKEHCEKLEKAILEYETQFGEWKLKWTTYHTEYCTKYDEI IKARHDW Y I EYLRSQY I CANNSTELTDEQKAKLAEVQKECDEKRTAAVEAYKLKL AE I LLECATKFSTS 1 1 DYRTKAKSY I QS I GDNFDACQKKRSED I KAYK EKLEAKG I SAKQSLFDSMNKAKKSHLTKYLD I LKLHHDOFVVNGDVCD GPTD I VTMANKYSEKLSEYCAAVLLECDKYWED 1 1 RTGRTSSLGWYST IPAATSARNRSASCPDSAGWITHGA 65033 D 3.00 0.00 -3.00 i 141 281 421 561 701 Si Q. g - m s. o 2.3kb Figure 2. Characterization of the CiEmlsl gene. (A) The predicted sequence of 650 amino acids of CiENDSl . The predicted signal peptide sequence is shown by red capitals, and putative N-linked glycosyla- tion sites by green capitals. The nucleotide sequence for CiEmlsl will appear under the accession number of ABO 10895 in the DDBJ, EMBL, and GenBunk nucleotide sequence databases. IB) Mean hydropathy index of the CiENDSl calculated across a window of 19 residues according to the method of Kyle and Doolittle (1982). The N-terminus of the protein is characterized by a 16-amino-acid-long hydrophobic region that contains the predicted signal peptide sequence (see text for details). This suggests that CiENDSl is a secretory protein. (C) Distribution of CiEndsl transcript in tissues and organs of the adult. Northern blots of poly(A) + RNA prepared from the endostyle (En), pharyngeal gill (PhG). body-wall muscle (BWM). intestine (Int), and gonad (Gonad) were hybridized with the random-primed ["P]-labeled DNA probes, and the membrane was washed under high-stringency conditions. The CiEndsl transcript of about 2.3 kb in length was detected only in the endostyle. Each lane was loaded with 8 /j.g of poly(A) + RNA. (D, E) Localization of CiEndsl transcript, as revealed by in situ hybridization. IS. incurrent siphon; OS, outcurrent siphon. (D) A whole-mount specimen of a 1 -month-old young adult and (E) a cross-section of an adult showing that the signal is restricted to the endostyle (D. arrow) and to zone 6 of the endostyle (E, arrows). containing the short side chain (position 16; A, Ala). A predicted cleavage site of the signal peptide was evident behind the Ala (position 16). However, the results of the PSORT Program calculation showed that CJENDS2 seems to have an uncleaved N-terminal signal sequence. In addition, CiENDS2 contained a putative /V-linked gly- cosylation site (Fig. 3A). CJENDS2 showed 48.3% iden- tity at the amino acid level with CiENDSl, a rinding that will be discussed later. The //i situ hybridization of whole-mount specimens demonstrated that the signals were restricted to the endo- style (Fig. 3D), whereas that for sectioned specimens demonstrated that the signal was restricted to zone 6 (Fig. 3E). No signals above the background level were found in the other zones. Characterization of cDNA clone for CiEnds3 The nucleotide sequence of cDNA clone for CiEndsS will appear under the accession number of ABO 10897 in the DDBJ, EMBL. and GenBank nucleotide se- quence databases. The insert of the CiEndsS cDNA was 64 M. OGASAWARA AND NOR1YUKI SATOH AMK I LFVLLAALAVTNAYSYTHCGCKRFLVYPKPQAFCYH I QYKSCWCS YKYYEPVLHVYPGKDCGTLGWTETP I EOLQTEMENLLKESLLE I TKKM MLRKWFVRQLTKTADEYKEEYKKN I TRYYAYY I ASADS I LRKEHL I K ERNEL I EEYNEQLDQKVTAAVEKCTTO I LAK I K I I AAYHEKLVKSAVG CLETREQK I EEY I TTLENKCKLYVGRFVSKHLA I LEQNKKYYRATLAK VHGSALWQKAKVDAV I EVYHEQEAKK I SVLAKEYAOTLNTCKAKL I TN YRCAYKCYMSNSCLRFYKKTYYSYCRNLGCWYKYTPSYCVVRKTLCPF YYPYKP I SFSCLRTCVVPAVVRNGAT I I KELEEKLEKA I KE YVV I FTA WKTKWTQYHTEYCDKYNE I LKERHEWY I RRV I ARFVVENNSTELTALQ KAE I AKLKKELNERRTEAVLTYKKKLLNLLLECVAKFNEN I EEYKQKA LDL I KG I AKAFDACLTKRKAO I LAYRFKM I KHSFSEKE I MRKDM I KSK LVHLRHYKEMLKTYHDGEEFPEEVNAMI EAYEDKLENYCED I LEECTK DWSAA I PKLTHHYACSYTCREGRFCMPKFCFGGYFRf VVKYPTAKCYT YYYRCYRTTCNYRVFYQKGCYRYGCH gen gg B 3.00 OS c C C. > * O 111 n m C en 2.3kb Figure 3. Characterization of the CiEnds2 gene. (A) The predicted sequence of 650 amino acids of CiENDS2. The predicted signal peptide sequence is shown by red capitals, and a putative N-linked glycosyla- tion site by green capitals. The nucleotide sequence for CiEiulx2 will appear under the accession number of ABO 1 0896 in the DDBJ. EMBL. and GenBank nucleotide sequence databases. (Bl Mean hydropathy index of the OENDS2. calculated as in the case of CiENDSl. (C) Distribution of CiEnds2 transcript in tissues and organs of the adult. The CiEnds2 transcript of about 23 kb in length was detected only in the endostyle. Each lane was loaded with 8 fjg of poly(A)* RNA. (D, E) Localization of CiEnds2 transcript, as revealed by HI situ hybridization. IS, incurrent siphon. (D) A whole-mount specimen of a 1-month-old young adult and (E) a cross-section of an adult showing that the signal is restricted to the endostyle (D, arrow) and to zone 6 of the endostyle (E, arrows). 1319 nucleotides, including 17 adenyl residues at the 3' end. The Northern blot analysis demonstrated the occurrence of a 1 .4-kb long CiEnds3 transcript, which was detected only in the endostyle (Fig. 4C). This sug- gested that the cDNA was close to full-length. The clone contained a single ORF of 945 nucleotides, which predicted a polypeptide of 315 amino acids (Fig. 4A). The calculated Mr of the CiEnds3-encoded protein (CiENDS3) was 35.3 k. CiENDS3 may also be a secretory protein. As shown in Figure 4B, the mean hydropathy profiles of CiENDS3 showed that the N-terminus was highly hydrophobic. This region had a typical signal peptide sequence that consisted of a positively charged residue (amino acid position 2; R), a hydrophobic (3-13) region of 10-15 residues, a charged residue (position 15; T), and a residue containing the short side chain (position 16; C). A predicted cleavage site of the signal peptide was evident behind the Cys (position 16). This sequence motif strongly suggests that C1ENDS3 is a secretory protein, with a probability of 58% determined by the PSORT Program. In addition, CiENDS3 contained a unique repeat of 10 amino acids (Fig. 4A). The repeat consisted of R(QPCI)(RRPC)I. This type of repeat has not been re- ported to date in the PDB, SWISSPROT. and PIR data- bases surveyed. The //; situ hybridization of whole-mount specimens demonstrated that the signals were restricted to the endostyle (Fig. 4D). In addition, the ;/; ,v//i/ hybridiza- tion of sectioned specimens demonstrated that the sig- nal was restricted to zone 2 (Fig. 4E). No signals above the backuroimd level were found in the other zones. ASCIDIAN ENDOSTYLE-SPECIFIC GENES 65 B MRFL I LLCLLFTTATCQE I SVSASAETLANE I LOG I TLDD I ELPE I NLPSQP I SVTQ I PSVQR I RQPSIR QP 1 1 RQPC I RQPCQPC I RKPC I RQPC I RRPCPPCV RKPC I RRPCQPC I RRPGPPC I RKPCQPC I RRPCPP C I RKPC I RRPCQPC I RRPCPPC I RKPC I RQPGQPC I RKPCQPC I RRPGQPC I RRPCQPC I RRPCQPCVRQ PCQPC I RRPCQPC I RRPCQPC I RRPCLPLKRPC I T PTPT I EPP I TEPAPEVDGTTTASATATVTTDGTTT VTETTTDGNAEAVAE I AEALSGVTRQ I FLTSFK I L 315aa RQPCI RRPC I 3.00 0.00 -3.00 65 129 193 257 321 c j > - o ui a. m JE ' -.. 1.4kb Figure 4. Characterization of the CiEnds3 gene. (A) The predicted sequence of 315 amino acids of C1ENDS3. The predicted signal peptide sequence is shown by red capitals. The sequence contained a characteristic repeat [R(QPCI)(RRPC)I]. The underlined amino acids are identical to those of the consensus sequences. The nucleotide sequence for CiEnds3 will appear under the accession number of ABO 10897 in the DDBJ, EMBL. and GenBank nucleotide sequence databases. (B) Mean hydropathy index of the CiENDS3 calculated as in the case of CiENDSl . (C) Distribution of CiEndsS transcript in tissues and organs of the adult. Northern blots of poly(A)* RNA prepared from the endostyle (En), pharyngeal gill (PhG). body-wall muscle (BWM). intestine (Int), and gonad (Gonad) were hybridized with the random-primed ["P]-labeled DNA probes, and the membrane was washed under high-stringency conditions. The CiEnds3 transcript of about 1 .4 kh in length was detected only in the endostyle. Each lane was loaded with 8 //g of poly( A)* RNA. (D, E) Localization of CiEnds3 transcripts, as revealed by in situ hybridization. IS. incurrent siphon; OS, outcurrent siphon. (D) A whole-mount specimen of a 1 -month-old young adult (scale bar is 1 mm) and (E) a cross-section of an adult (scale bar is 100 /urn) showing that the signal is restricted to the endostyle (D, arrow) and to zone 2 of the endostyle (E. arrows). Characterization of cDNA clone for CiEnds4 The in xitu hybridization to isolate endostyle-specific cDNA clones demonstrated that the transcript of another clone (CiEnds4) was restricted to the zones 3, 5, and 7 (Fig. 5A). Strong signals were evident in zones 3 and 5, supporting elements of the endostyle; weak signals were also evident in zone 7, a putative iodine-concentrating element (Fig. 5 A). The insert of the CiEnds4 cDNA was 1541 nucleotides, including 19 adenyl residues in the 3' end. The clone contained a single ORF of 1 125 nucleotides. which pre- dicted a polypeptide of 375 amino acids (data not shown). A cDNA clone for C. intestinalis cytoplasmic actin has been isolated and characterized in the laboratory of Dr. Takahito Nishikata of Konan University. Kobe, Japan (pers. comm.). The determination of the partial nucleotide sequence of CiEnds4 cDNA clone revealed that CiEnds4 encodes a cytoplasmic actin. The above-mentioned results suggested that the gene or genes for one or more cytoplasmic actin are actively 66 M. OGASAWARA AND NORIYUKI SATOH C. savignyi B 1.6kb o_ m . I o C. intestinalis Figure 5. Characterization of the CiEnds4 gene. The predicted amino acid sequence of CiENDS4 suggested that CiENDS4 is a cytoplasmic actin. (A) Localization of CiEnds4 transcripts, as revealed by in MIII hybridization. A cross-section of a young adult showing signals in zones 3, 5. and 7 of the endostyle. (B) Distribution of CiEnds4 transcript in tissues and organs of the adult. Northern blots of poly(A)* RNA prepared from the endostyle (En), pharyngeal gill (PhG). body-wall muscle (BWM), intestine (Int). and gonad (Gonad) were hybridized with the random-primed |' : P]-laheled DNA probes, and the membrane was washed under high-stringency conditions. The CiEmls4 transcript of about 1. 6kb in length, or closely related molecules, was detected in the organs examined. Each lane was loaded with 8 /ug of polylAT RNA. (C, D) Cross-reactivity of CiEnds4 probe with zone 3 (arrows) of the endostyle of (C) C. savignyi and (D) Styela clava. as revealed by in .\itii hybridization (scale bars in A, C, and D are 100 pm). expressed in the supporting elements of the endostyle. We therefore examined, using the CiEmls4 probe, whether the cytoplasmic actin gene is expressed in the supporting elements of the endostyle of two other ascidian species, C. savignyi and Styela clcmi. As shown in Figure 5C and D, the CiEmls4 probe detected cytoplasmic actin tran- scripts in zone 3 of both species. In addition, the Northern blot analysis demonstrated the occurrence of a 1 .6-kb-long CiEmls4 transcript not only in the endostyle but also in other organs including the pharyn- geal gill, body-wall muscle, intestine, and gonad (Fig. 5B). Discussion In the present study, we isolated cDNA clones for four uenes (CiEndsl, CiEmls2, CiEndsJ, and CiEnds4), which are expressed in different zones of the ascidian endostyle. CiEndsl and CiEnds2 are expressed in zone 6 and encode peptides with similar amino acid sequences. CiEndsB is expressed in zone 2 and encodes a polypeptide with a novel repeat of 10 amino acids, tentatively called "ends- repeat." CiEnds4 encodes a cytoplasmic actin that is ex- pressed mainly in zones 3 and 5. In a previous study, we characterized cDNA clones for two endostyle-specific genes. HrEndxl and HrEnds2. from the ascidian H. roretzi (Ogasawara el til.. 1996). Both genes are expressed in zone 6 and encode secreted proteins. Transcripts of both genes are abundant in the endostyle library; each represents about 10% of the cDNA clones of the library. As revealed by the present study. CiEndsl is also expressed in zone 6 and encodes a se- creted protein. This transcript also represents the most ASCIDIAN ENDOSTYLE-SPECIFIC GENES 67 abundant species in the library. These results strongly suggest that zone 6 plays a major role in the secretion of mucus by the endostyle and has functions different from those of zones 2 and 4. This notion is consistent with a previous ultrastructural observation that the size and structure of the secretory granules in zone 6 differ from those of the other glandular zones 2 and 4 (Aros and Viragh, 1969). Zone 6 occupies the largest area in the endostyle and is characterized as the most developed glan- dular zone, containing abundant endoplasmic reticulum (Fujita and Nanba. 1971). Aros and Viragh (1969) and Fujita and Nanba (1971) reported that zone 6 contains at least two types of secretory granule a large electron- dense granule and a smaller granule. Directly facing the pharynx in zone 6 is a wide exit for secretion; in contrast, zones 2 and 4 have only a very limited exit for secretion (Thorpe et aL 1972). When the amino acid sequences are compared for CiENDSl and CJENDS2 (Fig. 6) and for CiENDSl, HrENDS2, and C1ENDS2 (Fig. 7), these three polypep- tides closely resemble each other: the sequence identity was 48.3% between CiEndsl and CiEnds2, 22.2% be- tween CiENDSl and HrENDS2 (similarity 43.7%). and 22.8% between CiENDS2 and HrENDS2 (similarity 42.6%). Our previous genomic Southern blotting analysis of HrEnds2 suggested that there are some other genes in the H. mretzi genome that contain a sequence similar to that of HrEnds2. In the present study, we isolated two cDNA clones from C. intestinalis that contained a se- quence similar to that of HrEmls2. It is therefore likely that CiENDSl, CiENDS2, and HrENDS2 are members of the same protein family, and it is possible that these genes were derived from a common ancestral gene. The first aim of our studies is to isolate genes that are expressed in certain zones of secretory function, these genes being common in different species that be- long to different orders of uscidians. In the present study, we first attempted the screening of C. intestinalis homologs of HrEndsl and HrEnds2 with low-strin- gency hybridization conditions, using these genes as probes. Unfortunately, we could not isolate any homo- logs with the conditions we adopted. Therefore, we next tried the differential screening we used in a previ- ous study (Ogasawara et al., 1996), because we thought that if the nature of the endostyle is the same between C. intestinalis and H. roret-i, we might isolate homolo- gous genes easily using this method. In the present screening, we were able to isolate an endostyle-specific cDNA clone for CiEndsl; this clone was highly ex- pressed in zone 6 and contained a sequence similar to that of HrEnds2. In the further screening, we isolated CiEnds2, which was also expressed in zone 6 and con- tained sequences similar to that of HrEnds2. Ciendsl, CiEnds2, and HrEnds2 may be related to each other and play an important role in the ascidian endostyle, and therefore they are good candidates for future stud- ies. Interestingly, the CiEnds3 encodes a polypeptide with a novel repeat of 10 amino acids [the core repeat is 8 amino acids (QPCI)(RRPC)|. We tentatively call this the "ends-repeat." Because of its uniqueness and conserva- CiEnds! CiEnds2 CiEndsl CiEnds2 CiEndsl CiEnds2 CiEndsl CiEnds2 CiEndsl CiEnds2 CiEndsl CiEnds2 CiEndsl CiEnds2 CiEndsl CiEnds2 WYSTIPAATSA-RNRSASOSDSAG YTCREERFCMPKFCFGGYFRWVVKYGTAKC 559 Dl 537 E 644 VVITHGA 623 YTYYYRCYRTTCNYRVFYQKGCYRYGCH Figure 6. Alignment of the amino acid sequences of CiENDSl and CiENDS2. Identical amino acids are enclosed by boxes. Gaps were induced to obtain the maximal similarity. 93 71 186 164 279 257 372 350 465 443 558 536 643 622 650 650 68 M. OGASAWARA AND NORIYUKI SATOH CiEndsl aa HrEnds2 aa CiEnds2 aa CiEndsl aa HrEnds2 aa CiEnds2 aa CiEndsl aa HrEnds2 aa CiEnds2 aa CiEndsl aa HrEnds2 aa CiEnds2 aa CiEndsl aa HrEnds2 aa CiEnds2 aa 315 CiEndsl aa HrEnds2 aa CiEnds2 aa CiEndsl aa HrEnds2 aa MKVLLILLAFIAAASAFSY6N MKILIVLLSCLAVASAFGYGGYSRYSRYGRViNKYPSYGGYSKYSSYGSYKY-PSye MKILFVLLAALAVTNAYSYTHCGC-KlFLvl -RKYKQ 60 61 NIPYKKSCSYKYYEPVLHVYP;GCDCGTEG1TEKTVADLE|EMTNI gg KGLFKNE|CTTAFYSDVLSYYPpDAKNlLP-KANATDQVV|---A| ft HIQYKSCISCSYKYYEPVLHVYPGKDCGTLGWTETPIEDLQtlEMENi nm fragum, I', iiucilo), and an azooxanthellate cardiid clam (Vasticardium flavum) were collected in the Repub- lic of Palau, Western Caroline Islands, during the cruises of the R/V Sohgen-mani in 1993-1994. T. gigas, T. de- rasa, and H. porcellanus were purchased from the PMDC (Palau Mariculture Demonstration Center). Live speci- mens of a zooxanthellate cardiid, Corciiliim cardissa, which had been collected from mariculture tanks for tri- dacnid clams, were gifts from PMDC. A live specimen of Fragnin fragum collected in Okinawa, Japan, was a gift from Mr. Osumi of Ryukyus University. An azooxan- thellate cardiid clam. Fulvia tnutica, was purchased from a local fishery market in Shimizu, Japan. The shells were saved (except for those of T. gigas, which were lost during transportation) and were kindly identified by Prof. Oku- tani of Nihon University. Extraction of DNA from the clams Live clams were dissected, and the organs were sepa- rated and frozen at -20C until used. The white part of the gill or foot tissue (0. 1-0.2 g), which was devoid of zooxanthellae, was homogenized in a glass homogenizer in 2-3 ml TE buffer ( 10 mM Tris-HCl, 100 mM EDTA, pH 8.0) containing 0.5% sodium dodecyl sulfate, and digested with proteinase K (100/ug/ml) at SOT for 3 h (Wada et ai, 1992). The DNA was then extracted with TE buffer-saturated phenol and washed twice with a mix- ture of chloroform and isoamyl alcohol (24:1). An equal volume of 5 M ammonium acetate was then added, and the DNA was precipitated with cold ethanol, washed once with cold 70% ethanol, and dried. Amplification of 18S rDNA and DNA sequence determination A polymerase chain reaction (PCR) kit (Takara Shuzo, Kyoto, Japan) was used to amplify the 18S rDNA from 1 pg of genomic DNA. The manufacturer's instructions were followed; i.e.. 30 cycles comprising 93C for 1 .5 min. 58C for 1 .5 min, and 72C for 2 min. The exten- sion reaction at 72C in the final cycle was prolonged to 10 min, and the PCR products were then frozen at -20C until used. The universal eukaryotic primers used in the amplification of the 18S-rDNA were 5'-GGTTGAT- CCTGCCAGTAGTCATATGCTTG-3' (ss5) and 5'- GATCCTTCCGCAGGTTCACCTACGGAAACC-3' (ss3). These sequences were reported to be located four nucleotides from the 5' and 3' ends, respectively, of the 18S-rRNA of Prorocentrum micans, a dinoflagellate (Herzog and Maroteaux, 1986; Rowan and Powers, 1992). Amplified DNAs were cloned in pT7 plasmids (Nova- gene. USA) with E. coli JM 109 as the host. At least three cloned DNAs amplified from the genomic DNA of an individual clam were sequenced for each clam species, using custom-made 20-b DNA primers (Japan BioSer- vice, Saitama), a Takara PCR sequence kit with an ABI- type 373A DNA sequencer. Analysis of the sequence data The 18S rDNA sequences were aligned using Clustal W (Thompson et ai, 1994) and gap and insertion regions removed with the software program RMINSDEL in MOLPHY-2.2 (Adachi and Hasegawa, 1994). The aligned sequences were then analyzed by the neighbor- joining (NJ) method (Saitou and Nei, 1987) using the DNADIST and NEIGHBOR programs (using the Kimura 2-parameter model; Kimura, 1980) in PHYLIP 3.57c (Felsenstein, 1995). The statistical significance of each cluster in the tree was evaluated with 1000 iterations of bootstrap resamplings and tree reconstructions (Felsenstein, 1985) using DNADIST, NEIGHBOR, SEQ- BOOT, and CONSENSE in PHYLIP 3.57c. The same sequences were analyzed by the maximum parsimony 72 T. MARUYAMA ET AL (MP) method using the DNAPARS program in PHYLIP 3.57c. Decay analysis (Bremer, 1988, 1994; Winnepen- ninckx el al., 1996) was also undertaken by using PHY- LIP 3.57c. The sequence data of nine selected clams were further analyzed by the maximum likelihood (ML) method using the NUCML program adopting the Alpha/ Beta ratio 2.0 model (Hasegawa et al., 1985 ) in MOLPH Y 2.2 (Adachi and Hasegawa, 1994). The tree with minimal AIC (Akaike Information Criterion), which is defined as -2 (log-likelihood) + 2 (number of free parameters) (Akaike, 1974), was considered the most appropriate tree. The statistical significance of each cluster in the ML tree was evaluated by the bootstrap analysis with 1000 itera- tions. Phylogenetic trees were drawn with TreeView (Page, 1996) on a Macintosh computer. Results The PCR products Lengths of the amplified 18S rDNAs (between the primers ss5 and ss3) from the zooxanthellate and azoo- xanthellate cardiid clams were 1780-1875 bp longer than the corresponding lengths in the other clams exam- ined (1745-1772 bp) (Table I). Those sequences were deposited in the DDBJ (DNA data base of Japan) under the accession numbers shown in Table I. The clam shells (except T. gigas) were deposited in The National Science Museum in Tokyo with the specimen numbers listed in Table I. Phvlogenetic trees The alignment of 18S rDNA sequences of the clams is available on the web site of this journal at the following address: http://www.mbl.edu/html/BB/home.BB.html After removing the gaps and insertions, we analyzed 1698 sites within the sequences by the NJ and MP methods. Figure 1 shows a phylogenetic tree constructed by the NJ method. The 18S rDNA sequence of LiinicoUirici kambeitl (Gastropoda, Pulmonata. Achatinidae) was chosen as the outgroup. We also analyzed the data with the 18S rDNA Table I Length of amplified IXS-rDNAs Length of the PCR Accession Specimen Species Place of collection products' number number' Zooxanthellate bivalves (present work) Tridacna gigus Palau 1780 bp D84189 n.d. Tridacna derasa Palau 1803 D84658 NSMT-Mo70918 Tridacna crocea Okinawa ( Japan I 1803 D88908 NSMT-Mo70917 Tridacna maxima Palau 1805 D84659 NSMT-Mo70919 Tridacna sqiiamosa Palau 1804 D84190 NSMT-Mo70916 Hippopus hippopus Palau 1813 D84660 NSMT-Mo70914 Hippopus pOTcellanus Palau 1813 D84661 NSMT-Mo70915 Fragum fragum Palau 1865 D84662 NSMT-Mo70921 Fragum fragum Okinawa (Japan) 1865 D84663 NSMT-Mo70920 Fragum unedo Palau 1865 D84664 NSMT-Mo70922 Corcuhim cardissa Palau 1875 D88909 NSMT-Mo70923 Azooxanthellate Cardiidae bivalves (present work) Vasticai'diiim fiavuni Palau 1852 DS8910 NSMT-Mo70913 Fulvia nuitica Mikawa-bay (Japan) 1840 D88911 NSMT-Mo70912 Other bivalves (from database) Spisula solidissima 1772 LI 1270 Tresu* nuttali 1772 LI 1269 Placii/wti'n magellanicus 1745 X53899 Chlamvs islandiCQ 1746 LI 1232 Mytilux edulis 1757 L24489 Gastropod (from database) Limicolariu kamhcul 1770 X66374 1 Length of the amplified DNA belween but excluding primers ss5 and ss3. ; Accession number of the DNA sequence data in DNA Data Base of Japan. ' Deposit number of the shell specimen in National Science Museum, Tokyo; n.d., not deposited. PHYLOGENY OF CLAMS WITH ZOOXANTHELLAE 73 0.98 r Spisuk 1.000 L Tresus 0.870 1.000 solidissima nuttali 1.000 ^Vasticardium flavum \Fulvia mutica |- 1.000 JfHippopus hippopus 1.000 |1 1}IW ^H 1.0001 1.000 0.993 0.677 1 Hippopus porcellanus ! Tridacna gigas Tridacna derasa Tridacna crocea Tridacna maxima \ Tridacna squamosa 000 r\ Corculum cardissa 1 Fragum fragum UKI Fragum unedo 0.1 s |" Placopecten magellanicus .000 L Chlamys islandica Figure 1. A phylogenetic tree of zooxanthellate and azooxanthellale clams, calculated by the neighbor-joining method adopting the Kimuru 2-parameter model. Numbers at the nodes are the bootstrap values for the clades in 1000 replications. Box. zooxanthellate clam. Bar, 0.10 substitutions per site. sequence of Symbiodinium corculorum, the symbiotic alga of Corcitlwn cardissa (accession number LI 37 17) as the outgroup; the results were the same as those with L. kambeul (data not shown). Clams belonging to the Pteriomorphia (Mytilus edulis in Mytilidae, and Placopec- ten magellanicus and Chlamys islandica in Pectinidae) made a clade that was clearly separated from those of the Heterodonta. In the heterodont branch, a clade including the families Tridacnidae and Cardiidae and comprising all of the zooxanthellate clams examined, as well as two azooxanthellate clams, V. flavuin and F. mntica was clearly separated from another clade containing two spe- cies in the family Mactridae (Spisula solidissima and Tre- at m nuttali). This clade was further resolved into three lineages: Corculum-Fragum, Vasticardium-Fulvia, and Tridacna-Hippopus. The NJ tree indicates that the zoo- xanthellate cardiids and azooxanthellate cardiids diverged before the zooxanthellate tridacnids. The bootstrap value at the node uniting the Vasticardium-Fulvia and Tri- dacna-Hippopus clades was 0.870, and that at the node uniting the Vasticardium-Fulvia-Tridacna-Hippopus and Corculum-Fragum clade was 1.000. This indicates that tridacnid clams are more likely to form a clade with clams of Vasticardium-Fulvia than with those of Fragum-Cor- culiim. Three tridacnid clams. T. maxima, T. squamosa. and T. crocea, are evidently very closely related, whereas T. gigas and T. derasa are relatively distant from them. Fragum fragum and F. unedo are very closely related, the identity between their 18S rDNA sequences being 99.8% (1861 out of 1865 bp). The maximum parsimony (MP) method gave three equally most parsimonious trees of similar, but distinct, topologies (Fig. 2, A-C). The lengths of the trees were Limicolaria kambeul Mytilus edulis Chlamys islandica Placopecten magellanicus Tresus nuttali Spisula solidissima Fulvia mutica Vasticardium flavum Tridacna crocea 0.957] i JTridacna maxima 0.326 [ Tridacna squamosa 1 .000 ^IHippopus porceWanusI fiippopus hippopus [ [CorcuJum cardissa \ unedo 0.999 fragum 1.000 Tridacna gigas Tridacna derasa Tridacna maxima Tridacna crocea \ Tridacna squamosa 1.000 0.326 Tridacna gigas Tridacna derasa Tridacna squamosa Tridacna maxima Tridacna crocea Figure 2. Phylogenetic trees of zooxanthellate and azooxanthellate clams, calculated by the maximum parsimony method. Three topologies were selected as the best tree; the differences were found in Triilucim .ii/iiainnsa, T. crocea, and T. maxima. (A) One of the best trees. (B and C) Parts of the two other trees showing the dissimilarities to A. Numbers at the nodes are the bootstrap values tor the clades in 1000 replications. *, decay index. Box, zooxanthellate clam. Consistency index, 0.76: retention index, 0.88. 74 T. MARUYAMA ET AL. 1019 steps. Consistency and retention indices of the three trees were 0.76 and 0.88, respectively. These trees had essentially the same topology as that obtained by the NJ method, except for the positions of the three closely re- lated clams in the genus Tridacna (T. squamosa, T. cro- cea, and T. maxima). These differences suggest that the three species are closely related. In this tree, bootstrap value for the clade containing the tridacnid clams and Vasticardium-Fulvia was high (0.901). The decay index, which is the number of extra steps required before this clade collapsed, was calculated to be 6. These data sug- gest that the azooxanthellate curdiid lineage is more likely to form a clade with the zooxanthellate tridacnid clam lineage than with the zooxanthellate cardiid clam lineage. Lineages Tridacna- Hippopus- Vasticardium-Fulvia and Corculum-Fragum formed a clade, but the bootstrap value was relatively low (0.519). Due to the computational limitations, we selected nine clam species for a maximum likelihood analysis; one from subclass Pteriomorphia (Mytilus edulis) as an outgroup. two from zooxanthellate Cardiidae (Coirnlum cardissa and Fragum fragum), two from azooxanthellate Cardiidae (Vasticardium flavum and Fulviu niutica), two from the genus Tridacna (Tridacna gigas and T. derasa), and two from Hippopus (H. hippopus and H. porcellanus). A total of 1713 sites were used for the ML analysis. The tree topology (Fig. 3), was essentially the same as that ob- tained by the NJ method. The bootstrap value at the node ofTridacna-Hippopus and Vasticardium-Fulvia was 0.76, and that of Tridacna-Hippopus-Vasticardium-Fulvia and Corculum-Fragum was 1 .00. The differences between the minimal AIC (Akaike Information Criterion, as defined in the Materials and Methods) and the AICs of the other possible trees were greater than 15.5 (1.0), which suggests that the tree topology shown in Figure 3 is statis- tically significant. To examine the genetic difference between individuals from two distant locations, 18S rDNA sequences of F. fragum collected from Okinawa and Palau were com- pared. The identity between them, 99.9% (1864 out of 1865bp). indicated no significant difference between them. Insertions and gaps Table II shows the major gaps and insertions found in 18S rDNAs of cardiids and tridacnids when aligned with those of other clams, especially Limicolaria kambeiil, listed in Table I. Designations of the regions of the 18S rRNA are shown according to Winnepennickx ct al. ( 1992). The table includes any insertions and gaps longer than 10 b in any of the cardiids and tridacnids. Corre- sponding smaller gaps and insertions in other zooxanthel- Mytilus edulis 1.00 L Corculum cardissa Fragum fragum i.oo Hippoppus hippopus 0.76 i.oo Hippopus porcellanus 1.00 Tridacna derasa Tridacna gigas 1 .00 L Vasticardium flavum Fulvia mutica 0.1 Figure 3. A phylogenetic tree of nine selected bivalves, including zooxanthellate and azooxanthellate clams, calculated by the maximum likelihood method. Bar indicates 0.1 base substitutions per site. Numbers at the nodes are the bootstrap values for the clades in 1000 replications. Box. zooxanthellate clam. late clams are also shown. Insertions in the El 0-1 region are restricted to the azooxanthellate cardiid calms, V. fla- vum and F. mutica. On the other hand, zooxanthellate clams have gaps in this region. Some of those insertions were aligned and are shown in Figure 4. The identity between El 0-1 insertions of V. flavum and F. mutica is low (70%). While the inserts in Corculum and Fragum are similar to each other, those in Hippopus are different from those of Corculum and Fragum. Inserts in region 47 are restricted to Tridacna, but T. gigas lacks this insertion. Those of T. squamosa and T. maxima are the same: one base different from that of T. crocea, and two bases differ- ent from that of T. derasa. Discussion The tree topologies based on 18S rDNA sequences (Figs. 1-2) are consistent with the morphological taxon- omy of Mytilidae, Pectinidae, and Mactridae (Morton. 1996). The family Tridacnidae comprises two genera. Tri- dacna and Hippopus (Rosewater. 1965, 1982). The genus PHYLOGENY OF CLAMS WITH ZOOXANTHELLAE 75 Table II Major insertions and gaps in 1SS rDNAs in tridacnid and cardiid clams in comparison with those of Limicolaria kambeul Regions* Positions in the alignmentt Size (b) Species:): Insertions E10-1 247-269 23 Vf 22 Fm E21-2 768-782 15 Cc 12 Ff, Fu 11 Hh, Hp 4 Vf. Fm 3 Tg 4! 1484-1513 30 Ff. Fu 27 Cc 4 Vf. Fm. Hh. Hp. Tg. Td. Tc. Ts. Tm 47 1879-1910 32 Ts. Tc. Tm 31 Td 8 Cc 6 Fm 5 Fu. Ff, Vf, Tg Gaps E10-1 232-281 25 Hh, Hp. Tg. Td, Tc, Ts, Tm 232-246 14 Cc, Fu, Ff Gaps and insertions were detected in the aligned 1 8S rDNAs listed in Table I. * Regions in 18S rRNA of Limicolaria kambeul designated in Winnepennickx el al. (1992). t The alignment is available at the following URL: http://www.mbl.edu/htrnl/BB/home.BB.html t Cc, Corcitlum cardissa: Fu, Fragitm unedo; Ff, Fragum fragitm; Vf, Vasticardiinn flavum; Fm, Fulvia inutica; Hh, Hippopits hippopus; Hp, Hippopus porcelhmus; Tg, Tridacna gigas; Td, Tridacita derasa: Tc, Tridacna crocea: Ts, Tridacna squamosa; Tm, Tridacna maxima. S Very close gaps or insertions were combined. Tridacna is further divided into three subgenera, Tri- dacna, Persikima, and Chametrachea. Tridacna gigas be- longs to subgenus Tridacna; T. derasa to Persikima; and three species, T. squamosa, T. maxima, and T. crocea, belong to Chametrachea. This classical taxonomy also agrees well with the present tree topologies (Figs. 1-3). However, the present data are also in marked disagree- ment with the classical taxonomy: i.e., the tridacnid clams, genera Tridacna and Hippopus, are more closely related to the cardiids Vasticardium flavum and Fulvia mulica than these clams of Fragum and Corculum, sug- gesting either that the family Cardiidae is paraphyletic or that tridacnids belong to the family Cardiidae. The correct interpretation -either the traditional taxonomy of Tridac- nidae and Cardiidae or the present molecular view of these groups-is obscure. Molecular phylogenetic analy- ses using some other genes are underway and may answer this question. The present results indicate that the divergence between the Corculum-Fragum lineage and that of Vasticardium- Fulvia is deeper than that between Corculum-Fragum and Tridacna-Hippopus. Because there is no evidence of sym- biosis in clams of Vasticardium and Fulvia, clams in the tridacnid and Corculum-Fragum lineages might have ac- quired symbiotic relationships with zooxanthellae inde- pendently after their divergence from the lineage of Vasti- cardiiim-Fulvia. An alternative explanation is that the clam ancestral to the three lineages acquired the symbiotic relationship with zooxanthellae, and only the lineage of azooxanthellate Cardiidae lost this characteristic. If the latter explanation is correct, there may be some traces of symbiosis, not yet reported, in some of the azooxanthel- late Cardiidae. Further studies are necessary to distinguish between these two hypotheses. Masuda et al. (1994) reported that extracts of mantle homogenates from tridacnid clams (Tridacna derasa, T. maxima, T. crocea, and H. hippopus) are much more stimulatory to the excretion of photosynthate by T. derasa zooxanthellae than are tissue extracts of zooxanthellate Fragum clams, other azooxanthellate bivalves, or gastro- pods. These may reflect the relative phylogenetic distance between tridacnids and zooxanthellate cardiids, although the active substance in the mantle homogenate is still not known. Tridacna crocea larvae develop through several mor- phological stages: straight-hinge and prodissoconch. 76 T. MARUYAMA ET AL. Inserts in E10-1 region 247 269 1 : T-TG-TTTCAGCGTCCGCAGG-GCGG 1 : TCCGCTCTC-GGGT-GGC-GGCCCCG Inserts in E21-2 region 768 782 C. rardi aga . unedo H. hi pnopu.q 1 : TCCT-TACTAG-CTCCG 1 : -CC CTTGCCTCCG 1:-CC CTTGCCTCCG 1: -CCTGTA G-C-AAA H. porrpl laniig 1 : -CCTGTA G-C-AAA Inserts in 41 region 1484 1513 I 1 : GGGACCCAGG GACCGCTCT-CGAGCGGA 1 : GGGACCTAGGTCCGTCCGCTCTGCGGG-GGA 1 : GGGACCTAGGTCCGTCCGCTCTGCGGG-GGA Inserts in 47 region 1879 1910 I 1 : GTTG-GTTTCATCTCCTCGCGGGGTGTGCCTT 1 : GTTGCGCTCCATCTCCTCGCGGGGTGGGCCTT 1 : GTTGCGCTTCATCTCCTCGCGGGGTGGGCCTT 1 : GTTGCGCTTCATCTCCTCGCGGGGTGGGCCTT Figure 4. Inserts in I8S rDNAs of zooxanthellate tridacnids and cardiids, as well as in azooxanthellate cardiids, when aligned with 18S rDNA sequences ot Limicolaria kambeiil, Mytilus ethilis. Placo^ci ten magellanicus, Clilumys ixluinlifii. S/'ixii/a xolUlixxiimi, and 7Vr,v\ nut- itili. *, identical base sequence. Numbers at both ends of the sequence indicate the position of the base in the aligned sequences, which are available at the following URL: http://www.mbl.edu/html/BB/home.BB.htmJ cardiid, proto-tridacnid, and pre-hippopus, before reach- ing the final tridacnid stage (Kawaguti. 1983b). This sug- gests that the tridacnid clams evolved from the ancestral cardiid clam. The present phylogenetic topologies (Figs. 1-3) are consistent with this idea. Insertions in E10-1 in azooxanthellate cardiids are found in the variable region. The identities between these insertion sequences are relatively low, but they might appear after divergence from both of the zooxanthellate cardiids and tridacnids. In region 47, the insertions are restricted to tridacnid clams, except T. gigas. This inser- tion might appear after divergence between T. t,'M,'650 nm) light. The evoked depolarization consisted of a transient component, followed by a steady plateau in which the amplitude of the depolarization was well correlated with the log of the stimulus intensity. The evoked depolariza- tions induced action potentials in the photoreceptor cells, with the frequency of firing being well correlated with the stimulus intensity. The morphologies of individual photoreceptor cells were visualized by intracellular injec- tions of the fluorescent dye Lucifer yellow, and the path of the epistellar nerve across the stellate ganglion, into the pallial nerve, toward the brain was traced using the lipophilic dye Di-I. This pathway was confirmed physio- logically by recording light-evoked responses from the cut end of the pallial nerve. Introduction Most cephalopods have, in addition to their retinal pho- toreceptor system, extraocular photoreceptors or photo- Received 24 December 1997; accepted 21 April 1998. * To whom correspondence should be addressed. sensitive vesicles (PSVs). The PSVs of octopods such as Eledone moschata are located inside the mantle sac and appear as a small pigmented vesicle on the ventral poste- rior margin of the stellate ganglion (Bauer, 1909; Young, 1936, 1971; reviewed in Mauro, 1977; Fig. 1). These PSVs have also been termed the 'epistellar bodies' (Young, 1929, 1936). The ultrastructure of the epistellar body of E. moschata shows packed arrays of photorecep- tor cells with microvilli, reminiscent of rhabdomeres, but without dioptric apparatus (Nishioka et al.. 1962). to- gether with an epistellar nerve running into the stellate ganglion (Young. 1936; Perrelet and Mauro. 1972). The epistellar photoreceptor system in E. moschata has been shown to contain the visual pigment rhodopsin. with a maximum absorption wavelength of 475 nm; this is very close to 470 nm, which is the maximum absorption wave- length of retinal rhodopsin in this octopod species (Nishi- oka et til.. 1966; Mauro, 1977). This wavelength is also close to electrophysiological estimates of spectral sensi- tivity in E. moschata (Nishioka et al., 1966; Mauro, 1977). The accessory pigment retinochrome is also pres- ent in cephalopod PSVs (Hara and Hara. 1980; Ozaki et nl.. 1983). Although preliminary evidence has indicated that the photoreceptor cells within the epistellar body of octopus give depolarizing responses to light (Mauro and Bamnann, 1968; reviewed in Mauro. 1977), there has been no detailed study of these responses. Previously we have shown that extracellular, light-in- duced generator potentials in the epistellar bodies of Ele- done cirrhosa can be produced by light flashes of different intensity and duration, transmitted through the mantle sac wall of the octopus (Cobb et al., 1995a,b). In addition, the normal circadian behavioral rhythm entrained by a 78 OCTOPUS PHOTORECEPTOR ELECTROPHYSIOLOGY AND INNERVATION 79 Figure 1. Diagrams showing the position of the photosensitive vesi- cle or epistellar body in an octopus (not to scale). Top left: dorsal right view of octopus showing position of stellate ganglion (arrow) attached to the inner surface of the dorsal mantle sac wall. Bottom right: ventral view of left stellate ganglion in the same orientation as top left, with epistellar body (E), stellate ganglion (SG), stellar nerve (SN), and mantle connective or pallial nerve (MC) indicated. light-dark cycle is maintained after removal of the epistel- lar body in sighted octopus (Cobb el ai, 1995a,b). In the present study, we examine the capacity of the octopod extraocular photoreceptor system for light detection by characterizing the extracellular and intracellular responses to light flashes of increasing intensity and duration in the octopus Eledone cirrhosa. Lucifer yellow was used to stain iontophoretically and identify the photoreceptor cells in the octopus epistellar body; there was no evidence for dye coupling between these extraocular photoreceptor cells. It is also established that the epistellar body sends afferent nerves that pass across the stellate ganglion and connect with the central nervous system, via the mantle connective (pallial) nerve. Materials and Methods Collection and maintenance of experimental animals The lesser or northern octopus, Eledone cirrhosa (dor- sal mantle length 5-14 cm) used in this study were caught offshore near the coast of Plymouth, UK, at a depth of 10-15 m and transported to laboratory holding tanks with flow-through, aerated seawater at 12-18C. To prevent the octopus from escaping, the side walls of the tank were lined with 1-cm-thick sheets of plastic foam (Boyle, 1981). Octopus were supplied with live crab (Carcinus maenits) food ad libitum and were maintained in the tank system for up to 9 months until required. Electrophysiology For electrophysiological recordings, an octopus was anesthetized with 3% ethanol in seawater and decapitated. The paired stellate ganglia and attached epistellar bodies were then removed from the dorsal mantle wall. The epi- stellar body and attached stellate ganglion were washed in fresh seawater and pinned out, ventral side up. in a Sylgard-lined recording dish filled with filtered artificial seawater (ASW: NaCl 470 mM, KC1 lOmM, MgCl : 50 mM, CaCl : 10 mM, MOPS (3-[N-Morpholine]pro- pane-sulfonic acid) 10 mM, pH adjusted to 7.8, osmolarity 1010 mmol/kg). All subsequent electrophysiological re- cordings were made at room temperature (between 18 and 23C). Illumination stimuli were provided by aSchott KL1500 cold light source, with a quartz halogen bulb (Thorn EMI 15 V. 150 W). The light was passed through a standard heat filter into a glass fiber light guide and then, via an electronically controlled shutter and a second fiber light guide, to the ventral side of the stellate ganglion and epistellar body. The duration of the light flash was set by the electronic shutter (Uniblitz TI32. Optilas Ltd, UK) to between 10 ms and 10 s. The light intensity was varied by inserting neutral density filters into the light path and was measured at the preparation level using a portable calibrated radiometer (Haling Electro-optics, UK: model 27-5479). An additional, uncalibrated photocell was used to record the precise timing and duration of the stimulus light flashes. In some experiments, a red filter (Kodak 1A, UK, wavelength >650nm) was introduced into the light path. Suction electrodes were used to take extracellu- lar recordings from the mantle connective (pallial) nerve of the stellate ganglion in response to flashes of white or red light that were applied to the epistellar body of the preparation from two octopus. The extracellular receptor potentials were recorded by inserting a low-resistance microelectrode (2 M?2 resis- tance when filled with ASW) into the epistellar body wall. Intracellular recordings from photoreceptor cells in isolated intact epistellar bodies were made using high- resistance microelectrodes of borosilicate glass capillaries with inner filaments (Clark Electrochemical, UK, GC- 150F, 1.5 mm OD X 0.86 ID), filled with 3 M KC1 and having tip resistances of 30-150MS1 A conventional 80 C. S. COBB AND R. WILLIAMSON microelectrode amplifier (AxoClamp 2B amplifier, Axon Instruments, Inc. USA) was used for recording resting, venerator, and receptor potentials and for injecting current pulses through the intracellular microelectrode. In some experiments, octopus photoreceptor cells were injected iontophoretically with the fluorescent napthalimide dye Lucifer yellow CH (Stewart. 1978; Sigma, UK). For this the microelectrodes were back-filled with 3% Lucifer yel- low in 1 M LiCl and had resistances of 150-200 MO. Injections of hyperpolari/ing current ( 1.0 nA at 1 Hz) re- sulted in rapid movement of Lucifer yellow into the cells, and dye filling was considered complete after 30 min. Resting potential was recorded during cell impalement and generator potentials were recorded in response to lieht flashes, before switching to current injection in these experiments. Immediately after dye injection, the prepara- tion was photographed in whole mount under an epiflu- orescent microscope, using color film (400 ASA). For normal recordings, the signal from the microelec- trodes was amplified and, together with the signal from the photocell monitor, was passed to a computer-con- trolled signal averager (CED 1401 computer interface running Sigavg software, Cambridge Electronic Design, UK). Typically, 5-10 responses were averaged to im- prove signal-to-noise ratios, and repeated light flash stim- uli were separated by at least 20 s. The octopus prepara- tions remained viable for at least 6 h. Illumination of the experimental 'darkroom' was provided by red safelight (>650nm). Illumination of 0.1 ^W/cnr with this red light was found not to cause a decrement in photore- sponse. Epistellar body iniiervutio/i Innervation of the epistellar body was studied by ortho- dromic filling from the epistellar body using the lipophilic dye Di-I (Honig and Hume, 1989) in 10 animals. With the aid of a dissecting microscope, the ASW was removed from the dish and a crystal of the fluorescent carbocyanine lipophilic dye Di-I ( l,l'-dioctadecyl-3,3,3',3' tetramethyl- indocarbocyanine perchlorate, D-282 (Di-I C18(3)). Mo- lecular Probes, Inc. USA) was placed inside the epistellar body of each stellate ganglion through an incision made with a tine steel pin or razor blade. The tissues were then fixed in 2.5% paraformaldehyde in 0.1 M phosphate buffer. A microscope with epitluorescence and a rhoda- mine filter (Nikon DM580) was used to map the progress of the orange Di-I fluorescence from the epistellar body, across the stellate ganglion, through axons and into the pallial nerve over several days. Tissues were cleared in 60% (w/v) meglumine lothalamate with 0.01% (w/v) so- dium calciumedetate and 0.01% (w/v) sodium acid phos- phate (product: Conray 280. May & Baker Ltd (Rhone- nc Group). UK; Zill el cii. 1993), before the Di-I stained axons were photographed (400 ASA Kodak black- and-white film), under the epifluorescence microscope with rhodamine filter set. Additional white light illumina- tion was sometimes used to observe and photograph the relative positions of fluorescent Di-I-filled axons and the stellate ganglion tissue. Results Ancitonn- mid innervation of the octopus epistellar body Figure 2 shows the size, shape, and position of the epistellar body that contains the extraocular photorecep- tors on the stellate ganglion of Eledone cirrhosa. The epistellar body is located at the ventral posterior margin of the ganglion and is commonly spherical (Fig 2A). al- though it sometimes appears divided into two or more compartments (Fig 2B). It is confined within the common capsule of connective tissue surrounding the ganglion, but is separated from the ganglionic neuronal cell bodies by one or more sheath layers. The epistellar body is usu- ally orange in freshly dissected preparations, but the color may fade with time after dissection or be absent in some animals, particularly larger, older specimens. In the course of the intracellular recordings described below, some photoreceptor cells (/; = 4) were injected Ki(juri' 2. Epistellar body (arrows), stellate ganglion (SGl, and stel- lar nerves (SN) of the octopus Eledone cin-linxii. The epistellar body is commonly spherical (A), but sometimes appears to be divided into two compartments (B). Scale bars are 1 mm. OCTOPUS PHOTORECFPTOR ELECTROPHYSIOLOGY AND INNERVAT1ON 81 with Lucifer yellow, a fluorescent dye (Fig. 3). The dye fills showed that the photoreceptors have a cell soma (50 fj.m) that is located in the wall of the epistellar body and gives rise to a single axon, extending towards the periphery of the epistellar body. The axons from separate photoreceptor cells converge outside the epistellar body to form the 'epistellar' nerve. In addition, a process that may be as long as lOO/um projects towards the center of the epistellar body, sometimes dividing or branching toward its distal end. No dye coupling between photore- ceptors was observed in any of the cells that were well filled with dye. Figure 4A shows an epistellar body, the attached stel- late ganglion, and the connection of the mantle connective (pallial) nerve to the ganglion. For all cases (n = 8) in which the epistellar body and attached stellate ganglion preparations were filled with fluorescent Di-1 (Fig. 4B, C, D), orthodromic fills indicated that the epistellar body gave off an 'epistellar' nerve (Fig. 4B, C, D) that passed across the stellate ganglion, into the mantle connective (pallial) nerve (Fig. 4B, C), and then, presumably, to the brain. The nerve bundle was more shallow in its path across the stellate ganglion in some preparations (n = 2) than in others. In these preparations, the epistellar nerve remained intact and branched, sometimes into three sepa- rate fiber bundles (Fig. 4B), only before entry to the man- tle connective (pallial) nerve. In addition, these prepara- tions showed a single nerve branch, which occurred close SO^im Figure 3. lontophoretic microinjection of Lucifer yellow into an epistellar body photoreceptor cell. Detail of photoreceptor cell filled with Lucifer yellow showing cell soma (1) and axon process (2). Note long, branched process (arrows) that extends toward the center of ihc epistellar body. Figure 4. Innervation of the epistellar body. (A) Epistellar body and stellate ganglion of the octopus Eledone cirrhosa. The ovoid epistel- lar body (E) lies at the posterior end of the stellate ganglion near the origin of several stellar nerves (SN), opposite the mantle connective (pallial) nerve (MC). Scale bar is 1 mm. (B) Whole mount of epistellar body and stellate ganglion with orthodromic fluorescent Di-I-filled 'epi- stellar' nerve (arrow), epistellar body (E), stellar nerves (SN). and mantle connective (pallial) nerve (MC). Scale bar is 1 mm. (C) Whole mount of stellate ganglion with orthodromic fluorescent Di-I-filled epistellar body photoreceptor cell axons (arrows) connecting the epistellar body (E) and the mantle connective (pallial) nerve (MC). Scale bar is 1 mm. (D) Whole mount as in (C) under rhodamine-filtered light only, showing detail of Di-I-filled axons. Scale bar is I mm. to the epistellar body. This branch separated from the epistellar nerve and passed deep into the stellate ganglion, to an unidentified destination (Fig. 4B). However, in most preparations examined (n = 8), the Di-I-filled epistellar nerve branched close to the epistellar body, forming nu- merous nerve bundles that passed into the mantle connec- tive nerve and then toward the central nervous system (Fig. 4C. D). In addition to the epistellar nerve, these preparations showed two Di-I-filled nerves running from the side of the epistellar body in parallel to and on each side of the epistellar nerve (Fig. 4C. D). These parallel 82 C. S. COBB AND R WILLIAMSON nerve bundles then appeared to focus on the central path of the epistellar nerve bundles and pass into the center of the stellate ganglion (Fig. 4C, D). perhaps to the neuropil. The destination remains conjecture, however, because none of the preparations examined showed conclusive evidence of interaction between a Di-I-filled nerve fiber and the neuropil of the stellate ganglion. Recordings from the mantle connective (pallial) nerve To test whether light-evoked activity from the epistellar body could be observed in the mantle connective (pallial) nerve, suction electrode recordings were made from the cut end of the nerve as controlled light flashes were ap- plied to the epistellar body. When white light was flashed, a compound action potential was observed, mainly com- prising a large downward trough (Fig. 5). To test for artifacts and to determine the spectral sensitivity of the suction electrode response, a flash stimulus of red light was produced by using a >650 nm filter. Whereas a flash of white light evoked an extracellular voltage response in the mantle connective (pallial) nerve, a flash of red light, of the same duration, evoked no response (Fig. 5). The relatively long delay between stimulus and response does not permit any conclusions to be drawn about whether this is a direct or post-synaptic response; clearly the former would be expected. Electrophysiology of the epistellar body photoreceptors Generator potential responses induced by light flashes of constant duration but increasing intensity were re- corded extracellularly from the epistellar body photore- ceptors of the octopus, Eledone cirrhosa (Fig. 6A). The generator potential appeared as a short-latency downward deflection of the voltage trace, and the amplitude of the evoked response increased with increasing flash intensity. A graph of response against stimulus intensity (Fig. 6B) shows that the amplitude of the evoked response was well correlated to the log of light intensity across the entire range examined (Fig. 6B). The latency between the start Red Hash 400ms White flash Figure 5. Light-evoked extracellular responses in the mantle con- nective (pallial) nerve of the stellate ganglion preparation recorded by suction electrode in response to flashes of white and red light. Duration of the light Hash applied to the epistellar body is indicated in the bottom 250ms 50 uV B 150 -i 3- ioo - Si I 50H Flash intensity (u\V/cm ) I 300 1 200 ' ^ 100 - 100 " 1 50 I U 25 2 80 Flash intensity (uW/cm ) Figure 6. Epistellar body responses to change in intensity of the light stimulus. (A) Upper traces show generator potentials recorded extracellularly from photoreceptor cells in the epistellar body in response to a series of 250-ms-duration light flashes, as indicated in the lower trace, of increasing intensity. (B) Graph of log light-flash intensity against extracellular response amplitude. (C) Latency and rise time of the extracellular response with increasing stimulus intensity. of the stimulus and the start of the photoreceptor response was between 40 and 25 ms, and this latency decreased with increased stimulus intensity (Fig. 6C). Increasing stimulus intensity was also linked with a decrease in the time from the start of the stimulus to the peak of the response or rise time (Fig. 6C). The epistellar photorecep- tor cells needed some time to recover from each light flash: the extracellular response generated by a second flash given 2.5 s after the first was reduced by almost 80% (Fig. 7A). This decrease in response was evident up to 30 s after the first light flash. A plot of the response amplitude to the second flash (Fig. 7B) showed that the recovery had an exponential time curve with a time con- stant of almost 6 s. Similarly, the size of the extracellular response to a single light flash was affected by the inten- sity level of ambient illumination (Fig. 8). Figure 8A shows the extracellular responses to constant light flashes given after at least a I -min exposure to three different OCTOPUS PHOTORECEPTOR ELECTROPHYSIOLOGY AND INNERVATION 83 50 mV _n B 5. 100 i 80 - 60 - I I U 40 - 20 - T = 5.9s 10 15 20 25 30 Delay between flashes (s) 35 Figure 7. Adaptation or recovery responses between light flashes to the epistellar body. (A) Upper traces show examples of photoreceptor cell generator potentials recorded extracellularly from the octopus epi- stellar body in response to a series of 200-ms-duration light flashes with increased delay between flashes, but constant intensity (34 /vW/cnr), as indicated in the lower trace. (B) Amplitude of the extracellular response with increasing delay between flashes. intensity levels of 'background' illumination. It can be seen that the greater the level of background illumination, then the smaller the extracellular response to the light flash; Figure 8B shows that the relationship is more-or- less exponential. Intracellular recordings from individual photoreceptor cells demonstrated that, in the dark, these had membrane resting potentials of about 49 7 mV (mean SD, n = 43); such recordings could be maintained for up to 45 min. Epistellar body photoreceptors responded to a short flash of white light with a depolarization that often re- sulted in the firing of a burst of action potentials (Fig. 9). Although not studied in detail, this response was depen- dent on the wavelength of the light stimulus, for stimuli at wavelengths greater than 650 nm evoked no response (Fig. 9). This also provided confirmation that the response was not artifactual or related to other stimuli, such as the noise of the mechanical shutter. The amplitude of the intracellular receptor potential response varied with the intensity of the light flash (Fig. 10A). A graph of these responses (Fig. 10B), demonstrated that the amplitude of the depolarization was well correlated to the log of the light-flash intensity across the intensity range examined. Similarly, Figure 10B illustrates that the peak firing fre- 250ms SuV _n B v 1 I o. i 10- 20 Background illumination 40 Figure 8. Effect of background illumination on the response of the epistellar body to light-flash stimuli. (A) Upper traces show examples of photoreceptor cell generator potentials recorded extracellularly from the octopus epistellar body in response to a 200-ms-duration light flash (intensity 167 //W/cnr), as indicated in the lower trace, while subjecting the epistellar body to increased background illumination. (B) Amplitude of the extracellular response with increasing background illumination. In all cases each point represents the mean SEM, n = 10 separate receptor responses. quency of the resulting action potentials was well corre- lated to the log of the stimulus intensity and the conver- sion factor from receptor potential to action potentials or 'spikes' is about 2 spikes/mV depolarization; that value is within the range of other invertebrate spike encoding photoreceptor cells for example 1 spike/mV in eccen- Figure 9. Intracellular receptor potential's response to white and red light (>650 nm) stimuli recorded from the same photoreceptor cell in an epistellar body. The lower trace indicates the time and duration of 250-ms light flashes. Light-flash intensity was 167 ^iW/cnr and resting potential was 53 mV in both cases. 10 mV C. S. COBB AND R. WILLIAMSON Flash intensity (uW/cm) Figure 10. Typical intracellular receptor potentials recorded in a photoreceptor cell from an epistellar body, in response to change in light- Hash stimulus intensity. Resting potential was \2 mV to 47 mV. (A) The upper traces show the photoreceptor potentials in response to the series of 250-ms-duration light flashes, as indicated in the lower trace in response to increased intensity. (B) Graph of light-flash intensity against response receptor potential amplitude and peak firing frequency. (C) Rise time and latency of receptor potentials against increased light stimulus intensity. In all cases each point represents the mean SEM. n = 10 separate receptor potential responses to light-flash stimuli from a single photoreceptor cell. In some cases the SEM is too small to be visible. slowly decaying back to the normal resting potential for the photoreceptor cell (Fig. 11); this probably indicates that the action potentials do not actively invade the cell soma and are thus attenuated or absent in some soma recordings. Discussion A number of morphological and biochemical studies have shown that octopod and decapod cephalopods have extraocular photoreceptors (reviewed in Messenger, 1991). Young (1936) first described epistellar structures in several octopod genera: from histological considera- tions, he hypothesized that these might have a neurosecre- tory function. The epistellar body of Octopus vulgaris was later re-investigated in detail using electron micros- copy and found to contain cells that have microvilli very like those found in the rhabdomeres of the extraocular photoreceptors seen in other molluscs and arthropods (Nishioka et al.. 1962). This work was later extended to include the epistellar bodies of Eledone moschata (Nishi- oka et al., 1966); in it. electron microscopy gave further evidence for a rhabdomeric ultrastructure, and biochemi- cal evidence indicated the presence of the photopigment rhodopsin. These studies thus implied that the neuronlike cells of the epistellar body were not neurosecretory. but photoreceptor cells. This agreed with comparative studies showing the presence of extraocular photosensitive cells in the central nervous systems of many other inverte- brates for example, in the caudal ganglion of the cray- fish (Kennedy, 1963) and the molluscs Aplysiu californica (Arvanitaki and Chalazonitis, 1961) and Onchidiwn ver- ruculatiim (Hisano et til.. 1972). Preliminary electrophysi- ological evidence demonstrating a light-evoked response from cephalopod epistellar body photoreceptor cells was first obtained by Mauro and Baumann ( 1968) in an octo- pus, Eledone moschatu. Decapod cephalopods such as the trie cells from Linuilns polypheniiis (Behrens and Wulff. 1965). As already seen for the extracellular compound generator potentials, the time-to-peak depolarization for individual photoreceptor cells decreased with increasing stimulus intensity (Fig. IOC). However, stimulus-to-re- sponse latency for individual photoreceptor cells was fairly constant at around 25 ms for flashes of intensity greater than 5 /jW/cm 2 , but longer for very weak flashes (Fig. IOC). The effect of long-duration (5 s and l()s) but constant-intensity stimuli on the intracellular photore- sponse was examined. There was an increase in the dura- tion of the receptor potential as the light stimulus was lengthened from 5 s to 10s, with the long-lasting plateau maintained for the duration of the stimulus and then 5s SmV Figure 11. Intracellular receptor potentials recorded in a single pho- toreceptor cell from an epistellar body in response to constant intensity (77 ^/W/cnr) and increased long-duration light stimuli. The receptor potentials recorded from a 5-s and 10-s light flash are shown as indicated by the stimulus marker in the bottom trace. Resting potential was -50 mV. OCTOPUS PHOTORECEPTOR ELECTROPHYSIOLOGY AND INNERVATION 85 squid Loligo vulgaris and the cuttlefish Sepia officinalis also have PS Vs. or 'parolfactory vesicles': these are not located on the stellate ganglia, but lie underneath the cranial cartilage casing on and below the optic tract (Young, 1936; Perrelet and Mauro, 1972). These PSVs also have neuronlike cells with a rhabdomeric ultrastruc- ture containing rhodopsin (Nishioka e! al., 1966; Bau- mann ct til.. 1970) and their responses to light stimuli have been briefly reported for Todarodes sagittatus (Mauro and Sten-Knudsen, 1972; Mauro. 1977). Loligo pealei and Loligo forbesi (Sperling et al., 1973; Cobb and William- son. 1998). Electrophysiology of extraocular photoreceptor cells Using extracellular and intracellular recordings from extraocular photoreceptors in Eledone cirrhosa, the pres- ent study extends our understanding of the electrophysiol- ogy of epistellar body photoreceptors in cephalopods. The results show that the photoreceptors have cell resting po- tentials of about -49 7 mV and respond to a flash of light with a depolarization consisting of a transient component, often accompanied by a burst of action poten- tials, followed by a steady-state or plateau depolarization. The amplitude of the evoked depolarization and the peak firing frequency of the cell were directly correlated with the intensity of the light flash. No 'quantum bumps' were observed in these recordings, although these have been reported in recordings from the extraocular photorecep- tors of Eledone moschata (Mauro and Baumann. 1968). as well as from other invertebrates (Lisman and Brown, 1975). The extracellular generator potentials recorded from the epistellar body of Eledone cirrhosti showed adapta- tional changes when the level of ambient illumination was increased in this study. This agrees well with the adaptational changes seen in retinal photoreceptors for cephalopods (Weeks and Duncan, 1974) and other inver- tebrates (Laughlin. 1989). It should be noted that there are no reports of screening pigments in cephalopod extra- retinal photoreceptors, so the mechanism of screening pigment migration cannot be invoked here. The observed decrease in the stimulus-to-response latency of the evoked intracellular depolarization (from about 40 ms to 25 ms) with increasing intensity is similar to that seen in photore- ceptor latency measurements from the epistellar bodies of both Eledone moschata and Eledone cirrhosti (Mauro, 1977; Cobb et al., 1995b) and also from the retina of Sepiola atlantica, a sepiolid cephalopod (Duncan and Weeks, 1973). The small differences between the latency values reported in this study (40 ms) and in the others (30 and 23 ms, respectively) are probably due to the different stimulus intensities used, for as shown above, the onset delay decreases with stimulus intensity. When two extraocular photoreceptor cells from the same cluster in a parolfactory vesicle from a squid (Loligo pealei) were impaled simultaneously, partial electrical coupling was recorded in 1 out of 4 pairs of cells (Sperling et al.. 1973). In the present study, which used extraocular photoreceptor cells from the epistellar body of an octopus (Eledone cirrhosa), no evidence of Lucifer yellow dye coupling was observed. The lack of dye coupling may perhaps indicate a lack of electrical coupling, for the two are often well correlated, as, for example, in the horizontal cells of the turtle retina (Stewart, 1978) and in the pineal gland photoreceptors of teleost fish, where numerous gap junctions are present (Omura, 1984), and Lucifer yellow dye coupling has been observed (Nakamura et al.. 1986). However, electrical coupling is not always associated with dye coupling, particularly in molluscan preparations (Williamson. 1989; Ewadinger et al., 1994). Light-flash stimuli transmitted through the red filter (Kodak 1 A, wavelength >650 nm) neither stimulated the PSVs from Eledone cirrhosa nor evoked an afferent re- sponse in the mantle connective nerve; therefore, this wavelength of red light O650 nm), at the intensities used in this study, did not stimulate the photoreceptors. This outcome was perhaps to be expected, for the photorecep- tors in both the retinal and extra-retinal systems of Ele- done moschata have a rhodopsin absorption maximum around 470 nm (Nishioka et al.. 1966; Hamdorf et al., 1968), and the red light stimuli are therefore likely to be outside the detection range of the photoreceptors, at the stimulus intensities employed here. Innen'ation of the epistellar body Orthodromic fills of the photoreceptor axons with fluo- rescent Di-I from the epistellar body of Eledone cirrhosa have shown nerve bundles passing from the epistellar body into the mantle connective (pallial) nerve. Microana- tomical studies, silver-staining techniques, and electron microscopy were used in studies of the octopuses Eledone moschata and Octopus vulgaris to trace the 'epistellar nerve' from the photoreceptor cells of the epistellar body, through the stellate ganglion, to the mantle connective (pallial) nerve (Young, 1936; Cazal and Bogoraze, 1944; Nishioka et al., 1966; Perrelet and Mauro, 1972). In addi- tion, degeneration of nerve fibers running to the epistellar body was shown in E. moschata after the mantle connec- tive nerve was cut (Young, 1936). The mantle connective (pallial) nerve is known to contain about 16,000 nerve fibers (Young, 1965), with two groups of efferent, and possibly two groups of afferent fibers (Young, 1971). In E. moschata, the epistellar nerve contains about 1500 afferent nerves (Perrelet and Mauro, 1972). Therefore, the selective nerve tracing in this study (using Di-I and electrophysiological recordings from the mantle connec- 86 C. S. COBB AND R. WILLIAMSON live (pallial) nerve) presents new evidence supporting the view that the epistellar nerve contains the afferent axons of extraocular photoreceptor cells, and that these axons conduct photoreceptive information to the central nervous system in the octopus. Whether such sensory information passes to the palliovisceral lobe of the octopus brain from nerves earned in the mantle connective (pallial) nerve remains to be determined. In summary, this study provides further evidence for the presence of an active extraocular photoreceptor sys- tem in adult cephalopod molluscs. However, the func- tional role of the epistellar body and parolfactory vesicles in octopus and squid still remains enigmatic. The lack of a structurally organized retina or optical apparatus such as a lens, and the relatively deep location of all these PSVs argues against any function in visual image forma- tion. Rather, it is generally assumed that in both octopods and decapods the extraocular photoreceptors play a role in monitoring ambient light levels over lengthy periods; that is, they function as a photometer (Mauro. 1977; Houck, 1977a.b), perhaps connected with seasonal repro- ductive activity (Baumann et ui. 1970), control of circa- dian activity rhythms in octopods (Houck, 1977ab, 1981, 1982), diel vertical migration in squid (Palmer and O'Dor. 1978). or even the feedback control of ventral photo- phores in midwater squid such as Abralia trigoniiru (Young, 1972, 1973; Young et . Eye. Res. 17: 183-192. Kwadinger, N. W., N. I. Syed, K. Lukowiak, and A. G. M. Bulloch. 1994. Differential tracer coupling between pairs of identified neu- rones of the mollusc Lvmnaea stagnalis. J. E.\p. Biol. 192: 291 297. Hamdorf, K., J. Schwemer, and U. Taiiber. 1968. Der Sehfarbstoff, die Absorption der Rezeptoren und die spektrale Empfindlichkeit der Retina von Eledone moschata. Z. Vergl. Physio/. 60: 375 H5. Hara, T., and R. Hara. 1980. Retinochrome and rhodopsin in the extraocular photoreceptor of the squid. Todarodes. J. Gen. Physiol. 75: 1-19. Hisano, N., H. Tateda, and M. Kmvabara. 1972. Photosensitive neurons in the marine pulmonate mollusc, Onchidium verruculatum. J. E.\p. Biol. 57: 651-660. Honig, M. G., and R. L. Hume. 1989. Oil and DiO: versatile fluo- rescent dyes of neuronal labelling and pathway tracing. Trends Neu- rosci. 12: 333-341. Houck, B. A. 1977a. A morphological and behavioral study of an extra-ocular photoreceptor in octopods. Ph.D. Thesis, University of Honolulu, Hawaii. Houck, B. A. 1977b. Photoreception in octopods: The role of ocular and extra-ocular photoreception in maintenance of locomotor activ- ity rhythms [Abstract], Am. Zool. 17: 969. Houck, B. A. 1981. Locomotor activity and home selection in three species of Hawaiian octopods [Abstract]. Am. Zool. 21: 967. Houck, B. A. 1982. Temporal spacing in the activity patterns of three Hawaiian shallow-water octopods. Nautilus 96: 152-156. Kennedy, D. 1963. Physiology of photoreceptor neurons in the ab- dominal nerve cord of the crayfish. 7. Gen. Phyxinl. 46: 551-572. Laughlin. S. B. 1989. The roles of sensory adaptation in the retina. J. E.\p. Binl. 146: 39-62. I isman, J. E., and J. B. Brown. 1975. Light-induced changes of sen- sitivity in Limulus ventral photoreceptors. 7. Gen. Physiol. 66: 473- 488. Mauro, A. 1977. Extra-ocular photoreceptors in cephalopods. Symp. Zool. Soc. Ltiiul. 38: 287-308. Mauro, A., and F. Baumann. 1968. Electrophysiological evidence of photoreceptors in the epistellar body of Eledone moschata. Nature 220: 1332-1334. Mauro, A., and (). Sten-Knudsen. 1972. Light-evoked impulses OCTOPUS PHOTORECEPTOR ELECTROPHYSIOLOGY AND INNERVATION 87 from extra-ocular photoreceptors in the squid Todarodes. Nature 237: 342-343. Messenger, J. B. 1991. Photoreception and vision in molluscs. Pp. 364-397 in Evolution of the Eye and Visual System. J. R. Cronly- Dillon and R. L. Gregory, eds. Macmillan, London. Nakamura, T., G. Thiele, and H. Meissl, H. 1986. Intnicellular re- sponses from the photosensitive pineal organ of the teleost. Phoxintis phoxinux. J. Comp. Physio/. A. 159: 325-330. Nishioka, R. S., I. Yasumasu, and H. A. Bern. 1962. Ultrastructure of the epistellar body of the octopus. Z. Zellforsch. Mikrosk. Anat. 57: 406-421. Nishioka, R. S., I. Yasumasu, A. Packard, H. A. Bern, and J. Z. Y'oung. 1966. Nature of vesicles associated with nervous system of cephalopods. Z. Zellforsch. Mikrosk. Anat. 75: 301-316. Omura, Y. 1984. Pattern of synaptic connections in the pineal organ of the ayu. Plecog/ossus altivelis (Teleostei). Cell Tissue Res. 236: 611-617. Ozaki, K., R. Hara, and T. Mara. 1983. Histochemical localization of retinochrome and rhodopsin studied by fluorescence microscopy. Cell Tissue Res. 233: 335-345. Palmer, B. W., and R. K. O'Dor. 1978. Changes in vertical migration patterns of captive lllex illecebrosus in varying light regimes and salinity gradients. Tech. Rep. Fish. Mar. Sci. Canada 833: 23.1- 23.12. Perrelet, A., and A. Mauro. 1972. Ultrastructure of nerves associated with the epistellar body of the octopod Eledone moschata and the parolfactory vesicles of the squid Todarodes sagittatus. Brain Res. 37: 161-171. Sperling, L., J. E. Lisman, and A. Godfrey. 1973. Light-evoked responses from the ventral parolfactory vesicles of Lot/go pealei [Abstract]. Biol. Bull. 145: 456. Stewart, W. W. 1978. Functional connections between cells as re- vealed by dye-coupling with highly fluorescent naphtalimide tracer. Cell 14: 741-754. Sundermann, G. 1990. Development and hatching state of ectodermal vesicle organs in the head of Sepia officinalis, Loligo vulgaris and Loligo forbesi (Cephalopoda. Decabrachia). Zoomorphologv 1(19: 343-352. Weeks, F. I., and G. Duncan. 1974. Photoreception by a cephalopod retina: response dynamics. Exp. Eye Res. 19: 493-509. Williamson, R. 1989. Electrical coupling between secondary hair cells in the statocyst of the squid Alloteuthis sitbiiluta. Brain Res. 486: 67-72. Young, J. Z. 1929. Sopra un nuovo organo dei cefalopodi. Boll. Soc. Iinl. Biol. Sper. 4: 1022-1024. Y'oung, J. Z. 1936. The giant nerve fibres and epistellar body of ceph- alopods. Q. Jl. Microsc. Sci. 78: 367-386. Y'oung, J. Z. 1965. The diameters of fibres of the peripheral nerves of Octopus. Proc. R. Soc. Land. B. 162: 47-79. Young, J. Z. 1971. The Anatomy of the Nen'ous system of Octopus vulgaris. Clarendon Press. Cambridge. Young, R. E. 1972. Function of extra-ocular photoreceptors in bathy- pelagic cephalopods. Deep-Sea Res. 19: 651-660. Young, R. E. 1973. Information feedback from photoreceptors and ventral countershading in mid-water squid. Pac. Sci. 27: 1-7. Young, R. E., C. F. E. Roper, and J. F. Walters. 1979. Eyes and extraocular photoreceptors in midwater cephalopods and fishes: Their roles in detecting downwelling light for counterillumination. Mar. Biol. 51: 371-380. Zill, S. N., S. F. Frazer, D. L. Maclarland, and S. E. Fish. 1993. Characterization of insect sense organs and optical clearing of whole- mount preparations using Dil in fixed tissues. J. Exp. Biol. 175: 299-303. Marine Biological Laboratory Woods Hole Massachusetts One Hundreth Report for the Year 1997 One-Hundred and Ninth 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 James D. Ebert, President of the Corporation John E. Burris, Director and Chief Executive Officer Mary B. Conrad, Treasurer Neil Jacobs, Clerk of the Corporation Contents Report of the Director and CEO Rl Report of the Treasurer R7 Financial Statements R8 Report of the Library Director R19 Educational Programs Summer Courses R22 Short Courses R26 Other Programs R31 Summer Research Programs Principal Investigators R33 Other Research Personnel R34 Library Readers R36 Institutions Represented R37 Year-Round Research Programs R42 Honors R53 Board of Trustees and Committees R59 Administrative Support Staff R62 Members of the Corporation Life Members R65 Members R66 Associate Members R76 Certificate of Organization R79 vi tides of Amendment R79 .Bylaws . R79 Photo credits: W. Amos R19 Beth Armstrong R2 (top), R34, R37, R43 Ken Foreman R5 (bottom), R31 Linda Colder R59, R65 Roger Hanlon R3 (top), R62 Richard Howard R4 (top), R5 (top), R22, R26, R30, R78 Tom Kleindinst R45 Alan Kuzirian R53 Beth Liles, R49 George Lower R4 (bottom), R7 Chris Neill, R42 Bruce Peterson R47 Janice Reed Rl Report of the Director and Chief Executive Officer The Marine Biological Laboratory had another exciting and successful year in 1997. We set our sights high and achieved a number of major goals for the Laboratory. We launched the first Semester in Environmental Sciences program for undergraduates from liberal arts colleges; recruited new investigators for our resident research programs; planned for and established new advanced summer courses; and found new and expanded ways to capitalize on the flexibility and sophistication of the Marine Resources Center. These endeavors were supported by the tangible and intangible resources and energies generated by the laboratory's new fundraising campaign. Discovery: The Campaign for Science at the Marine Biological Laboratorv, which was launched with a public celebration and street fair on August 8. 1997. In my report last year. I discussed the concerted planning process that set the stage for this campaign. By focusing our research and educational directions and priorities, we were able to announce last August a campaign goal of $25 million to be raised by December 31, 2000. I am pleased to say that, as of the spring of 1998, we have already raised $14.5 million towards that goal. I have every reason to believe that we will meet, if not exceed our goal within the timeframe of the campaign. Research at the MBL We made great strides in the MBL's resident research centers and programs during 1997. The avenues for independent research broadened and diversified in each of MBL's programmatic areas, while our investigators found new ways to work together in various interdisciplinary pursuits. The Josephine Bay Paul Center Dr. Mitchell Sogin and his colleagues in the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution continued their important studies on the evolution of eukaryotes, most recently focusing on those found in extreme environments. An exciting development is a major grant from the National Institutes of Health to sequence the genome of Giurdia lamblia. a water-borne human pathogen. Giardiasis is a major contributor to the enormous burden of human diarrhea! diseases, which are second only to respiratory infections as causes of mortality and morbidity worldwide. While Giardia can be a deadly organism, it is manageable in terms of genome analysis because it has a relatively modest genome size of 12 million base pairs distributed onto five chromosomes. Dr. Michael Cummings was recruited to the Bay Paul Center in 1997. He comes to the Center to study Mycobacteria as a means of understanding more clearly the evolution of pathogenicity and how various strains of tuberculosis develop drug resistance. Another member of the Bay Paul Center, Dr. Monica Riley, continues to travel world-wide sharing her expertise on the E. coli genome. She was a co-author in September on a paper published in the journal Science which reports the successful sequencing of the genome of the K-12 strain of the bacterium. In 1997, Center scientist Dr. Neal Cornell and his co-workers conducted a molecular phylogenetic analysis for 5-aminolevulinate synthase, the first enzyme of heme biosynthesis. The analysis supports the suggestion that alpha-purple bacteria are the closest contemporary eubacterial relatives of eukaryotic mitochondria. The study also indicates that the massive gene duplication required for the evolution of true vertebrates had occurred when non-vertebrate jawless fish appeared, 400 to 500 million years ago. The Ecosystems Center The MBL's Ecosystems Center, co-directed by Drs. John Hobbie and Jerry Melillo, continues its leadership in environmental research on local and global scales. Rl R2 Annual Report For more than a decade, MBL scientists have led a Long-Term Ecological Research (LTER) project on the arctic tundra around Toolik Lake in Alaska. In 1997. scientists at the Center secured another LTER project grant of nearly $3.4 million over six years from the National Science Foundation to study the Plum Island Sound system in northeastern Massachusetts. An LTER grant is important scientifically and institutionally because it allows long-term data collection and analysis in the same location and provides a solid base of funding with which to recruit scientists and research technicians for the project. We are proud of the Ecosystems Center team because they were the only group that was funded out of 2 1 proposal submissions nationwide. In the southern hemisphere, our scientists have also been compiling data for more than a decade on the effects of deforestation on soil fertility and trace gas emissions in the rainforests in Brazil. Their work continues as they evaluate what effects changing and more aggressive agricultural practices are having on the Amazon Basin and the world. In 1997. the MBL received a challenge grant of $1 million from The Clowes Fund. Inc.. of Indiana, to provide support for new and expanded facilities for The Ecosystems Center. Thanks to this grant, the MBL is now in the early stages of planning renovations and expansion that will nearly double the Center's existing laboratory. Held staging, and office space. The plans also call for the creation of a new computer teaching laboratory in the Center. The Marine Resources Center Dr. Roger Hanlon has advanced his research agenda in the Marine Resources Center (MRC) with the introduction of the Program in Sensory Biology and Neuroethology and the Program in Scientific Aquaculture. An MRC Advisory Committee has been constituted to review research directions and scientist recruitment strategies. Members of the Committee include Drs. Gerald Fischbach, Harvard Medical School; Irwin Levitan, Brandeis University; and Vilayanur Ramachandran, University of California, San Diego. In the program on neuroethology. Dr. Hanlon and his colleagues study vision, balance, chemical sensing, and signaling to gain a better understanding of the output of neural activity, i.e., the resultant behavior of an organism that has evolved through natural selection. Towards that end. Dr. Hanlon has recently been awarded research grants that will enable molecular and cellular studies on squid and cuttlefish. These marine organisms possess very large neurons and have evolved dramatic and rapidly changing skin patterning displays as evidence of aggressive and reproductive behaviors. Last summer, Hanlon and his colleagues performed a series of experiments in the MRC in which they discovered that squids are able to detect transparent prey at greater distances thanks to a specialized way of seeing known as polarization vision. The result is enhanced predution and feeding by the squid. Hanlon' s findings appeared in the journal Nature in May 1998. The second MRC initiative, the Program in Scientific Aquaculture. seeks to select organisms with defined genetic lines or at precise developmental stages for use as biomedical models, to attract industry and Report of the Director and CEO R3 commercial ventures to develop and test products in the MRC. and to study molecular and cellular mechanisms of disease processes and prevention. Architectural Dvnamics in Living Cells Program MBL scientists continue as leaders in the design and use of advanced imaging systems to study the molecular definition and physical forces that affect the structure and function in living cells. Dr. Shinya Inoue, MBL Distinguished Scientist, received additional honors in 1997 for his contributions to light microscopy and was awarded the Ernst Abbe Award for Microscopy by the New York Microscopical Society. Inoue and his colleagues announced at the 1997 meeting of the American Society of Cell Biology the development of the Centrifuge Polarizing Microscope. This new microscope allows researchers to view architectural changes in cells at the subcellular level when exposed to high gravitational forces. This technical feat was the fruit of collaboration between scientists at the MBL, Olympus Optical Co., and Hamamatsu Photonics Corp. Dr. Rudolf Oldenbourg and his associate Kaoru Kato, together with Dr. Peter Smith and Katherine Hammar of the BioCurrents Research Center, reported in 1997 on their observations of the dynamic behavior of actin molecules at the tip of growing nerve cells. These observations were made possible by the exceptionally high sensitivity and sharp image quality of Oldenbourg' s Pol-Scope, which was developed in collaboration with Cambridge Research and Instrumentation, Inc. Laboratoty for Reproductive Medicine Dr. David Keefe, of the Laboratory for Reproductive Medicine of Brown University and the Women and Infants Hospital in Providence, R.I., established a resident laboratory at the MBL in 1997. Here he investigates the mechanisms underlying age-related female infertility. Keefe uses technologies developed at the MBL to seek out clues that indicate whether an egg has been damaged with age and is therefore not viable. With the ion selective probe, developed in Peter Smith's BioCurrents Research Center, Keefe can detect the flow of certain ions in and out of the cell. This flow may reflect an egg's potential to develop successfully. He is also using Oldenbourg's Pol-Scope to study the egg's spindle; flaws in the spindle may be responsible for the faulty transfer of genetic material during cell division. Laboratory of Aquatic Animal Health and Pathology In 1997, scientists from the University of Pennsylvania's Laboratory of Aquatic Animal Health and Pathology, which is based at the MBL, continued their efforts to identify and treat diseases affecting marine organisms. They identified in toadfish an abcess of the abdominal organs caused by Pseudomonas sp. bacteria and a bacterial pericarditis disease caused by Edwardsiella sp. Dr. Roxanna Smolowitz continued her effort to culture QPX and describe the pathogenesis of the disease in susceptible strains of clams, while Dr. Robert Bullis and colleagues worked on ways to detect illegal chemical scrubbing of eggs in female lobsters. Boston University Marine Program (BUMP) BUMP Postdoctoral Fellow Frank Grasso organized and co-hosted an international workshop on "Plume Tracing" last year. Twenty-five scientists presented and debated the state-of-the-art in our knowledge of animals R4 Annual Report following odor plumes to their source of release. Lobsters are animals that locate odor sources (for example, baited traps) under water without visual or auditory signals. Scientists in Dr. Jelle Atema's laboratory have developed a tool known as Robo-Lobster to perform the plume tracing task and to learn about the sensory input and signal processing used by lobsters. Summer Research The MBL's laboratories were again tilled with scientists from all over the world during the summer of 1997. We welcomed back investigators with whom we have worked for a number of seasons and were able to share in the enthusiasm of scientists working here for the first time, either as fellows or as new principal investigators. More than 400 scientists came to the MBL last summer to do their research, including 23 research fellows who received more than $171,000 in fellowship awards. The summer research season is framed by the Poster Session in June and is capped off by the General Scientific Meetings in August. These events attest to the breadth and diversity of the multidisciplinary work at the MBL where presentations range from papers in ecology and population biology to cellular and molecular biology to vision and biophysics. One of the highlights of the summer research season was Dr. Stephen Highstein's (Washington University) continuing work on the vestibular system of toadfish. The toadfish is an excellent marine model for studying how changes in pressure affect balance and equilibrium, because its vestibular system is similar to humans. Such studies teach us about motion sickness and dizziness, which are often signs of vestibular organ dysfunction. Highstein and his colleagues are focusing on the problems of nausea and dizziness experienced by astronauts when they get out beyond the earth's gravity, a malady known as "space adaptation syndrome." As I write, four toadfish are passengers aboard the space shuttle Columbia, participants in an experiment designed to help scientists understand what happens in the vestibular system during an extended period in space. Another area of research that is becoming increasingly important at the MBL is evolution and development or "evo-devo." Researchers from all over the world come to the MBL to learn more about how the dividing cells in an embryo decide which physiological role they will assume as the organism grows. For example, Drs. Mark Q. Martindale (University of Chicago), Jonathan Q. Henry (University of Illinois), and Barbara Boyer (Union College) take advantage of the wide variety of marine organisms available at the MBL to compare the developmental patterns of different kinds of embryos. They are studying relatively primitive animals like ctenophores whose development is simple enough that scientists can trace how a change in one cell or set of cells affects the whole organism as it grows. Ultimately, they hope to understand more clearly when and how organisms have diverged from their evolutionary ancestors. The MBL's General Scientific Meetings are held annually in mid-August. This three-day meeting is an opportunity for summer and resident scientists to report on their most recent research results. One highlight from the 1997 meeting is the finding from the laboratory of Robert Barlow (SUNY Health Science Center) that the lateral eyes of two species of horseshoe crabs the Woods Hole species Liiuuhis polvpliennis and its Japanese counterpart Tachypleus triilcnuitus are similar, but not alike. Both show the same nighttime circadian rhythms, but Barlow and co- workers found that the mechanism responsible for those rhythms differs between the two. The eye of Limn/its is larger and contains more ommatidia than that of Tuchvpleiis. As a result, the Limulus lateral eye Report of the Director and CEO R5 becomes more sensitive at night by capturing more photons. The smaller lateral eye of Tachypleus is nearly as sensitive, but due to its size is not able to catch as many photons as its Woods Hole relative. Instead, its photoreceptors make up for this deficiency by increasing gain, or the amount of information absorbed by the photoreceptors. Education at the MBL Summer Courses The MBL's educational program in biology and biomedicine continues to be preeminent, with almost 400 students and 400 faculty coming to the MBL each summer for a superb hands-on scientific experience. The MBL now offers 20 courses each summer, three of which were developed in 1997. One of these, Molecular Mycology: Current Approaches to Fungal Pathogenesis, was taught for the first time during the summer of 1997. The others. Frontiers in Reproduction: Molecular and Cellular Concepts anil Applications, and Neural Development and Genetics of Zebrafish, will be offered for the first time during the summer of 1998. Competition for admission to these and all MBL courses is intense. The 368 students enrolled during the summer of 1997 were selected from 833 applications that were culled from 1521 inquiries. Students continue to come to MBL courses from throughout the world, with about 35% to 40% of them from foreign countries. The popularity of the courses is one indication of their excellence. Another is the peer review process that suiTounds the awarding of many of the funds that support the courses. For the first time in my and many of my colleagues' recollections, one of our summer courses, Neural Systems and Behavior, received a perfect score of 100 in its N1H review; Neurobiology also received near perfect marks from the NIH. This attests to the vision of the course directors and faculty and the quality of the curriculum that is offered at the MBL. The generous support of a number of foundations, as well as the Federal government and a large cadre of individual donors is crucial to our ability to offer the best courses to the best students. Admission to many of the courses continues to be on a need-blind basis because of the scholarships we provide from these gifts and grants and from our endowment income. Five course directors retired in 1997, including David Kleinfeld, University of California, San Diego, and David Tank, Lucent Technologies (Methods in Computational Neuroscience); Steve Hajduk, University of Alabama, Birmingham (Biology of Parasitism); Mark Mooseker, Yale University (Physiology); and Pierre Drapeau. Montreal General Hospital (Neurobiology and Development of the Leech). They will be replaced by William Bialek and Rob de Ruyter, NEC Research Institute (Methods in Computational Neuroscience); Ed Pearce, Cornell University (Biology of Parasitism); Kerry Bloom, UNC, Chapel Hill (Physiology); and Christie Sahley, Purdue University (Neurobiology and Development of the Leech). I thank all retiring course directors for their efforts on behalf of the MBL, and welcome our newest faculty members to the Laboratory. Semester in Environmental Sciences As I mentioned above, we are buoyed by the success of our first semester-long program in environmental science for undergraduates from liberal arts colleges. We welcomed 1 6 students for the inaugural semester which was held in the fall of 1997. Twenty-four liberal arts colleges and universities now participate in the consortium of institutions from which these students are drawn. The Ecosystems Center scientists who served as R6 Annual Report faculty were impressed by the dedication and intellect of the group. I attended the student presentations before a large audience at semester-end and was impressed by the range and creativity of the research projects that they undertook. The semester proved a satisfying experience for all participants in this innovative new educational program, and it attests to the flexibility and commitment of the MBL in designing new and creative ways to further education in the life sciences. MBL/WHOI Library The Library is constantly responding to the challenges of straddling the traditional and the electronic modes of information transfer. This requires both technical achievement and renewal as well as the ongoing commitment to provide access to the best information essential to scientific research and teaching in the diverse Woods Hole scientific community. This charge translates into maintaining the unique, internationally acclaimed archival collection while, at the same time, being responsive to the fast-paced, complex, and demanding forces shaping electronic information delivery. So adept is the MBL/WHOI Library at this synthesis, that its director, Catherine Norton, is serving as President of the Boston Library Consortium, a group of academic, research, and hospital libraries. Facilities Our physical plant received significant attention during 1997. Major grants from the National Science Foundation and Colonial Gas Company provided nearly $600,000 to renovate classrooms and laboratories in the Loeb building. Additional monies were spent to reconfigure and modernize research laboratories to accommodate newly appointed MBL scientists and expanded research needs. MBL Trustees At their November 1997 meeting, the Board of Trustees of the Marine Biological Laboratory elected Dr. John E. Dowling to serve as President of the MBL Corporation. Dowling succeeds Dr. James D. Ebert, who retired after serving as President for seven years. In recognition of his "outstanding service to the development of American science in general and his long-standing dedication to the Marine Biological Laboratory," the Board has designated Ebert an Honorary Member of the Board of Trustees. Dr. Dowling is the Maria Moors Cabot Professor of Natural Science at Harvard University, a long-time MBL summer investigator, and former MBL Trustee. His tenure as President of the MBL Corporation began with the March 1998 meeting of the Board. Two New Trustees Join Board At that same meeting, Mr. Sydney M. Cone, III, was elected a member of the Class of 2002. He is a partner with the law firm of Cleary, Gottlieb, Steen & Hamilton in New York City. He is also Starr Professor of Law of International Trade and Finance and the Director of the Center for International Law at New York Law School. He has served as a member of the MBL's Council of Visitors since 1996. Mrs. Robert W. (Jean) Pierce was elected as a member of the Class of 1999. She is a resident of Wellesley, Woods Hole, and Boca Grande. Florida. She is an incorporator of the Heritage Plantation in Sandwich, and serves on the boards of the Women's Club of Boca Grande and the Penzance Point Road Trust. Her late husband. Robert, served as a member of the MBL's Board of Trustees from 1990 to 1993. Five Board Members Reappointed Mr. John R. Lakian, Chairman of the Board of the Fort Hill Group. Inc.; Dr. Joan Ruderman. Marion V. Nelson Professor of Cell Biology at Harvard Medical School; Dr. Sheldon J. Segal. Distinguished Scientist at the Population Council; Dr. William T. Speck. President and CEO of Columbia-Presbyterian Medical Center; and Mr. Alfred Zeien. Chairman and CEO of Gillette, were also elected in 1997 as members of the Class of 2002. Council of Scientific Advisors I owe a debt of thanks to my colleagues here in Woods Hole and to the international network of scientific, philanthropic, and business colleagues who share their time, interest, leadership, and support to keep the Marine Biological Laboratory at the forefront of biology. During 1997. I was especially fortunate to receive input and advice from the Council of Scientific Advisors, a newly constituted group of eminent scientists. They include: Drs. Bruce Alberts. Thomas Eisner. Walter Gilbert. Eville Gorham, Marc Kirschner. and Carla Shatz. With their counsel and that from our Board of Trustees, the MBL Science Council, and all of you, I look forward to a bright and dynamic future for the Marine Biological Laboratory. John E. Bums Report of the Treasurer The year 1997 was a successful one for the Marine Biological Laboratory. The net assets of the Laboratory increased by $6.2 M in 1997. from $54.5 M to $60.7 M. This increase was due to operations, the efforts of the capital campaign, and the success of our investment management activities. In accordance with generally accepted accounting principles, the Laboratory reported the results of operations after depreciation of $1.5 M, resulting in unrestricted net assets decreasing by $.8 M. This practice has masked a comparatively strong and healthy result of operations for the year. The capital campaign was responsible for a $3.1 M increase in net assets and permanently restricted net assets through increased gifts; and our long-term investments increased $4.9 M, resulting in a total return of 18.2% on our portfolio. The 1997 balance sheet revealed an increase in liquidity as cash and short-term investments increased by $.6 M to $5.0 M and account for 50% of current net assets. Long-term investments increased by $5.8 M. This increase was the result of $2.1 M of new gifts, market value increase of $4.9 M, and a withdrawal of $1.2 M to pay for scholarships and fellowships in accordance with the donors' instructions. Land, buildings, and equipment, net of accumulated depreciation, decreased by $.7 M as a result of the depreciation expense exceeding capital expenditures. This resulted in our inability to recover depreciation expense on federally funded capital projects. The Laboratory continues to demonstrate the ability to attract funds from the federal government, from foundations, and from individuals. We are in the process of significantly upgrading the physical plant, making the Laboratory an even more attractive facility at which to do science. Our housing budget continues to generate surplus cash. With long-term investment growth and the strength of our capital campaign, the Marine Biological Laboratory will continue to provide a productive scientific experience for its community. Mary B. Conrad R7 Financial Statements Coopers & Lybrand L.L.P. &Lybrand a professional services firm REPORT OF INDEPENDENT ACCOUNTANTS To the Board of Trustees of Marine Biological Laboratory Woods Hole, Massachusetts We have audited the accompanying balance sheet of Marine Biological Laboratory (the "Laboratory") as of December 31, 1997 and the related statements of activities and cash flows for the year then ended. 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 generally accepted auditing standards. 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 as evaluating the overall financial statement presentation. We believe that our audit provides a reasonable basis for our opinion. In our opinion, the financial statements referred to above present fairly, in all material respects, the financial position of Marine Biological Laboratory as of December 31, 1997, and the changes in its net assets and its cash flows for the year then ended in conformity with generally accepted accounting principles. 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, 1997 is presented for the puipose 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. Boston, Massachusetts f^ . ~7 .r~-~J / / . fi March 27, 1998 ** ^ ...... ~7 .r~-~J &*** Coopers & Lybrand L.L.P. is a member of Coopers & Lybrand International, a limited liability association incorporated in Switzerland. R8 MARINE BIOLOGICAL LABORATORY BALANCE SHEETS December 31, 1 997 (with comparative totals as of December 31, 1996) ASSETS Cash and cash equivalents Short-term investments, at market (Note Cl Accounts receivable, net of allowance for doubtful accounts of $36,782 in 1997 and $15,000 in 1996 Current portion of pledges receivable (Note H) Receivables due for costs incurred on grants and contracts Other assets 1997 $ 560,801 4,408.046 1,221,781 2,219,056 1,157,165 560,269 1996 $ 391,528 3,918,194 762,860 1,826.494 1,114.082 482,992 Total current assets 10.127,118 8,496.150 Long-term investments, at market (Notes C and D) Pledges receivable, net of current portion (Note H) Plant assets, net (Notes E and F) 35,614,151 2,238,826 20,026,580 29.763,495 2.406,350 20,695,624 Total long-term assets Total assets 57,879,557 $68,006,675 52.865,469 $61,361,619 LIABILITIES AND NET ASSETS Current portion of long-term debt and capital leases (Note E) Accounts payable and accrued expenses Deferred income and advances on contracts 229,657 1,494,948 384,258 204.108 1,536,015 321,998 Total current liabilities 2.108.863 2.062.121 Annuities and unitrusts payable Long-term debt and capital leases, net of current portion (Note E) Advances on contracts 1.213,583 2.567.370 1.433,208 959,513 2,591,973 1,210,950 Total long-term liabilities Total liabilities 5.214,161 7.323.024 4.762,436 6,824.557 Commitments and contingencies (Notes F and H) Net assets: Unrestricted Temporarily restricted Permanently restricted 18,729,311 25,596,656 16,357.684 19,371.978 21,484,748 13.680,336 Total net assets (Note B) 60,683,65 1 54,537,062 Total liabilities and net assets $68,006,675 $61,361.619 The accompanying notes are an integral part of the financial statements. R9 MARINE BIOLOGICAL LABORATORY STATEMENT OF ACTIVITIES for the year ended December 31, 1997 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 B) Total expenses Change in net assets before nonoperating activity Nonoperating revenue: Total investment income and earnings Less: investment earnings used for operations Reinvested investment earnings Total change in net assets Net assets, beginning of year Net assets, end of year Temporarily Permanently 1997 Unrestricted Restricted Restricted Total $ 9,986.800 $ 9,986,800 1.178,192 1.178,192 1.478,757 1.478.757 399.703 399,703 3,085.616 3,085,616 615,882 $4.434,938 $1,390,609 (,,441,429 471.832 1.238.151 1 .709,983 322.667 322.667 (165.504) 1.057 (164.447) 3.811.922 (3.871,922) 60,000 21.351.371 1,635,663 1.451.666 24,438.700 11, 03 1, 1 ) 14 11.031.914 4,144,508 4.144,508 1,487.705 1.487.705 5.440.808 5.440,808 22.104.935 22,l()4. t )35 (753.564) 1,635.663 1.451.666 2.333.765 152.543 3,490,810 1.225.682 4.869,035 (41.646) (1.014.565) (1.056.211) 110,897 2.476.245 1.225.682 3.812.824 (642,667) 4.1 1 1.408 2.677.348 6.146,589 19,371.978 21.484.748 13,680,336 54,537,062 $18.729.311 $25,596.656 $16,357,684 $60,683,651 Tin' notes an- tin intci;nil part of the financial statements. Kill MARINE BIOLOGICAL LABORATORY STATEMENTS OF CASH FLOWS for the year ended December 31. 1997 (with comparative totals for the year ended December 31, 1996) Cash flows from operating activities: Change in net assets Adjustments to reconcile change in net assets to net cash (provided by) from operating activities: Depreciation Unrealized (gain) loss on investments Realized (gain) loss on investments Present value adjustment to annuities 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 Loan proceeds Payments on long-term debt and capital leases Net cash provided by financing activities Net increase (decrease) in cash and cash equivalents Cash and cash equivalents at beginning of year Cash and cash equivalents at end of year 1997 6,146,589 1,483,203 (1,740,501) (1,728.792) 164.447 (1,390.609) 21,781 89.620 (480,702) (314.658) (43.083) (77,277) (71,564) 62,260 120,052 222.258 2,463,024 (814,159) 23,450,218 (26.321.432) (3.685.373) (30.430) 1,321,302 69,307 250.000 (218.5571 1,391.622 169,273 391.528 $ 560,801 1996 6,375,604 1.473.878 (1,765,532) (932,530) (118,076) (1,042,945) 5.000 (25,489) (1,726,570) 502,596 (15,940) (409,962) (59,539) 757.875 3.018,370 (730,966) 7,293,473 (11.347.575) (4.785,068) (23.369) 954.739 88.206 500,000 (205.878) 1,313.698 (453,000) 844.528 $ 391,528 The uc nii nines are an integral pun of the financial statements. Rll R12 Annual Report Marine Biological Laboratory Notes to Financial Statements A. 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. B. Significant Accoiintinx 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, 1996. from which the summarized information was derived. The Laboratory classifies net assets, revenues, and realized and unrealized gains and losses based on the existence or absence of donor-imposed restrictions and legal restrictions imposed under Massachusetts State law. Accordingly, net assets and changes therein are classified as follows: Unrestricted Unrestricted net assets are not subject to donor-imposed restrictions of a more specific nature than the furtherance of the Laboratory's mission. Revenues from sources other than contributions are generally reported as increases in unrestricted net assets. Expenses are reported as decreases in unrestricted net assets. Gains and losses on investments and other assets or liabilities are reported as increases or decreases in unrestricted net assets unless their use is restricted by explicit donor stipulations or law. Expirations of temporary restrictions on net assets, that is, the donor-imposed stipulated purpose has been accomplished and/or the stipulated time period has elapsed, are reported as reclassifications between the applicable classes of net assets. Temporarily Restricted Temporarily restricted net assets are subject to legal or donor-imposed stipulations that will be satisifed either by the actions of the Laboratory, the passage of time, or both. These assets include gifts plus monies for which the specific, donor-imposed restrictions have not been met and pledges, annuities, and unitrusts for which the ultimate purpose of the proceeds is not permanently restricted. As the restrictions are met, the assets are released to unrestricted net assets. Also, realized/unrealized gains/losses associated with permanently restricted gifts which are not required to be added to principal by the donor are classified as temporarily restricted 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 gifts, 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. Investment income from short-term investments and investments held in trust by others is included in operating support and revenues. To the extent that nonoperating investment income and gains are used for operations as determined by the Laboratory's total return utilization policy (see below), they are reclassified from nonoperating to operating on the statement of activities as "Investment earnings used for operations." All other activity is classified as operating revenue. The Laboratory recorded net realized gains of $1,728,792, net unrealized gains of $1,740,501 and dividend and interest income of $2,053,514 in 1997. Cash and Cash Equivalents Cash equivalents consist of resources invested in overnight repurchase agreements and other highly liquid investments with original maturities of three months or less. Financial instruments which potentially subject the Laboratory to concentrations of risk consist primarily of cash and investments. The Laboratory maintains cash accounts with one banking institution. Investments are maintained primarily with two institutions. Investments 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 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 are classified as short-term. All other investments are considered long-term. In 1924, the Laboratory became the beneficiary of certain investments, included in permanently restricted net assets, which are held in trust by others. The Laboratory has the continuing rights to the income produced by these funds in perpetuity, subject to the contractual restrictions on the use of such funds. Accordingly, the trust has established a process to conduct a review every ten years by an independent committee to ensure the Laboratory continues to perform valuable services in biological research in accordance with the restrictions placed on the funds by the agreement. The committee met in 1994 and determined that the Laboratory has continued to meet the contractual requirements. The market Financial Statements R13 values of such investments are $7,440.158 and $6,214,477 at December 31, 1997 and 1996, respectively. The dividend and interest income on these investments totaled $254,898 and $224.324 in 1997 and 1996. respectively. Investment Income and Distribution For the master pooled investments, the Laboratory employs a total return utilization policy that establishes the amount of the investment return made available for spending each year. The Finance and Investment Committee has approved a standing policy that the withdrawal will be based on a percentage of the latest three-year average ending market values of 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 1%, can be utilized if approved in advance by the Finance and Investment Committee of the Board of Trustees. For fiscal 1997, the Laboratory obtained approval to expend 6% of the latest three-year average ending market values of the investments. The 6% includes a l'A% administration fee for endowments. This fee was approved by the Laboratory's Board of Trustees for the years 1996-2000. 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 D). Plant Assets Buildings and equipment are recorded at cost. Donated facility assets are recorded at fair market value at the date of the gift. Depreciation is computed using the straight-line method, beginning the month after the asset is placed in service, over the asset's estimated useful life. Estimated useful lives are generally three to ten years for equipment and 20 to 40 years for buildings and improvements. Depreciation expense for the year ended December 31, 1997 amounted to $1,483,203 and has been recorded in the statement of activities in the appropriate functionalized categories. When assets are sold or retired, the cost and accumulated depreciation are removed from the accounts and any resulting gain or loss is included in unrestricted income for the period. Annuities and Unitrusts Payable Amounts due to donors in connection with gift annuities and unitrusts are determined based on remainder value calculations, as of December 31, 1997, 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 utilized within the next year. Advances on contracts includes funding received for grants and contracts before it is earned. In certain circumstances, long-term advances are invested in the master pooled account until they are expended. Revenue Recognition Revenue is recognized at the time it is earned. The 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 tuition income is net of student financial aid of $536,097 and $479,000 in 1997 and 1996, 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 received 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 using various methodologies. 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 1997 is $1,226,360. 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. Reclassification Certain prior year amounts have been reclassified to conform with current year presentation. The reclassifications had no effect on net assets. Tax-Exempt Status The Laboratory is exempt from federal income tax under Section 501(c)(3) of the Internal Revenue Code. R14 Annual Report C. Investments: The following is a summary of the cost and market value of investments at December 31, 1997 and 1996: Market Cost 1997 1996 1997 1996 Certificates of deposit $ 40.000 $ 55,350 S 40.000 $ 55,350 Money market securities 2,168,958 3.469,588 2.168.958 3.469.589 U.S. Government securities 1,292,600 1,233,690 1. 098,526 998,829 Corporate fixed income 2,587,861 3,431.333 2.472.653 3.278.396 Common stocks 5,279.266 3.793,156 4.271.853 2.742,841 Mutual funds 23,223.812 17.371.972 19,317,499 14.555,958 Limited partnerships 5.429.700 4.326,600 3,309.994 3,108,464 Total investments $40,022.197 $33.681.689 $32,679.483 $28.209.427 Investment portfolios for the years ended December 31, 1997 and 1996 are as follows: Marke r Cost 1997 1996 1997 7996 Short- Term Investments Certificates of deposit $ 40,000 $ 55 ISO S 40 000 $ 55 350 Money market 1784 Fund 1,759,589 2,755.593 1,759,589 2,755,593 Common stocks 55 1 .780 50.608 530.936 50,608 Mutual funds 2.056.677 1 .056.643 2.056.679 1.063.089 Total 4.408,046 3.918,194 4.387,204 3,924,640 Long-Term Investments Pooled investments: Master pooled investments $26,163.702 $21,858,658 $20,201.962 $17,889,881 Separately invested: General Chase trust 5.846.916 4,898,630 4,986.443 3,926,959 Library Chase trust 1,593,242 1,315.847 1,358.149 1.065.008 Annuity and unitrust investments -, QJQ TQJ 1.690,360 1,745.725 1 .402.939 Total 35,614,151 29,763,495 28,292.279 24,284.787 Total investments $40,022.197 $33.681,689 $32,679,483 $28,209,427 D. Accounting fur 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. 1997 and 1996 is as follows: 7997 7996 Unrestricted 4.192 4.415 Temporarily restricted 42.693 41.426 Permanently restricted 65.41 1 66.442 Advances on contracts 6.506 6.493 i is. so: 118.776 Financial Statements R15 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 7997 $ 186.35 220.30 $ 33.95 1996 $ 159.37 186.35 $ 26.98 E. Long-Term Debt: Long-term debt consisted of the following al December 31: Variable rate (5.50% at December 31, 1997) Massachusetts Industrial Finance Authority Series 1992 A 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 Capital leases with various rates and due dates /997 $ 960,000 1,280,000 325,210 231,817 1996 t 990,000 1.330,000 418,821 57,260 $2,797,027 $2,796,081 The aggregate amount of principal due on long-term debt for each of the next five fiscal years and thereafter is as follows: 1998 1999 2000 2001 2002 Thereafter $ 229,657 243,274 267,404 173,664 148,028 1.735,000 Less current portion of long-term debt 2,797,027 (229,657) Long-term debt net of current return $2,567,370 In 1992, the Laboratory issued $1,100,000 Massachusetts Industrial Finance Authority (MIFA) Series 1992A Bonds and $1,500,000 MIFA Series I992B. These bonds pay varying annual interest rates ranging from 3.48% to 6.63%. Interest expense on this debt totaled $141,899 for the year ended December 31, 1997. The Series 1992 A and B Bonds mature on December 1. 2012 and are collateralized by a first mortgage on certain Laboratory property. 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 (before transfers), interest, expense and transfers from the quasi-endowment for debt service must equal or exceed all debt service payments, as defined by the agreement. The Laboratory was in compliance with these covenants and restrictions at December 31. 1997. In 1996, the Laboratory borrowed $500,000 with an interest rate of 5.8% from the University Financing Foundation, Inc. The interest expense for the year ended December 31, 1997 was $21.829. The loan matures in 2000 and is collateralized by 69,440 shares of a fixed income mutual fund with a fair value of $933,690 at December 31, 1997. In 1997, the MBL borrowed $250,000 with an interest rate of 5.8% per annum from the University Financing Foundation, Inc. The interest expense for the year ended December 31. 1997 was $5,866. This loan matures 2002 and is collateralized by 69,440 shares of a fixed income mutual fund with a fair value of $933,690 at December 31, 1997. The Laboratory has a line of credit agreement with BankBoston from which it may draw up to $1,000.000. No amounts were outstanding under this agreement as of December 31, 1997 and 1996. R16 Annual Report F. Plant Assets: Plant assets consist of the following at December 3 1 : Land Buildings Equipment 1997 $ 702.908 32.419.072 4.300.932 1996 $ 702.908 3 1 .730,830 4.270,278 Total Less: Accumulated depreciation 37.422.912 (17.396.332) 36,704,016 (16.008.392) Plant assets, net $20.026.580 $20,695.624 G. Retirement Plan: The Laboratory participates in the defined contribution pension plan of TIAA-CREF (the "Plan"). The Plan is available to permanent employees who have completed two years of service. Under the Plan, the Laboratory contributes 10% of total compensation for each participant. Contributions amounted to $715.858 and $661.089 for the years ended December 31, 1997 and 1996. respectively. 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 live years After live years 1997 $2,219.056 2,485,851 80,000 1996 $1,836,874 2,780,000 Total 4,784,907 4.616,874 Less: discount of $227,025 in 1997 and $373.650 in 1996 and allowance of $100.000 in 1997 and $10,380 in 1996 (327,025) (384,030) $4.457,882 $4,232.844 Pledges receivable at December 31 have the following restrictions: Research and education Permanently restricted net assets $3,787,882 670,000 $4,232,844 $4,457,882 $4.232.844 I. Pu.ttri'tirement Benefils: The Laboratory accounts for its postretirement benefits under Statement No. 106. "Employers' Accounting for Postretiremen! Benefits Other than Pensions." which requires employers to accrue, during the years that the employee renders the necessary service, the expected cost of benefits to be provided during retirement. As permitted, the Laboratory has elected to amortize the transition obligation over 20 years commencing on January 1. 1994. The Laboratory's policy is that all current retirees and certain eligible employees who retired prior to June I, 1994 will continue to receive postretirement health benetits. 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 postretirement medical benefit plan. Financial Statements R17 Net postretiremen! benefits for 1997 and 1996 include: Service cost (benefits earned during period) Interest cost (on projected benefit obligation) Actual return on plan assets Net amortization and deferral Net postretiremen! benefits cost 1997 $ 29,095 135,892 (31.976) 59.07 1 $ 192,082 1996 $ 61,712 149.149 (20.700) 92.176 $ 282,337 Below is a reconciliation of the funded status of the Plan at December 31. 1997 and 1996: Accumulated postretirement benefit obligation: Retirees and dependents Fully eligible active participants Other active participants $1,439,899 163.188 316.778 $1.369.787 230,290 608.796 Total Market value of plan assets Unfunded obligations Unrecognized prior service cost (credit) Unrecognized net (gain) loss Unrecognized transition obligation Accrued postretirement benefit cost 1,919,865 701.140 1,218,725 (191,078) 1.389.159 $ 20,644 2,208.873 588.337 1,620,536 136.158 1.475.981 $ (8,397) The health care cost trend rate assumptions used in determining the projected benefit obligation begin at 9.5% in 1997 and gradually decrease to 5.0% in the year 2006 and thereafter. The effect of raising the assumed health care cost trend rate by one percentage point in each year would be to increase the accumulated postretirement benefit obligation as of December 31. 1997 by $173.001 and to increase the aggregate of the service and interest cost components of net periodic postretirement benefit cost for the year then ended by $14.528. The discount rate used in determining the accumulated postretirement benefit obligation is 7.5%, and the expected return on plan assets was 8.0%. During 1997, the Laboratory contributed $192,082 to fund the Trust for these postretirement benefits. >H Oi O < ct O CO < J J < u O S UJ Z 5 00 1 X w z (J U. O D Q UJ U CO tu U-l si CL D CO ON ON -o I 3 1) in r*-, O m oo so ri ^ r - ON n n m c J m ffi ^ rj m ON N *t CO r "i n"i r OS ON 1 OO OO 00 r-i r-l so "j O r- o co r- T 3 O n h rj o f . T " sO i (N t -T ' 3 1 O od r J _ ^- - -' ri ri r 1' * ri rj 11 8 oo rj >n ON n ON in Tt ON m SO P "-1 00 SO o S ""* Tt ON ON ^> r" ON -t \ ri ri sO ^o* la ri oo r-4 O 11 ON Tj- IT, ON r*"i (N "~1 ON O "* n si O K en S r ?3 rj pvj r- r^ 00 oo oo oo ^ r W", oo' r-l r-4 ? ^ -rf ON in ON ON O- QV ^ to Z fi -f ^ ^ ^ m r- c ^ f- O so -t ^~ ' ^ r- r- O m m ON r- o - O r- - - sO -t ON S In 1 i \O' rn r*^ 1 in M 5 * ON r- r-' "t ON 1 r o o -3 en t "d ON 1 O in ^ f S 3 ri >/~) O J- Tj- ON ^ od ri rn - -.-)[ r*"j Os ON ri ^_J oo in so c ri i/- ( ri o ^ ; CO C N in m m, ri oo O r*-* r-' rK ' ^ 6^ onal ^ 4J O .^- "D. ^ * S ^ , 9- 4J (rt rj ^ | c2 - i5'S"aS2FS5 tL.a.onWooHiioDQO I o R18 Report of the Library Director One of the goals of the MBL's recently announced Discovery Campaign is to raise $1 million in support of the MBLAVHOI Library. This is excellent news indeed for the library. Funding at this level will be a good start towards protecting for future generations of scientists one of the world's most comprehensive and valuable collections of books and journals in the biological, oceanographic, and ecological sciences. This support will also help in the development of information technology, which promises to play a vital role in the future of the library. The way the library will serve scientists in the next decade will be very different from the way it serves them today. More and more the library is becoming a conduit rather than a repository of information, providing on-line access to the burgeoning electronic literature. But even as electronic delivery alters the library and the way it is used, the MBL's irreplaceable book and journal collection continues to be a national treasure that must be conserved. The Library Committee has spent considerable time over the past year evaluating our collection as a whole, the library's physical plant, and the future of both the paper and electronic library. The committee found that even with the advent of more electronic resources in the library, still more room will soon be needed to house all the early monographic series now available in the stacks and the hardware required to serve the growing electronic resources. With fellow members of the Boston Library Consortium, we are looking into the feasibility of having an off-site storage facility. If this plan is realized, we can look forward to a cooperatively run depository that will offer electronic retrieval and courier service as well as warehousing for some of our lesser used volumes, making room for other, ever expanding information resources. Special collections A new Special Collections area has been created in the third floor book section of the library through a generous gift made in memory of Ivor Cornman. In this area, the expeditions, bound collected reprints, and books from the open shelves published before 1900 are being protected. Earlier works, currently on the open shelves in the stacks, are being cataloged and placed in the Rare Books Room. Digital library As we preserve the older collection, we are expanding the electronic collection. The library currently supports more than 300 full-text electronic journals on its web site (www.mbl.edu). We provide access to more than 35 commercial databases and provide public access to databases created here at the MBL on marine animals, E. coli, women-in-science, the Leuckart charts, and other educational material of interest to both scientists and students. From the Community of Science we have purchased funding and patent databases including the Expertise Database, which contains information on individual scientists and their projects. Woods Hole scientists who enter information about their work in the data bank are rewarded with timely information on available funding sources in their field. The library continues to update to the latest versions of Internet Grateful Med and PubMed and InfoBank Search. The latter service includes newspaper, magazine, academic, and general reference searching targeted to our student and visitor populations. Bibliographies of relevant resources for the many summer courses held at the MBL have been created and can be accessed via the web. The Don Flescher photo collection of fish is a new database available from the library home page. It is comprised of 1500 pictures searchable by species, common name, catch area, or taxonomy. Journal issues One of the newest issues facing libraries, along with the continued rise in subscription prices, is the new R19 R20 Annual Report phenomena of publishers collecting electronic information at the "article level" when library users log into their sites. Vendors and publishers can now gather specific user data right down to the exact article, phrase, and words that someone pulls up or orders. What happens to this information? If this user information should be sold to the right company, it could become important for determining who else in the world is interested in, or perhaps working on, the same research problem. Will investigators hesitate to look at publishers' electronic sites for fear of competitors getting a hint of what they are working on? What protections can the library provide? A cloak of anonymity, possibly; but right now information on article use is transmitted almost exclusively to the publishers and not provided to the library. Like other libraries, the MBLAVHOI Library is now managing licenses covering information content as opposed to purchasing the contents. At the same time, we are trying to negotiate protections for our users. Our newly appointed Technical Services Coordinator carries the responsibility of "watchdog" for these licenses. From the arena of fair use, we now are venturing into the arena of ethical use of information. Ensuring access to today's electronic resources tomorrow is another major concern. Since 1991 the library has canceled more than 500 journal subscriptions. In 1991 we borrowed 1858 articles for our patrons. In 1997 we borrowed 3259, representing a 42% increase over six years. This practice will continue to increase as it becomes necessary to cancel titles in order to meet publishers' price increases for "must have" journals. As a result, electronic document delivery, whether providing documents for other libraries or retrieving materials for our own scientists, is becoming an ever-increasing component of the library's services. Instruction The new Science Reference Librarian has quickly expanded and improved the library's instructional programs on all four campuses (MBL. WHOI, Fisheries, and USGS). Training and course instruction is now provided for researchers and students, and new on-line bibliographies and reserve lists have been created for MBL summer courses. There continues to be high demand for new courses in how to search scientific databases, JAVA, Web page design, Windows 95, Powerpoint, etc. Branch libraries Jacqueline Riley, the Science Reference Librarian at 'he Fisheries, has implemented many new services including launching a NEFSC library web page. The contract that provides library services for the National Marine Fisheries, while bringing a collegia! interchange at the local level, presents some technological problems at the national level that resulted in different automated library systems. The marriage of two different library systems seemed to be impossible until a method was devised by Laurel Duda (MBL) and Maggie Rioux (WHOI) that allows both libraries to share bibliographic records without redundant effort (Duda, L.E. and M.A. Rioux, One Library, One Bib Record- Two OPACs, Two Systems, Information Outlook Vol. 2, No 3: 31-36). A new library was incorporated into the design of the new WHOI Research Vessel Atlantis. Space was designed for a computer workstation with Internet access for the researchers and staff to fulfill their information needs while at sea. A reference collection, popular works, and a wonderful collection of World War II monographs donated by Ken Parda are also available aboard ship. The Data Library, using the Library's integrated computer systems, has installed a new image server, providing photographic images of the WHOI scientific instrument collection. The new ALVIN archive catalog provides retrieval of individual ALVIN dive records through our local on-line catalog. MARINER, including all related media the data archive holds and hot links to images from the dives. More than 700 of the photographs in the MBL archives, including the late 1800 and early 1900 collection of MBL's Baldwin Coolidge photos, have also been cataloged and are available through MARINER and the Web. A growing debate focusing on numerical data in the Earth and Environmental sciences and the issues relevant to the management of all non-text information was the focus of the Scientific Data Advisory Committee which met this past year at WHOI. The major problem identified was access to the data and how the library must make it easier for users of data sets to find, select, and retrieve that data. In order to do this the Data Library needs to encourage submission of data sets in a useable standard metadata format, with appropriate documentation. Because getting scientists to do the latter is referred to as "herding cats," the community needs to provide assistance and incentives. The future The National Library of Medicine awarded the library $1,033,278 in support of the project titled, "Professional Services in Support of NLM's Outreach Efforts to Encourage the Use of Computers and Information Science in Medicine." With this support, Library Director's Report R21 the library is able to plan for "change" over the next five years and to offer services that will fit evolving needs. However, all of the members of the scientific community must promote the value of academic access to information for research and teaching. They will also need to use innovative technology to improve scholarly communications without operating in an economic model tied to increasing costs from large commercial publishers. Libraries and administrators have realized that spiraling costs cannot be solved by strategies that merely include canceling titles and collective purchasing. Change must be effected through partnerships in new technologies, and the development of new models of publication and distribution. While the new electronic environments offer opportunities to provide new services, we also need to think about the sustainability of the information ecosystem: managing the life cycle of information for preservation and access. This year the Florence Gould Foundation awarded $30,000 to the library for the preservation and conservation of the French scientific rare books in our collection. The library will make these books available electronically and will use some of the high-end translation dictionaries now available on our Web page that allows French to English translation. As noted, one of the goals of the Discovery Campaign is to help preserve the library facility and the book and journal collections for future scholars. Managing this traditional collection will be much easier than managing the new electronic collection, which presents a number of new issues, not the least being the lack of planning for digital archival storage and migration to new technologies by the publishers. As we approach the 21st Century, "change" will be the MBL/ WHOI Library's primary challenge. Catherine Norton Educational Programs Summer Courses Biology of Parasitism: Modern Approaches (June I2-August 15) Directors Steven Hajduk. University of Alabama. Birmingham Edward Pearce, Cornell University Facult\ Robert Bullis. Marine Biological Laboratory Harry Dickerson. University of Georgia Steve Ealick, Cornell University Christopher Hunter, University of Pennsylvania School of Veterinary Medicine Susan Little. University of Georgia Phil LoVerde, State University of New York, Buffalo David Russell. Washington University School of Medicine Phillip Scott. University of Pennsylvania David Sibley, Washington University School of Medicine Christian Tschudi, Yale University School of Medicine Buddy Ullman, Oregon Health Sciences University Ehsabetta Ullu, Yale University School of Medicine Teaching Assistants Laura Rosa Brunei. Cornell University Nino Campobasso, Cornell LIniversity Dai-rick Carter, Oregon Health Sciences University Jaime Dant, Washington University Medical School Jerome Drain. University of Alabama. Birmingham Merle Elloso. University of Pennsylvania Wendy Freebern, State University of New York. Buffalo Maren Lingnau. Washington University School of Medicine Carol Lopez-Estrano. Yale University Joao Pedras-Vasconcelo, Cornell University Veterinary School Elizabeth Sabin. Cornell University Lecturers Norma Andrews, Yale University School of Medicine James Bangs, University of Wisconsin, Madison Stephen Beverley, Harvard University School of Medicine John Boothroyd, Stanford University Karen Day, Oxford University, United Kingdom John Donelson, University of Iowa College of Medicine Paul Englund. Johns Hopkins University School of Medicine Daniel Goldberg, Washington University Richard K. Grencis, University of Manchester. United Kingdom Kasturi Haldar, Stanford University School of Medicine John Heuser. Washington University School of Medicine Stephen Hoffman. Naval Medical Research Institute Peter Hotez, Yale University School of Medicine William Jeffery, Pennsylvania State University Patricia Johnson. University of California. Los Angeles Richard Komuniecki. University of Toledo Manfred Kept", Basel Institute for Immunology, Switzerland Jean Langhorne. Imperial College of Science and Technology. United Kingdom Timothy Nilsen. Case Western Reserve University Marilyn Parsons. Seattle Biomedical Research Institute William Petri. University of Virginia Health Sciences Center Meg Phillips. University of Texas Southwest Steven Reed. Infectious Disease Research Institute Steve Reiner. University of Chicago David Sacks, National Institutes of Health Alan Sher, National Institutes of Health Mitchell Sogin. Marine Biological Laboratory Kenneth Stuart, Seattle Biomedical Research Institute Sam Turco, University of Kentucky College of Medicine C. C. Wang. University of California, San Francisco Gary Ward, University of Vermont Tom Wellems, National Institutes of Health R22 Educational Programs R23 Dyann Wirth. Harvard University School of Public Health Maria Yazdanbakhsh, Leiden University, The Netherlands Course Coordinator Robert Sabatini, University of Alabama. Birmingham Course Assistant Brian Hondowicz, University of Pennsylvania Laboratory Assistant Joseph Paton, Tufts University Students Kimberly Brouwer, Johns Hopkins School of Public Health Maristela Camargo, Federal University of Minas Gerais, Brazil Soren Gantt, New York University School of Medicine Michelle Gleeson, University of Technology, Australia Meike Hensmann, Yale University David Jiang, Johns Hopkins University School of Medicine Andreas Lingnau, Washington University Medical School Gunnar Mair, Queen's University of Belfast. United Kingdom Antoinette Marsh, University of California. Davis Lillian Ouko, University of Pennsylvania Lisl Shoda, Washington State University Kevin Tan, National University, Singapore Carl Johan Treutiger, Karolinska Institute, Sweden Elisabeth Triplet!, Cornell University Eric Villegas, University of Pennsylvania Ulrike Wille, University of Tubingen, Germany Embryology: Concepts and Techniques in Modern Developmental Biology (June 15-July 26) Directors Marianne Bronner-Fraser, California Institute of Technology Scott Fraser, California Institute of Technology Faculty Andre Adoutte, Universite Paris-Sud, France Rosa Beddington, National Institute for Medical Research, United Kingdom R. Andrew Cameron, California Institute of Technology Andrea Collazo, California Institute of Technology Eric H. Davidson, California Institute of Technology Richard Harland, University of California, Berkeley Lee Niswander. Memorial Sloan-Kettering Cancer Center James Posakony, University of California, San Diego Nadia Rosenthal, Massachusetts General Hospital Joel Rothman, University of California, Santa Barbara Joshua Sanes, Washington University School of Medicine John Saunders, Jr.. Marine Biological Laboratory Martin Shankland, University of Texas Judith Venuti, Columbia University Teaching Assistants Maria Ina Arnone, California Institute of Technology Guillaume Balavoine. Wellcome/CRC Institute, United Kingdom Rebecca Beach, Hollins College Steve Gendreau, University of California. Santa Barbara Gabrielle Kardon, Duke University Carmen Kirchhamer, Harvard University Anne Knecht, University of California. Berkeley Catherine Krull. California Institute of Technology Julie Kuhlman, Memorial-Sloan Kettering Cancer Center Eric Lai. University of California. San Diego Nicholas Lartillot. Universite Paris-Sud, France David Nellesen. University of California. San Diego Craig Neville, Massachusetts General Hospital Robert Zeller, University of California, San Diego Lecturers Sean Carroll, University of Wisconsin Nancy Hopkins, Massachusetts Institute of Technology Michael Levine, University of California, Berkeley Tom Maniatis. Harvard University David McClay, Duke University Course Coordinator Linda Huffer. Marine Biological Laboratory Course Assistants Allison Freeman, Wheaton College Jason Rickles, Boston University School of Education Students Pia Aanstad, University of Newcastle, United Kingdom Dominique Bergmann, University of Colorado, Boulder Brian Cooper. National Institute for Medical Research. United Kingdom Wim Damen, Munchen University. Germany Gregory Davis. University of Chicago Eric Finkelstein. State University of New York Health Science Center Paola Giuliano, Zoological Station, Italy Tiffany Heanue, Harvard University Sharon Hesterlee, University of Arizona Christoph Kaufmann. Massachusetts General Hospital Peter Ladurner, University of Innsbruck, Austria Walter Lerchner, National Institute for Medical Research, United Kingdom Per Lindahl, University of Goteborg, Sweden Matthew Marlowe, University of California, Berkeley Sean Megason, Harvard University Raffaella Melfi, Universita Degli Studi di Palermo, Italy Axel Meyer, State University of New York Bennett Novitch, Harvard Medical School Catherine Olsen, University of California. Davis Eva Reissmann. Max-Planck-Institut, Germany Elaine Seaver. University of Texas. Austin James Spotts. California Institute of Technology Xin Sun, University of California. San Francisco Christina Takke, University of Koln, Germany Emily Walsh. University of California, San Francisco Microbial Diversity (June 15-July 31) Directors Edward Leadbetter, University of Connecticut Abigail Salyers, University of Illinois R24 Annual Report Faculty Kurt Hanselmann. University of Zurich. Switzerland Bruce Paster, Forsyth Dental Center Rolf Schauder. University of Frankfurt, Germany Teaching Assistants Elena Barbieri. University of Urbino, Italy Daniel Gisi, Swiss Federal Institute of Technology, Switzerland David Graham, University of Illinois Irena Levin. Forsyth Dental Center Caroline Plugge, Wageningen Agricultural University. The Netherlands Lecturers Bianca Brahamsha. University of California. San Diego Colleen Cavanaugh, Harvard University Yehuda Cohen. Hebrew University of Jerusalem, Israel Sharon Danielson. Rensselaer Polytechnic Institute Daniel Distel, University of Maine Stephen Farrand. University of Illinois. Urbana-Champaign James Fleming. University of Tennessee Tillman Gemgross, Metabolix. Inc.. Cambridge Robert Haselkorn, University of Chicago Holger Jannasch. Woods Hole Oceanographic Institution Jared Leadbetter, Michigan State University Mark McBride, University of Wisconsin, Milwaukee Terry Miller, New York State Department of Health Duane Moser, Center for Great Lakes Studies. Milwaukee Sandra Nierzwicki-Bauer, Rensselaer Polytechnic Institute Thomas Pitta. Rowland Institute for Science James Russell, Cornell University Daad Saffarini, University of Massachusetts Anglica Seitz, Harvard University Nadja Shoemaker, University of Illinois Alfred Spormann, Stanford University David Stahl, Northwestern University Anne Summers. University of Georgia Andreas Teske, Woods Hole Oceanographic Institution Pieter Visscher. University of Connecticut, Avery Point John Waterbury. Woods Hole Oceanographic Institution Lily Young, Rutgers University Course Coordinator Madeline Vargas, College of the Holy Cross Course Assistant Noah Horst, Tufts University Students Ehud Banin, Tel Aviv University, Israel Fikry Barghuthy. Hebrew University, Israel Brendan Bohannan, Michigan State University Alfred Boyle, Rutgers University Scott Dawson, University of California, Berkeley Jeffrey Dugas, University of Connecticut Kelly Evans, Queen's University. Canada Deborah Hughes, Scripps Institution of Oceanography Elke Jaspers. Universitat Oldenburg. Germany Hope Johnson, Stanford University Sabine Krause, Max-Planck-Instiltit, Germany Thomas Lie, University of Connecticut David Long, Montana State University Junko Munakata Marr. Colorado School of Mines William Sobczak, Cornell University Alexandra Stone. Ohio State University Acharawan Thongmee. University of North Texas Crisogono Vasconcelos, Universidade Federal Fluminense, Brazil S. Wenuganen, Bogor Agricultural University, Indonesia Ludek Zurek. University of Alberta. Canada Neural Systems & Behavior (June 15-Augmt 8) Directors Janis Weeks, University of Oregon Harold Zakon, University of Texas, Austin Faculty Ronald L. Calabrese. Emory University Catherine Carr, University of Maryland Kathleen French, Llniversity of California, San Diego David Glanzman, University of California. Los Angeles Scott Hooper, Ohio University Richard Hyson, Florida State University Masashi Kawasaki, University of Virginia William Kristan. University of California. San Diego Richard Levine, University of Arizona Pierre Meyrand, Universite de Bordeaux, France Michael Nusbaum, University of Pennsylvania School of Medicine Glen Prusky, University of Lethbridge, Canada William Roberts, University of Oregon Gary Rose, University of Utah Angela Wenning, Universitat Konstanz. Germany Teaching Assistants Cecilia Armstrong. University of Oregon Dawn Marie Blitz, University of Pennsylvania School of Medicine Shanping Chen, University of California, Los Angeles Richard Dyck. University of Lethbndge. Canada Michael Ferrari, University of California, San Diego Evelyn Field, University of Lethbridge. Canada Matthew Friedman. Cornell University Jorge Golowasch, Brandeis University Andrew Hill. Emory University Maria Kubke. University of Maryland David Lenzi, University of Oregon Lynne McAnelly. University of Texas Tasha McMahon, University of California. Los Angeles Geoffrey Murphy, University of California, Los Angeles David Sandstrom, LIniversity of Arizona Daphne Scares, University of Maryland Laura Wolszon, Columbia University Lecturers George Augustine, Duke University Medical Center Rita Balice-Gordon, University of Pennsylvania School of Medicine David Clayton, LIniversity of Illinois Roger Hanlon, Marine Biological Laboratory Ron Hoy, Cornell University Edward Kravitz, Harvard Medical School Educational Programs R25 Eduardo Macagno. Columbia University Gina Turrigiano. Brandeis University Scholars-in-Residence Sascha du Luc, Salk Institute Alan Gelperin. Bell Labs Rebecca Johnston, Colby College Simon Laughlin. University of Cambridge. United Kingdom Course Assistants Sarah Doernberg, Emory University Sarah Gaines, Princeton University Students Emre Aksay, Bell Labs Eric Bauer. University of Texas. Austin Ben Bonham. University of California. San Francisco Holly Campbell, University of Arizona Randy Chitwood, University of Texas. San Antonio Virginia de Sa. University of California. San Francisco Teresa Esch. University of Virginia Joe Pass. Michigan Tech University Paul Gasser, Arizona State University Timothy Holy, Princeton University William Kargo. Allegheny University Christopher Kilroy, University of North Carolina, Wilmington Susan Laessig. University of Maryland, Baltimore Lynne Merchant, University of California, San Diego Leslie Osbome, University of California, Berkeley Dorit Polnaii, Northeastern University Rex Robison. Stanford University Sen Song. Brandeis University Ayako Yamaguchi. Columbia University Shih-Rung Yeh. Georgia State University Neurobiology (June 15- August 16) Directors Gary Banker, University of Virginia Medical School Daniel Madison. Stanford University Medical Center Section Directors Michael Greenberg. Childrens' Hospital Stephen Smith. Stanford University Medical Center Facult\ Susan Birren. Brandeis University Mark Bovvlby, Wyeth-Ayerst Research Gabriel Corfas, Childrens' Hospital Kerry Delaney, Simon Eraser University. Canada Donald Faber. Allegheny University of the Health Sciences Steven Finkbeiner, Childrens' Hospital Philip Haydon, Iowa State University Stephen Jones. Case Western Reserve University Maurice Kernan, State University of New York. Stony Brook Stephen Lin, Wyeth-Ayerst Research Ed McCleskey, Oregon Health Sciences University Adam Rory McQuiston, Stanford University School of Medicine Thomas Reese. National Institutes of Health Morgan Sheng. Howard Hughes Medical Institute Carolyn Smith, National Institutes of Health Stuart Thompson, Stanford University Li-Huei Tsai, Harvard Medical School Susan Wray, National Institutes of Health Joshua Zimmerberg, National Institutes of Health Teaching Assistants F. Woodward Hopt, Stanford University Medical Center Alane Murdock, Stanford University Medical Center Paul Pavlidis, Stanford University Medical Center Alberto Pereda, Allegheny University Marline Usdin, Stanford University Medical Center Lecturers Yadin Dudai, Weizmann Institute of Science, Israel Justin Fallon, Brown University Julie Ann Kauer, Duke University Medical Center Maurine Linder. Washington University Medical School Diane Lipscombe, Brown University Luis Parada, University of Texas Robert Rosenberg, University of North Carolina, Chapel Hill Gary Ruvkun, Massachusetts General Hospital Thomas Sudhof, University of Texas Karel Svoboda, Cold Spring Harbor Laboratory Roger Tsien, University of California, San Diego Course Assistants Tara Bennett, Dartmouth College Eleanore Edson, Stanford University Students Alberto Bacci. University of Milano, Italy Elizabeth Brown, Medical College of Virginia Benjamin Cravatt. Scripps Research Institute Samuel Hess, Cornell University Warren Kim. Yale University School of Medicine Stuart Licht. Massachusetts Institute of Technology Johanna Montgomery, Otogo University. New Zealand Craig Nelson, Harvard University Eric Norman, University of Pittsburgh Rita Saltier, University of Toronto, Canada Marilee Shelton. University of North Carolina. Chapel Hill Stephan Sigrist. Max-Planck-Institiit. Germany Physiology: Cellular and Molecular Biology (June 16-July 27) Directors Kerry Bloom, University of North Carolina, Chapel Hill Mark Mooseker. Yale University Faculty William Bement, University of Wisconsin Richard Cheney, University of North Carolina. Chapel Hill Jonathan Chernoff, Fox Chase Cancer Center Ruth Empson. University of Oxford, United Kingdom Antony Galione, University of Oxford. United Kingdom Tom Hays, University of Minnesota, St. Paul Daniel Kiehart, Duke University Medical Center Daniel Lew. Duke University Medical Center R26 Annual Report Mary Ann Sells. Fox Chase Cancer Center Edwin Taylor, University of Chicago Joseph S. Wolenski, Yale University Elaine Yeh. University of North Carolina, Chapel Hill Teaching Assistants Elaine Bardes, Duke University Medical Center Lisa Evans, Yale University Tama Hasson, Yale University Amanda Hayward-Lester. Yale University Gary Michael Idelchik, University of Wisconsin Min-gang Li, University of Minnesota, St. Paul Feng Liu, Fox Chase Cancer Center Craig Mandate, University of Waterloo, Canada Ruth Montague. Duke University Medical College Samara Reek-Peterson. Yale University Melissa Reeder, Fox Chase Cancer Center Rey Antonio Sia. Duke University Medical Center Jenny Sider, University of Wisconsin Andre Silvanovich, University of Minnesota. St. Paul Lecturers Sid Altman, Yale University Angelika Amon. Massachusetts Institute of Technology Van Bennett, Duke University John Chant, Harvard University Paul Forscher, Yale University Martin Hemler, Dana Farber Cancer Institute Laurinda Jaffe, University of Connecticut Health Center Jennifer Lippincott-Schwartz. National Institutes of Health Bruce Mayer. Harvard University Ben Neel. Beth Israel Hospital Terry Orr- Weaver, Massachusetts Institute of Technology Michael Snyder, Yale University Karl Swann. University of Connecticut Health Center Mark Terasaki, University of Connecticut Health Center Lew Tilney, University of Pennsylvania Pat Wadsworth. University of Massachusetts. Amherst Sandra Wolen. Yale University Tian Xu, Yale University Medical School Bruce Zetter, Harvard University Course Coordinator Dawn Grant, Louisiana State University Medical Center Course Assistant Rchekah Harrison, Stanford University Students Richard Bayliss, University of Cambridge. United Kingdom Dale Beach, University of North Carolina. Chapel Hill George Bell, University of Arizona Jill Broome, University of North Carolina, Chapel Hill Julie Canman, University of North Carolina, Chapel Hill Anthony DePass, University of Massachusetts. Amherst John Diggins, Providence College Jean-Emmanuel Faure, Centre National de la Recherche Scientitique. France Rip Finst, Emory University Gundula Ones, University of Pennsylvania Edward Guo, Columbia University William Heinz, Johns Hopkins University Maria Holzmann, University of Geneva. Switzerland G. Karthikeyan, Tata Institute of Fundamental Research, India Kaoru Katoh. Marine Biological Laboratory Reed Kelso, Yale University Tijs Ketelaar. Wageningen Agricultural University, The Netherlands Rosy Lee, Stanford University Laura Linz, Louisiana State University Medical Center Jonathan Lyon, Scripps Institute of Oceanography Paul Maddox, University of North Carolina. Chapel Hill Gregory McGillem, University of Alabama, Birmingham Spontaneous McKnight. University of Arizona Brian Nihhelink, University of Vermont Helen Nilsson, University of Goteborg, Sweden Nesrin Ozoren, University of Pennsylvania Omar Quintero, Duke University Medical Center Rachael Ream. Hopkins Marine Station Eric Reese, University of California, Riverside Yasushi Satoh, University of Tokyo. Japan Matthew Simmons. University of Florida Justin Skoble, University of California, Berkeley Aline Valster, University of Massachusetts. Amherst Gang Wang, University of Iowa Yihong Wang, Ohio University Kathryn White, Scripps Institute of Oceanography Short Courses Analytical & Quantitative Light Microscopy (May 8 -May 16) Directors Greenfield Sluder, University of Massachusetts Medical Center. Worcester Foundation Campus David Wolf. University of Massachusetts Medical Center. Worcester Foundation Campus Faculty William B. Amos. Medical Research Council, United Kingdom Richard Cardullo, University of California, Riverside Walter Carrington, University of Massachusetts Medical School Jeff Gelles, Brandeis University Shinya Inoue. Marine Biological Laboratory Rudolf Oldenbourg. Marine Biological Laboratory Edward Salmon, University of North Carolina, Chapel Hill Randi Silver, Cornell University Medical College Kenneth Spring, National Institutes of Health Educational Programs R27 Yu-li Wang. University of Massachusetts Medical Center, Worcester Foundation Campus Warren Zipsel, Cornell University Teaching Assistants Christine Thompson. University of Massachusetts Medical Center. Worcester Foundation Campus Elizabeth Thompson. University of Massachusetts Medical Center. Worcester Foundation Campus Clare Waterman-Storer, University of North Carolina. Chapel Hill Course Coordinator Frederick Miller, University of Massachusetts Medical Center, Worcester Foundation Campus Students Julia Avery. Yale University Margaret Clarke. Oklahoma Medical Research Foundation Carol Cogswell. University of Sydney, Australia David Ceilings, North Carolina State University Rossella Conti. Brandeis University Carol Gregorio. University of Arizona. Tucson Alexey Khodjakov, Wadsworth Center for Labs and Research Helmut Knapp. Swiss Federal Institute of Technology. Switzerland Stephen Lambert. University of Massachusetts Medical Center, Worcester Foundation Campus Jeannie Lee. Whitehead Institute Edwin Levitan, University of Pittsburgh Ilona Linnoila. National Cancer Institute Frank Macaluso, Albert Einstein College of Medicine Richard MacDonald, Massachusetts General Hospital Ivana Novak. August Krogh Institute. Denmark Joan Packenham, National Institute of Environmental Health Sciences Brigitta Peteri-Brunback. Astra Hassle AB. Sweden Jonathan Pines, The Wellcome Trust/Cancer Research Campaign. United Kingdom Guillermo Romero. University of Pittsburgh Ichiro Sase, Laboratory of Molecular Biophotonics. Japan Shuji Toyonaga. Laboratory of Molecular Biophotonics, Japan Shlomo Trachtenberg. National Institutes of Health Steven Treistman, University of Massachusetts Medical Center Stefan Wilhelm. Carl Zeiss, Inc., Germany Ginger Withers, University of Virginia Steven Wooding. Medical Research Council, United Kingdom Ji-Hong Zang, Stanford University Julianne Zimmerman. Payload Systems, Inc. Medical Informatics (June 1-June 8) Director Daniel Masys. University of California. San Diego Faculty Paul Clayton. Columbia University James Grigsby, University of Colorado Health Sciences Center George Hripcsak. Columbia-Presbyterian Medical Center Stephen Johnson. Columbia-Presbyterian Medical Center Lawrence Kingsland. National Library of Medicine David Landsman. National Library of Medicine Donald D.A.B. Lindberg. National Library of Medicine Richard Rodgers, National Library of Medicine Jay Sanders, Global Telemedicine Group Students Michael Ashburn. University of Utah Lynda Baker, Wayne State University Janet Bangma. East Carolina University Health Sciences Library Stephen Bartold, Texas Tech University Health Science Center David Boilard. Medical College of Ohio Sekum Boni-Awotwi. Howard University Neil Busis, University of Pittsburgh Laura Cailloux, Northwest Portland Area Indian Health Board Thomas Clay, East Carolina University School of Medicine May Dobal. Wayne State University Janice Dreyer, Community Hospitals of Central California Kathel Dunn. New York Academy of Medicine David George. Reading Hospital & Medical Center Gale Hannigan, Texas A&M University Health Science Center Conor Heneghan, New York Eye & Ear Infirmary Ruth Hoist. Columbia Hospital Arthur Huntley. University of California. Davis Charles Jaffe, American College of Allergy Juan Larach. Mission Neighborhood Health Center Clare Leibfarth, Doctors Hospital of Stark County Ethel Madden, Ochsner Medical Institutions Faith Meakin, University of Florida Peter Pevonka, University of Florida Health Science Center Ted Rosenkrantz, University of Connecticut Health Center Floyd Russell, West Virginia University Gerald Segal, New York State Psychiatric Institute Marcus Simpson, George Washington University Elaine Trzebiatowski, Allina Health System Amy Warner, University of Michigan Ken Wolf, Charles R. Drew University of Medicine & Science Methods in Computational Nenroscience (August 3 -Au gust 30) Directors David Kleinfeld, University of California, San Diego David Tank. Bell Laboratories/Lucent Technologies Faculty Lawrence Abbott, Brandeis University Moshe Abeles, Hebrew University of Jerusalem. Israel Richard Andersen. California Institute of Technology Robert Barlow, State LIniversity of New York Health Science Center William Bialek, NEC Research Institute Thomas Collett, University of Sussex. United Kingdom Rob de Ruyter van Steveninck. NEC Research Institute Kerry Delaney, Simon Fraser University. Canada Bard Ermentrout, University of Pittsburgh David Hansel, Ecole Polytechnique, France John Hopfield, Princeton University Daniel Johnston. Baylor College of Medicine Nancy Kopell. Boston University Kevan Martin, Swiss Federal Institute of Technology, Switzerland Kevin Martin. University of North Carolina. Chapel Hill David McCormick, Yale University School of Medicine Wolfram Schultz. Universite de Fribourg. Switzerland Terrance Sejnowski, Salk Institute R28 Annual Report H. Sebastian Seung, Bell Laboratories Arthur Sherman, National Institutes of Health Karen Sigvardt, University of California, Davis Frederick Sigworth, Yale University School of Medicine Haim Sompolinsky. Hebrew University of Jerusalem. Israel Mircea Steriade, Universite Laval, Canada David Terman. Ohio State University Naftali Tishby, Hebrew University of Jerusalem, Israel Misha Tsodyks, The Weizmann Institute, Israel Steven Zucker, Yale University Lab Instructors Roderick Jensen, Wesleyan University Terrance Kovacs, Bell Laboratories John White, Boston University Course Assistant Joseph Paton, Tufts University Students Thomas Adelman. Cornell University Yoram Ben Shaul, Hebrew University Hadassah School of Medicine, Israel Karl Deisseroth, Stanford University Reiko Maki Fitzsimonds. University of California. San Diego Galit Fuhrmann, Hebrew University of Jerusalem, Israel Mark Goldman, Harvard University Audrey Guzik, Allegheny University Richard Hahnloser, Swiss Federal Institute of Technology, Switzerland Robert Harris, University of Cambridge, United Kingdom Katrina MacLeod. California Institute of Technology William Miller, Brandeis University Jonathan Murnick, Harvard University Medical School Louis Neltner, Ecole Polytechnique, France Duane Nykamp, New York University Anitha Pasupathy, Johns Hopkins University Sharon Peled, Massachusetts Institute of Technology Daniel Reich, Rockefeller University Mark Schnitzer, Princeton University Oren Shriki, Hebrew University of Jerusalem, Israel Jonathan Simon. University of Maryland William Vinje. University of California, Berkeley Martin Wainwright, Harvard University James Zackheim, Rutgers University Microinjection Techniques in Cell Biology (May 20-May 27) Director Robert B. Silver, Marine Biological Laboratory Faculty Suzanne Klaessig. Cornell University Douglas Kline. Kent State University Gernot Presting, Institute of Plant Breeding, Germany Eric Shelden, University of Michigan Course Assistant Lisa Mehlmann, University of Connecticut Health Center Students Annie Borgne, CNRS. France Tao Cheng, Massachusetts General Hospital Goffredo Cognetti, University of Palermo, Italy Maxim Dorovkov, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School William Faught. Medical University of South Carolina Jennifer Hartt, University of Pennsylvania Lorayne Jenkins, Wyeth Ayerst Research Kathleen Jensen, U.S. Environmental Protection Agency Shuxian Jiang. Harvard Institutes of Medicine David Lagunoff. St. Louis University Yoshiaki Omura, Heart Disease Research Foundation Stelios Papaioannou. United Medical and Dental School of Guy's and St. Thomas' Hospitals. United Kingdom May Penarroyo, De La Salle University, Philippines Gloria Perez. Massachusetts General Hospital Margherita Randazzo. University of Palermo. Italy Vasantha Reddi, University of Virginia Carol Reinisch, Tufts University School of Veterinary Medicine Nancy Searby, NASA Ames Research Center/Stanford University Jennifer Stephens. University of Oklahoma Nirmala SundarRaj, University of Pittsburgh Molecular Mycology: Current Approaches to Fungal Pathogenesis (August 10-Augmt 30) Directors John Edwards, Jr., Harbor-UCLA Medical Center Paul T. Magee, University of Minnesota Aaron P. Mitchell, Columbia University Lecturers Arturo Casadevall, Albert Einstein College of Medicine Gary T. Cole, Medical College of Ohio Brendan Cormack, Stanford University School of Medicine Jim Cutler. Montana State University Judith Domer, Tulane Medical School Scott Filler, Harbor-UCLA Medical Center Gerald Fink, Whitehead Institute of Biomedical Research Carol A. Kumamoto, Tufts University School of Medicine Myra Kurtz, Merck Research Laboratories June Kwon-Chung, National Institutes of Health John McCusker, Duke University Medical Center John Perfect, Duke University School of Medicine Michael A. Pfaller, University of Iowa College of Medicine Judith Rhodes. University of Cincinnati Stewart Scherer, University of Minnesota School of Medicine Brian Wong. Veterans Administration Connecticut Healthcare System Teaching Assistants Janna Beckerman, University of Minnesota Maria Soushko, Columbia University Yang Xiao. Columbia University Educational Programs R29 Course Assistant Joseph Paton. Tufts University Students James Alspaugh, Duke University Kent Buchanan, University of Oklahoma Health Sciences Center Enda Clarke, Royal Postgraduate Medical School, United Kingdom Marianne De Backer, Janssen Research Foundation. Belgium Maurizio Del Poeta, Duke University Medical Center Tamara Doering, Cornell University Medical School Roy Hopfer, University of North Carolina. Chapel Hill Margaret Hosteller. University of Minnesota Linda Janusek, Loyola University of Chicago Herbert Mathews, Loyola University of Chicago Frank-Michael Miiller, National Cancer Institute Norbert Schnell, Zeneca Pharmaceuticals, United Kingdom Craig Thompson, Scriptgen Pharmaceuticals Jo-Anne van Burik. Fred Hutchinson Cancer Research Center Paul Verweij. University Hospital Nymegen. The Netherlands Mason Zhang, University of Nevada School of Medicine Neurobiology & Development of the Leech (August 9-August 30) Directors Pierre Drapeau. McGill University, Canada Martin Shankland. University of Texas, Austin Faculty Andreas Baader, University of Zurich. Switzerland Irmgard Dietzel-Meyer. Ruhr-Universitat Bochum, Germany Francisco Fernandez de Miguel. Universidad Nacional Autonoma, Mexico John Jellies. Western Michigan University Jorgen Johansen. Iowa State University Anna Kleinhaus. New York Medical College Eduardo Macagno, Columbia University Mark Martindale. University of Chicago Barbara Modney. Cleveland State University Kenneth Muller, University of Miami School of Medicine John Nicholls, University of Basel, Switzerland Christine Sahley, Purdue University Catherine Wedeen, New York Medical College David Weisblat, University of California Course Assistant Gabrielle Tomasky. Marine Biological Laboratory Students Istvan Albert, University of Notre Dame Dianne Allen, Louisiana State University Medical Center Maria Casanueva. Catholic University. Chile James Einum. Marquette University Chunta Jie. Iowa State University Lynette Nguyen. Smith-Kettlewell Eye Research Institute Elizabeth Perruccio, New York Medical College Aloysius Phillips. Columbia University Giulietta Pinato, International School for Advanced Studies, Italy Subhabrata Sanyal, Tata Institute of Fundamental Research, India Daniel Shain. University of California, Berkeley Patrick Wigge, Medical Research Council. United Kingdom Optical Microscopy and Imaging in the Biomedical Sciences (October 8-October 16) Director Colin Izzard. State University of New York. Albany Facitlt\ Joseph DePasquale, New York State Department of Health Robert Hard, State University of New York, Buffalo Brian Herman, University of North Carolina, Chapel Hill Shahid Khan. Albert Einstein College of Medicine Frederick Maxrield, Cornell University Medical College John Murray, University of Pennsylvania David M. Piston. Vanderbilt University Kenneth Spring. National Institutes of Health Jason Swedlow. Harvard University Medical School Teaching Assistants Ken Dunn. Indiana University Medical Center Lynda Pierini. Cornell University Medical College Wade Sigurdson. State University of New York, Buffalo Elizabeth Welnhofer, State University of New York, Buffalo Lecturers Jan Hinsch, Leica, Inc. Shinya Inoue, Marine Biological Laboratory H. Ernst Keller, Carl Zeiss. Inc. Rudolf Oldenbourg. Marine Biological Laboratory Martin Scott, Consultant in Scientific Imaging Course Assistant Kari Lavalli, Marine Biological Laboratory Students Diana Bartelt, St. John's University Michelle Burack, University of Virginia Dorthe Christensen, Biolmage, Denmark Conan Cooper, University of Calgary, Canada John Crocker. University of Pennsylvania Leanne Delbridge. University of Melbourne, Australia Mark Drew, Johns Hopskins University Tracey du Laney, University of North Carolina, Chapel Hill Lars Hansen, Hagedorn Research Institute, Denmark Brian Helmke, University of Pennsylvania Robert Hughes, University of Washington Eleanor Kable. Sydney University, Australia Peter Kaplan, University of Pennsylvania Timothy King. University of Texas Thomas Kinraide. United States Department of Agriculture David Marcey. Kenyon College Susanne Pedersen, University of Copenhagen, Denmark Marli Robertson, University of Calgary, Canada Pascal Stein, Harvard Medical School David Swift, University of Pennsylvania James Thomson. University of Wisconsin Sebastian Tille, Carl Zeiss Jena. Germany Mariko Tokito, University of Pennsylvania Frances Wang, National Institute of Standards & Technology Karen Zito. University of California. Berkeley R30 Annual Report Pathogenesis of Neuroimmunologic Diseases (August 17- August 29) Directors Celia F. Brosnan. Albert Einstein College of Medicine Jack Rosenbluth, New York University School of Medicine Faculty Barbara Barres, Stanford University School of Medicine Etty Benveniste, University of Alabama, Birmingham Joan Berman, Albert Einstein College of Medicine Peter Charles. Albert Einstein College of Medicine Patricia Coyle, State University of New York, Stony Brook Robert Darnell. Rockefeller University Judah Denburg. McMaster University, Canada David Felten, University of Rochester Robert M. Gould. New York State Institute of Basic Research Diane Griffin. Johns Hopkins University John Griffin, Johns Hopkins School of Medicine William Hickey. Dartmouth-Hitchcock Medical Center Gilla Kaplan. Rockefeller University Paul Knopf, Brown University Vijay Kuchroo, Brigham and Women's Hospital Jon Lindstrom. University of Pennsylvania School of Medicine James Martiney, Picower Institute for Medical Research Steven Pteifter, University of Connecticut Health Center Richard Ransohoff, Cleveland Clinic Foundation Bruce Ransom, University of Washington School of Medicine Anthony Reder. University of Chicago J. M. Ritchie, Yale University School of Medicine James Salzer, New York University Medical Center Clifford Saper, Beth Israel Hospital Moon Shin, University of Maryland, Baltimore Michele Solimena, Yale University Esther Sternberg, National Institutes of Health J. Wayne Streilein, Scheppens Eye Research Institute Byron Waksman. Foundation for Microbiology Students Amit Bar-Or, Massachusetts General Hospital Rita Baron-Faust. WCBS NewsRadio 88 Alexei Boiko. Russian State Medical University, Russia Emanuela Bonfoco, La Jolla Institute of Immunology Laurent Coscoy, Pasteur Institute, France Benedicte Dubois. Rega Institute, Belgium Urszula Fiszer, Institute of Psychiatry & Neurology. Poland Glen Greenough. Dartmouth-Hitchcock Medical Center Carolyn Hoban, Cambridge Neuroscience Inc. Liwei Hua. Albert Einstein College of Medicine Kee-Ching Jeng, Taichung Veterans General Hospital, Taiwan Pitagoras Justino. Universidade Federal de Uberlandia. Brasil Bernd Kieseier. University of Wurzburg. Germany Pia Kivisakk, Karolinska Institute, Sweden Dmitriy Labunsky. Institute of Neurology, Russia Igor Leykin, Weizmann Institute of Science, Israel Carrie McManus, Albert Einstein College of Medicine Mary McMenamin. Oxford University. United Kingdom Neelufar Mozaffarian, Albert Einstein College of Medicine Marcin Mycko, Medical Academy of Lodz, Poland Jitendra Patel, Zeneca Pharmaceuticals Barry Singer. New York Hospital-Cornell Medical Center Nevil Singh. Tata Institute of Fundamental Research, India Sulpicio Soriano. Children's Hospital/Harvard Medical School Workshop on Molecular Evolution (August 3-August 15) Directors Daniel B. Davison. Bristol-Myers Squibb PR1 Mitchell Sogin, Marine Biological Laboratory Faculty W. Ford Doolittle. Dalhousie University, Canada Douglas Eernisse. California State University Joseph Felsenstein. University of Washington Michael Gray. Dalhousie University, Canada Robert Haselkorn, University of Chicago David Hillis. University of Texas Mike Holder, University of Houston Richard Hudson, University of California, Irvine Thomas Kaufman, Indiana University David Maddison. University of Arizona, Tucson Geoffrey McFadden, University of Melbourne, Australia Lynn Miller, Genetics Computer Group, Inc. Michael Miyamoto, University of Florida Educational Programs R31 Rasmus Nielsen. University of California. Berkeley Gary Olsen, University of Illinois William Pearson, University of Virginia Kevin Peterson, California Institute of Technology David Roos. University of Pennsylvania Pamela Soltis, Washington State University David Swofford. Smithsonian Institution John Wakeley, Nelson Biological Labs Bruce Walsh, University of Arizona, Tucson Sam Ward. University of Arizona. Tucson Carl Woese, University of Illinois, Urbana-Champaign Teaching Assistant Steven Thompson. Washington State University Course Assistant Udeni Amit. Marine Biological Laboratory Students Michael Alfaro. University of Chicago Field Museum of National History John Archibald, Dalhousie University, Canada Pamela Arnofsky, Woods Hole Oceanographic Institution Vijay Aswani, Smithsonian Tropical Research Institute Andrew Baker, University of Miami Anne Bansemir, Rutgers University Elizabeth Barratt, Zoological Society of London, United Kingdom Mary Bateson, Montana State University Christiane Biermann. State University of New York, Stony Brook Carrine Blank, University of California, Berkeley Lisa Borghesi. Oklahoma Medical Research Foundation Adriana Briscoe, Harvard University Daniel Brumbaugh. University of Texas, Austin Nina Brunner. University of Essen, Germany Carmen Cadilla, University of Puerto Rico Susan Chien, University of Florida Manuela Coelho. University of Lisbon, Portugal Chris Conroy, University of Alaska Museum Colomban de Vargas. University of Geneva. Switzerland Joel Dore. 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University of Texas, Houston Lei Liu, University of Connecticut Frieder Mayer, University of Erlangen, Germany Damhnait McHugh. Harvard University Kirsten Nicholson. University of Miami Link Olson, University of Chicago Field Museum of National History Bertil Pettersson. Royal Institute of Technology. Sweden Mary Poss, University of Washington Linda Prince. University of North Carolina. Chapel Hill Anne Marie Quinn. Yale University Patrick Reynolds. Hamilton College Frank Rosenzweig. University of Idaho Marco Salemi. Rega Institute. Belgium Andrew Salywon, Arizona State University Nikolaos Schizas, University of South Carolina Alastair Simpson, University of Sydney, Australia James Robert Stevens, University of Bristol. United Kingdom Suzanne Sukhdeo. Rutgers University Jin Gou Tone. Chinese University, Hong Kong Catherine Walton. University of Leeds, United Kingdom Hui Wang, University of Houston Jacqueline Weicker. University of Alaska. Fairbanks Connie Westhoff, University of Nebraska Adrian Whatmore. University of Warwick. United Kingdom Kenneth Wurdack. University of North Carolina, Chapel Hill Other Programs Semester in Environmental Science (September 8-December 19) Directors Jerry M. Melillo, Director Kenneth H. Foreman. Associate Director Faculty Charles Hopkinson. Aquatic Course Director Knute Nadelhoffer, Terrestrial Course Director John Hobbie. Microbial Ecology Edward Rastetter, Mathematical Modeling Anne Giblin Linda Deegan Bruce Peterson Christopher Neill Joe Vallino Mathew Williams R32 Annual Report Paul Steadier Peter Siver, Faculty Fellow. Connecticut College Research Assistants Jeffrey Hughes James Laundre Jane Tucker Lori Soucy Kristin Tholke Kathleen Regan Beth Hooker John Helfnch Martha Downs Amy Nolin Kathleen Newkirk Neil Bettez Teaching Assistants Martha Peterson Michele Bahr Patricia Micks Bonnie Kwiatkowski Robert Garritt Nat Weston Students Toby Ahrens, Connecticut College Hyacinth Armstrong. Mt. Holyoke College Noah Bleich. Brandeis University Abbey DeRocker. Bates College Lynn Diener, Bard College Janice Glass, Lafayette College Sarah Jackson, Connecticut College Samuel Kelsey, Dickinson College Christy Meredith. Allegheny College Sophie Parker. Wellesley College Stephanie Parker, Middlebury College Rachel Poretsky. Brandeis University Shana Rapoport, Brandeis University Amy Townsend-Small, Skidmore College Marlene Tsie. Brandeis University Rebecca Weidman, Carleton College Teachers' Workshop: Living in the Microbial World (August 17 -August 23) Course Director Lorraine Olendzenski, University of Connecticut Course Assistant Andy Heaford, University of Connecticut Presenters Lynn Margulis, University of Massachusetts Ricardo Guerrero, University of Barcelona, Spain Steve Goodwin. University of Massachusetts, Amherst Robert Bullis, Marine Biological Laboratory Holger Jannasch. Woods Hole Oceanographic Institute Greg Hinkle, University of Massachusetts, Dartmouth Art Girard, Pfizer Central Research. Groton. CT Norman Wainwright, Marine Biological Laboratory Adrian Smith. Marine Biological Laboratory Participants Charles Anastasia, Mashpee High School Diane E. Arnold, Bennie Dover Jackson Middle School Catherine Baker, East Junior High School Marcia A. Benvenuti. Bennie Dover Jackson Middle School Florence Berdan, Parsippany Hills High School Wesley Blauss, Indian Head Middle School William Cerino, Lyme-Old Lyme High School Paul J. Chamberlin. Nauset Regional High School Elizabeth Check. Attleboro High School Howard C. Estes, East Junior High School Maryrose L. Flynn, Indian Head Middle School Marsha R. Folger. Lyme-Old Lyme High School Mary Johnson. Parsippany Hills High School. Brooklawn Middle School Lucy Lupinacci. Griswold Intermediate School Joreen Mattras. Griswold Intermediate School Sheila E. McTigue, Lyme-Old Lyme High School M. Susan O'Donnell, Indian Head Middle School Jane Shute, Indian Head Middle School Judith J. Trotta, Nauset Regional High School James Watson, East Junior High School Summer Research Programs Principal Investigators Armstrong, Clay, University of Pennsylvania Armstrong, Peter B., University of California, Davis Augustine, George J., Duke University Medical Center Barbieri, Elena, Marine Biological Laboratory Barlow, Jr.. Robert B., State University of New York Health Science Center Beauge, Luis, Instituto M. y M. Ferreyra, Argentina Bennett, Michael V. L., Albert Einstein College of Medicine Berlin, Joshua, Bockus Research Institute Bloom, George, University of Texas Southwestern Medical Center Bodznick, David, Wesleyan University Boron. Walter F., Yale University Medical School Borst, David, Illinois State University Boyer, Barbara, Union College Brady, Scott T., The University of Texas Southwestern Medical Center, Dallas Brown, Joel E.. Albert Einstein College of Medicine Browne. 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Roger D., Dartmouth College Spann, Timothy, Northwestern University Medical School Spiegel, Evelyn, Dartmouth College Spiegel. Melvin. Dartmouth College Standart, Nancy, University of Cambridge. United Kingdom Steffen. Walter. Institute of Biochemistry and Molecular Cell Biology. Austria Stuart, Ann E.. University of North Carolina, Chapel Hill Sugimon. Mutuysuki. New York University Medical Center Suszkiw, Janusz, University of Cincinnati Takahashi, Megumi, Kanagawa Psychiatric Center, Japan Telzer, Bruce, Pomona College Tokumam, Hiroshi, Duke University Medical Center Tran, Phong, University of North Carolina, Chapel Hill Troll, Walter, New York University Medical Center Tytell, Michael, Bowman Gray School of Medicine, Wake Forest University Wachowiak, Matt, University of California, Berkeley Weil, E. Jennifer. Marine Biological Laboratory Whittaker, J. Richard. University of New Brunswick. Canada Wicklein. Martina. University of Ari/ona Zigman. Seymour, University of Rochester Medical School Zottoli. Steven. Williams College Zukin, R. Suzanne. Albert Einstein College of Medicine Other Research Personnel Abe, Terno. Niigata University, Japan Altamirano, Anibal, University of Buenos Aires. Argentina Anderson. Erik, St. Francis Xavier University, Canada Antic. Srdjan, Yale University School of Medicine Araneda, Ricardo, Albert Einstein College of Medicine Armstrong, Clara. University of Pennsylvania Baikie, lain. Robert Gordon University. United Kingdom Barrera. Jose, New York University Medical Center Bearer, Elaine, Brown University Becker, Julie, Temple University Bezanilla. Francisco. University of California. Los Angeles Bobb. David, Huston-Tillotson College Boyle. Kim-Laura, Colby-Sawyer College Brackenbury. Robert, University of Cincinnati Breitwieser, Gerda E., Johns Hopkins School of Medicine Brown, Euan. Marine Biological Association. United Kingdom Burris, Jennifer. Carleton College Carnall, Nicola. 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Miguel, Massachusetts General Hospital Horner, Michael, University of Gottingen, Germany Jarchow. Janina. Friedrich Miescher Institute, Switzerland Johannes, Eva. North Carolina State University Jonas, Elizabeth. Yale University School of Medicine Kaftan, Edward, University of Connecticut Health Center Kamino, Kohtaro, Tokyo Medical and Dental University, Japan Katz. Benjamin, Williams College Keyoung. Jinsoo, Albert Einstein College of Medicine Khater, Kevin, Chicago Medical School Khuon, Satya. Northwestern University Medical School Klimov, Andrei. University of Pennsylvania Koroleva, Zoya, Hunter College Koulen. Peter, Max-Planck-Institute for Brain Research. Germany Krasne, Frank, University of California, Los Angeles Krasne. Sally, University of California. Los Angeles Kummer. Rebecca, Williams College Lam, Ying-Wan, Yale University Lasser-Ross, Nechama. New York Medical College Lema-Foley, Christine. 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Duke University Medical Center Tokumaru, Keiko, Duke University Medical Center Twersky, Laura, St. Peters College Vasandani. Veena. University of Virginia Wagg, Jonathan. The Rockefeller University White. Benjamin. Yale University Williams, Tracey, Howard University Wollert. Torsten. University of Rostock, Germany Womack, Mary, Duke University Medical School Wu, Peter. Columbia University R36 Annual Report Zakevicius, Jane M., University of Illinois, Chicago Zavilowitz, Joseph, Albert Einstein College of Medicine Zecevic, Dejan, Yale University School of Medicine Zeki, Semir, University College London, United Kingdom Zhao. Jinhua, Yale University Zigman. Bunnie R.. University of Rochester Medical Center Library Readers Abbott, Jayne, Marine Research Inc. Adelberg, Edward A.. Yale University Ahmadjiian. Vernon, Clark University Alkon, Daniel, National Institutes of Health Allen. Garland E.. Washington University Alliegro, Mark, Louisiana State University Medical Center Anderson, Everett, Harvard Medical School Baccetti, Baccio, University of Siena, Italy Barry, Susan R., Mount Holyoke College Benjamin, Thomas L., Harvard Medical School Bentham, Dolores A., SPC Associates, New York Berne, Rosalyn W., University of Virginia Bernhard, Jeffrey D., University of Massachusetts Medical Center Bernheimer, Alan W., New York University Borgese, Thomas A., Lehman College Boyer, John, Union College Burgess, David, Pittsburgh, PA Campbell, Robert K., Ares Advanced Technology Candelas. Graciela C., University of Puerto Rico Cariello, Lucio, Stazione-Zoologica, Italy Chaet, A. B., University of West Florida Child. Frank M., Trinity College Clark, Arnold M.. University of Delaware Clark, Denise, University of New Brunswick Clark. Eloise E., Bowling Green State University Clarkson. Kenneth L., Bell Labs, Lucent Technologies Cogswell, Carol, University of Sydney, Australia Cohen, Seymour S., American Cancer Society Cohen, Leonard A., American Health Foundation Cohen, Yehuda, Hebrew University of Jerusalem, Israel Collier, Marjorie M., St. Peters College Comoglio, Paolo. Institute for Cancer Research, Turino, Italy Cooperstein, Shenvin J.. University of Connecticut Health Center Copeland, Eugene C., Woods Hole, MA Cowling, Vincent F., SUNY, Albany D'Allessio, Giuseppe, University of Naples deToledo-Morrell. Leyla, Rush Medical College Duncan, Thomas K., Nichols College Epstein, Herman T.. Brandeis University Feldman, Susan C., New Jersey Medical School Finch, Caleb E., University of Southern California Frenkel, Krystyna, NYU Medical Center Gehrke, Lee, Massachusetts Institute of Technology German, James L., New York Blood Center Goldman, Robert, Northwestern University Medical School Goldstein, Moise H., Johns Hopkins University Groden, Joanna, University of Cincinnati Gross. Paul, University of Virginia Grossman, Albert. NYU Medical School Gruner, John, Cephalon, Inc. Haimo, Leah, University of California Halvorson, Harlyn, University of Massachusetts, Dartmouth Herskovits, Theodore T.. Fordham University Hunter. Robert. Gartnaval Royal Hospital Ilan, Judith, Case Western Reserve University Inoue, Sadayuki. McGill University. Canada Issodorides. Marrietta, University of Athens, Greece Jacobson, Allan S.. University of Massachusetts Medical Center Josephson, Beth, Ocean Arks International Kaltenbach, Jane C.. Mount Holyoke College Kaminer. Benjamin, Boston University School of Medicine Kamino, Kohtaro. Tokyo Medical & Dental University, Japan Kaplan, Arnold, University of Illinois at Chicago Karlin. Arthur. Columbia University Kelly, Robert E., University of Illinois Keynan, Alex, Israel Academy of Sciences and Humanities King, Kenneth, Falmouth. MA Klein. Donald, Colorado State University Kramer, Fred R., Public Health Research Institute 1997 Library Room Readers Daniel Alkon National Institutes of Health Lucio Cariello Stazione-Zoolociga A. Dohrn A. Chaet University of Connecticut Paolo Comoglio Institute of Cancer Research, Turino, Italy Giuseppe D'Alessio University of Naples Robert Goldman Northwestern Univ. Medical School Harlyn Halvorson Marine Biological Laboratory Alex Keynan Israel Academy of Science Hans Laufer University of Connecticut Joe L. Martinez University of Texas, San Antonio Michael Rabinowitz Marine Biological Laboratory George Reynolds Princeton University Gerald Weissman NYU School of Medicine Summer Research R37 Laderman. Aimlee D.. Yale School of Forestry & Environmental Studies Laster. Leonard, University of Massachusetts Medical Center Laut'er, Hans, University of Connecticut Lee. John J.. City College of CUNY Leighton. Joseph, Aeron Biotechnology, Inc. Levy, Arthur L., St. Vincents Hospital of New York Lisman, John, Brandeis University Long, Carol, Allegheny University Lorand, Laszlo, Northwestern University Medical School Luckenbill-Edds, Louise, Ohio University MacNichol. Edward F.. Boston University School of Medicine Major. Guy. Oxford University Masland, Richard. Massachusetts General Hospital Martinez. Joe L., University of Texas, San Antonio Mauzerall, David, Rockefeller University Michaelson. James, MGH Cancer Center Miller, Daniel, Rockefeller University Mills. Eric L., Dalhousie University, Canada Mmkoff, Charles G.. Duke University Medical Center Mitchell. Ralph. Harvard University Mizell, Merle, Tulane University Nagel, Ronald L.. Albert Einstein Institute Narahashi, Toshio, Northwestern University Medical School Nathans, Jeremy. Johns Hopkins University Naugle, John, NASA Nicaise, Mari-Luz, Universite de Nice, France Nicaise, Ghislain, Universite de Nice, France Nickerson. Peter A., SUNY, Buffalo Pappas. George D., University of Illinois Pollen, Daniel A., University Massachusetts Medical Center Porter, Mary E., University of Minnesota Prusch, Robert D.. Gonzaga University Przybyszewski. Andrew W., University of Massachusetts Medical Center Rabinowitz, Michael. Marine Biological Laboratory Rafferty, Nancy S.. Northwestern University Rafferty, Keen, Northwestern University Reynolds, George, Princeton University Rosenbluth. Raja, Simon Fraser University Rosenkranz, Herbert S., University of Pittsburgh Ryan, Terence E.. Regeneron Pharmaceuticals Sanger, Joseph W., University of Pennsylvania Medical School Sanger. Jean M.. University of Pennsylvania Medical School Schauder. Rolf. University of Frankfurt, Germany Segal. Rosalind, Harvard Institute of Medicine Shepro, David. Boston LIniversity Microvascular Research Siwicki. Kathleen K., Swarthmore College Spector, Abraham, Columbia University Spotte, Stephen. University of Connecticut Sundquist. Eric. US Geological Survey Sweet, Frederick, Washington University School of Medicine Tilney, Lewis, University of Pennsylvania Trager, William, The Rockefeller University Tweedell. Kenyon S., University of Notre Dame Tykocinski, Mark L., Case Western Reserve University Van Holde, Kensal E., Oregon State University Walton, Alan J.. Cavendish Lab, Cambridge University, UK Warren, Leonard, Wistar Institute Weidner, Earl, Louisiana State University Weissman. Gerald. NYU Medical Center Whittaker, Victor P.. Max-Planck-Institute for Biophysical Chemistry Yevick, George. Stevens Institute of Technology Domestic Institutions Represented AgBiotech Center Alabama, University of, Birmingham Alaska Museum. University of Alaska, University of Albert Einstein College of Medicine Allegheny University Allegheny University of the Health Sciences Allina Information Services American College of Allergy Arizona State University Arizona. University of Bancroft School Baylor College of Medicine Bell Labs Beth Israel Medical Center Bockus Research Institute Boston University Boston University School of Education Boston University School of Medicine Bowdoin College Brandeis University Brigham and Women's Hospital R38 Annual Report Bristol-Myers Squibb PRI Brown University California Institute of Technology California State University 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 Cambridge NeuroScience, Inc. Carl Zeiss, Inc. Carleton College Case Western Reserve University Center for Great Lakes Studies Chicago Field Museum, University of Chicago Medical School Chicago, University of Children's Hospital. Boston Cincinnati Medical Center, University of Cincinnati, University of Cleveland Clinic Foundation Cleveland State University Colby College Colby-Sawyer College Cold Spring Harbor Laboratories College of the Holy Cross Colorado Health Science Center, University of Colorado School of Mines Colorado, University of Columbia Hospital Library Columbia University Columbia-Presbyterian Medical Center Community Health Systems Connecticut College Connecticut Health Center, University of Connecticut. University of Cornell University Cornell University Medical College Cornell University Veterinary School Dana Farber Center Institute Dartmouth College Dartmouth-Hitchcock Medical Center Doctors' Hospital of Stark County Drew University of Medicine & Science Duke University Duke University Marine Laboratory Duke University Medical Center East Carolina University East Carolina University School of Medicine Emory University Eye Foundation Hospital Finch University of Health Sciences Florida State University Florida, University of Food and Drug Administration Fordham University College, Lincoln Center Forsyth Dental Center Foundation of Microbiology Fox Chase Cancer Center Fred Hutchinson Cancer Research Center Genetics Computer Group, Inc. George Washington University Georgia State University Georgia, University of Global Telemedicine Group Goucher College Hamilton College Hampshire College Harbor-UCLA Medical Center Hartford. University of Harvard Institutes of Medicine Harvard Medical School Harvard University Harvard University School of Public Health Heart Disease Research Foundation Hollins College Hopkins Marine Station Houston, University of Howard Hughes Medical Institute Howard University Hunter College Huston-Tillotson College Idaho. University of Illinois State University Illinois Wesleyan University Illinois, University of Indiana University Indiana University School of Medicine Infectious Disease Research Institute Institute for Basic Research in Developmental Disabilities Iowa College of Medicine, University of Iowa State University Iowa, University of Jester Center Jet Propulsion Laboratory Johns Hopkins University Johns Hopkins University School of Medicine Johns Hopkins University School of Public Health Kansas, University of Kent State University Kentucky College of Medicine, University of Laboratory of Kidney & Electrolyte Metabolism Leica, Inc. Louisiana State University Medical Center Loyola University of Chicago Maine, University of Marine Biological Laboratory Marquette University Marshall University Summer Research R39 Maryland, University of Massachusetts General AIDS Research Center Massachusetts General Hospital Massachusetts Institute of Technology Massachusetts Medical School, University of Massachusetts, University of Medical College of Georgia Medical College of Ohio Medical College of Pennsylvania Medical University of South Carolina Meharry Medical College Memorial Sloan-Kettering Cancer Center Merck Research Laboratories Metabolix. Inc., Cambridge Miami. University of Michigan State University Michigan Technological University Michigan, University of Millbrook School Minnesota Medical School, University of Minnesota, University of Mission Neighborhood Health Center Montana State University Museum of Comparative Zoology Labs NASA Ames Research Center National Cancer Institute National Institute of Environmental Health Science National Institute of Standards & Technology National Institutes of Health National Library of Medicine Naval Medical Research Institute Nebraska. University of NEC Research Institute Nelson Biological Labs Nevada, University of New Jersey, College of New Jersey, University of Medicine and Dentistry New York Academy of Medicine New York Eye & Ear Infirmary New York Medical College New York State Department of Health New York State Institute for Basic Research New York State Psychiatric Institute New York University Medical Center New York University School of Medicine North Carolina State LIniversity North Carolina, University of North Carolina, University of, Chapel Hill North Texas, University of Northeastern University Northwestern University Northwestern University Medical School Notre Dame, University of NW Portland Area Indian Health Board Ochsner Medical Library Ohio State University Ohio University Oklahoma Health Sciences. University of Oklahoma Medical Research Foundation Oregon Health Science University Oregon, University of Payload Systems, Inc. Pennsylvania School of Medicine, University of Pennsylvania School of Veterinary Medicine, University of Pennsylvania State University Pennsylvania, University of Picower Institute for Medical Research Pittsburgh School of Medicine, University of Pittsburgh, University of Pomona College Princeton University Providence College Puerto Rico, University of Purdue University Reading Hospital & Medical Center Rensselaer Polytechnic Institute Rochester Medical School. University of Rochester. University of Rockefeller University Rowland Institute for Science Rutgers University Salk Institute San Francisco State University Scheppens Eye Research Institute Scripps Institution of Oceanography Scripps Research Institute Scriptgen Pharmaceuticals Seattle Biomedical Research Institute Sloan Center for Theoretical Neurobiology Smith-Kettlewell Eye Research Institute Smithsonian Institution Smithsonian Tropical Research Institute South Carolina. University of Southwestern University Medical Center St. John's University St. Louis University School of Medicine St. Peters College Stanford University Stanford University Medical Center State University of New York Health Science Center State University of New York. Albany State University of New York, Buffalo State University of New York, Stony Brook Temple University Tennessee, University of Texas A&M University Texas A&M University College of Medicine Texas Christian University Texas Health Science Center, University of Texas M. D. Anderson Cancer Center, University of Texas Medical Branch, University of Texas Southern University Texas Southwestern Medical Center, University of Texas Tech University R40 Annual Report Texas, University of Toledo, University of Tufts University Tufts University School of Medicine Tufts University School of Veterinary Medicine Tulane University Medical School U. S. Army Natick RD&E Center U. S. Department of Agriculture U. S. Environmental Protection Agency Union College Utah, University of Vanderbilt University Vermont, University of Veterans Administration Connecticut Healthcare System Virginia Commonwealth University Virginia Health Sciences Center, University of Virginia School of Medicine, University of Virginia, University of Vollum Institute Wadsworth Center for Labs and Research Wake Forest University Wake Forest University, Bowman Gray School of Medicine Washington School of Medicine. University of Washington State University Washington, University of Wayne State University Wellesley College Wesleyan University West Virginia University Western Michigan University Wheaton College Whitehead Institute for Biomedical Research Williams College Wisconsin Regional Primate Research Wisconsin, University of Women & Infants Hospital Woods Hole Oceanographic Institution Worcester Foundation for Biomedical Research Wyeth-Ayerst Research Yale University Yale University School of Medicine Zanvyl Krieger Mind Brain Institute Zeneca Pharmaceuticals Foreign Institutions Represented Alberta, University of, Canada Astra Hassle, Sweden Auckland, University of. Australia August Krogh Institute. Denmark Basel. University of. Switzerland Bogor Agricultural University. Indonesia Bordeaux. University of. France Brazil. University of. Brazil Bristol. University of. United Kingdom Buenos Aires, University of, Argentina Calgary. University of, Canada Cambridge, University of. United Kingdom Carl Zeiss. Inc., Germany Catholic University of Chile, Chile CENA-USP. Brazil Centre National de la Recherche Scientitique. France Chinese University of Hong Kong. Hong Kong Dalhousie University. Canada De La Salle University, Phillipines Ecole Polytechnique, France Edinburgh. University of, Scotland Erlangen, University of. Germany Federal University of Minas Gerais, Brazil Frankfurt, University of, Germany Fribourg, University of, Switzerland Friedrich Meischer Institute, Switzerland Geneva. University of, Switzerland Goteborg, University of, Sweden Gottingen. University of, Germany Hagedorn Research Institute. Denmark Hebrew University of Jerusalem, Israel Hospital for Sick Children. Canada Huddinge University Hospital, Sweden I.V.I.C.. Venezuela Imperial College of Science, Technology and Medicine. United Kingdom Innsbruck. University of, Austria International School of Advanced Studies, Italy Institut Nationale de la Recherche Agronomique, France Institut Pasteur de Lille, France Institute for Marine Biosciences, Canada Institute of Biochemistry and Molecular Cell Biology. Austria Institute of Neurology of Moscow, Russia Institute of Plant Breeding. Germany Institute M. y M. Ferreyra. Argentina J. W. Goethe-Universitat, Germany Janssen Research Foundation, Belgium Julius-Maximilians-University. Germany Kangawa Psychiatric Center, Japan Karolinska Institute. Sweden Laboratory of Molecular Biophotonics, Japan Laval University School of Medicine, Canada Summer Research R41 Leeds. University of. United Kingdom Leiden, University of. The Netherlands Lethbridge. University of, Canada Lisbon, University of. Portugal Lund University. Sweden Manchester. University of. United Kingdom Marine Biological Association, United Kingdom Max-Planck-lnstitiit. Germany McGill University, Canada McMaster University, Canada Medical Academy of Lodz. Poland Medical Research Council, United Kingdom Melbourne. University of, Australia Milano. University of, Italy Montreal, University of. Canada Munich. University of, Germany Naples, University of, Italy National Institute for Medical Research, United Kingdom National University of Singapore, Singapore National Veterinary Institute. Sweden New Brunswick. University of, Canada Niigata University, Japan Oldenburg, University of, Germany Otago, University of. New Zealand Ottawa, University of, Canada Oxford. University of. United Kingdom Palermo. University of, Italy Poland Institute of Psychiatry & Neurology, Poland Queen's University, Canada Queen's University of Belfast. United Kingdom Rega Institute, Belgium Regina Elina Center Research Institute, Italy Robert Gordon University. United Kingdom Rostock, University of, Germany Royal Institute of Technology, Sweden Royal Postgraduate Medical School, United Kingdom Ruhr-Universitat Bochum, Germany Russian State Medical University, Russia School of Biological Sciences, United Kingdom Seville, University of, Spain Simon Fraser University, Canada St. Francis Xavier University. Canada Stazione Zoologica A. Dohrn, Italy Sussex, University of. United Kingdom Swiss Federal Institute of Technology, Switzerland Sydney, University of, Australia Taichung Veterans General Hospital, Taiwan Technion. Faculty of Medicine. Israel Tel Aviv University. Israel Tokyo Medical & Dental University. Japan Tokyo, University of, Japan Toronto. University of. Canada Tsukuba Life Science Center, Japan Tubingen, University of. Germany Universidad Nacional Autonoma, Mexico Universidad Nacional de Cuyo-Conicet, Argentina Universidade Federal Fluminense, Brazil Universitat GH Essen. Germany Universitat zu Koln, Germany Universite Paris-Sud. France University College London. United Kingdom University Hospital Nijmegen, The Netherlands University Laboratory of Physiology. United Kingdom University Newcastle upon Tyne. United Kingdom University of Technology. Australia Urbino, University of, Italy Utrecht University. The Netherlands Victoria. University of, Canada Wageningen Agricultural University. The Netherlands Waterloo, University of, Canada Weizmann Institute of Science, Israel Wellcome/CRC Institute. United Kingdom York University, Canada Zeneca Pharmaceuticals, United Kingdom Zoological Society of London, United Kingdom Zurich. University of. Switzerland Year-Round Research Programs Architectural Dynamics in Living Cells Program Established in 1992. this program focuses on architectural dynamics in living cells the timely and coordinated assembly and disassembly of macromolecular structures essential for the proper functioning, division, motility. and differentiation of cells; the spatial and temporal organization of these structures; and their physiological and genetic control. The program is also devoted to the development and application of powerful new imaging and manipulation devices that permit such studies directly in living cells and functional cell- free extracts. The Architectural Dynamics in Living Cells Program promotes interdisciplinary research and consists of resident core investigators and a cadre of adjunct members. Resident Core Investigators Danuser, Gaudenz, Postdoctoral Fellow Inoue. Shinya, Distinguished Scientist Katoh. Kaoru. Postdoctoral Research Associate Oldenbourg. Rudolf, Associate Scientist Staff Geer, Thomas, Research Assistant Knudson, Robert, Instrumental Development Engineer Maccaro, Jackie. Laboratory Assistant MacNeil, Jane, Executive Assistant Visiting Investigators Burgos. Mario H.. Universidad National de Cuyo, Conicet, Mendoza, Argentina Fukui, Yoshio, Northwestern University Medical School Inoue, Theodore D., Universal Imaging Corporation, West Chester, PA Okada. Naobumi. Olympus Corporation. Hachioji, Japan Suzuki, Keisuke. Olympus Corporation, Hachioji, Japan Takahashi, Hajime. Olympus Corporation. Hachioji. Japan Tran. Phong, University of North Carolina, Chapel Hill The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution Major emphasis in The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution is placed upon comparative/ phylogenetic studies of genes and genomes, molecular microbial ecology/biodiversity, evolution of pathogenesis 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 genes and genomes. The Marine Biology Laboratory has considerable strength in Comparative Molecular Biology and Evolution including Mitchell Sogin's studies of genome evolution and diversity of Eukaryotes, Monica Riley's Metabolic Database and evolutionary studies of protein sequences, Neal Cornell's comparative molecular studies of genes critical to heme biosynthesis. Norman Wainwright's studies of the molecular basis of host defense mechanisms in marine invertebrates, and Michael Cumming's work on evolution of pathogenesis in prokaryotes. Other collaborative projects include studies of P450 evolution between M. Sogin and John Stegeman's laboratory at Woods Hole Oceanographic Institution (WHOI). a molecular ecology component of the Long-Term Ecological Research project between M. Sogin's laboratory and J. Hobbie of the Ecosystems Center, and collaborative studies of molecular diversity among marine protists and bacteria with several eukaryotic microbiologists at WHOI. Excellent resources for studies of molecular evolution exist in the form of automated DNA sequencing, well-equipped research laboratories, and powerful computational facilities. The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution plays an active role in educational activities at the MBL. In addition to participating in the Parasitology and Microbial Diversity courses, it sponsors the Workshop in Molecular Evolution at the MBL which has gained an international reputation for excellence. This Workshop serves 60 students by offering a series of lectures and mini-symposia, which are complemented by a state-of-the-art computational facility. The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution includes the laboratories of Neal Cornell, Michael Cummings, Monica Riley, Mitchell Sogin. and Norman Wainwright. Resident Core Investigators Sogin, Mitchell. Director and Senior Scientist Cornell. Neal, Senior Scientist Riley, Monica. Senior Scientist Wainwright. Norman. Senior Scientist 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-aminolevulinate synthase. the first enzyme in the pathway. Because the ability to produce heme from common metabolic materials is a near universal requirement for living organisms, these genes provide useful indicators of molecular R42 Year-Round Research R43 aspects of evolution. For example, 5-aminolevulinate synthase in vertebrate animals and simple eukaryotes such as yeast and Plasmodium falcifianim have high sequence similarity to the enzyme from the alpha-purple subgroup of eubacteria. This supports the suggestion that alpha-purple bacteria are the closes! contemporary relatives of the ancestor of eukaryotic mitochondria. The analysis also raises the possibility that plant and animal mitochondria had ditterent 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 Dunlap. Rachel. Research Associate Faggart, Maura A., Research Assistant Kreiling, Jill, Research Assistant Macarro, Jackie, Laboratory Assistant O'Neil, Brendan, Laboratory Assistant Visiting Scientist Fox, T. O., Harvard Medical School 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 Linntlus pohphemus. Limiilus hemocytes exhibit a very sensitive LPS-triggered protease cascade which results in blood coagulation. Several proteins found in the hemocyte and hemolymph display microbial binding proteins that contribute to antimicrobial defense. Commensal or symbiotic microorganisms may also augment the antimicrobial mechanisms of macroscopic marine species. Secondary metabolites are being isolated from diverse marine microbial strains in an attempt to understand their role. Microbial participation in oxidation of the toxic gas hydrogen sulfide is also being studied. Staff Wainwright. Norman, Senior Scientist Child. Alice. Research Assistant Visiting Investigator Anderson, Porter, University of Rochester Molecular Evolution of Genomes The genome of the bacterium Escherichia coli contains all of the information required for a free-living chemoautotrophic organism to live, adapt, and multiply. The information content of the genome can be dissected from the point of view of understanding the role of each gene and gene product in achieving these ends. The many functions of E. coli have been organized in a hierarchical system representing the complex physiology and structure of the cell. In collaboration with Dr. Peter Karp of SRI International, an electronic encyclopedia of information is being constructed on the genes, enzymes, metabolism, transport 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 . coli in terms of their evolutionary origins. By grouping like sequences and tracing back to their common ancestors, one learns not only about the paths of evolution for all contemporary E. coli proteins, but one extends 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 proved to be valuable to the community of scientists sequencing microbial genomes. E. coli data serve as needed reference points. Staff Riley. Monica. Senior Scientist Pelligrini-Toole, Alida. Research Assistant II Kerr. Alastair, Postdoctoral Research Associate Program in Comparative Molecular Biology and Evolution: Laboratory of Mitchell L. Sogin The Program 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. We are 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. We take 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. We have discovered new evolutionary assemblages that are as genetically diverse and complex R44 Annual Report 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 revealed new habitats and major revelations about geographical distribution of microorganisms. This past year we initiated a new project designed to unlock the secrets of genome evolution in the parasite Giardia lamblia. We selected G. lamblia as a model organism for genome analysis because of its well-recognized impact on human health, its relatively modest- sized genome containing 12 million base pairs distributed onto five chromosomes, and the insights it will provide about the origins of nuclear genome organization. Comparisons of several different gene families demonstrate Giardia 's basal position in molecular phylogenies. which is consistent with the absence of several prominent organelles like mitochondria, peroxisomes. and mitotic spindles that characterize most eukaryotic cells. This genome project will complement an ongoing survey of genomic diversity from eukaryotic microorganisms that do not have mitochondria. We 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 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. Staff Sogin, Mitchell L., Director and Senior Scientist Amaral, Linda. Post Doctoral Research Associate Edgcomb. Virginia, Post Doctoral Research Associate Hmkle. Greg. Post Doctoral Research Associate Morrison. Hilary G., Research Associate Roger, Andrew, Post Doctoral Research Associate Silbennan. Jeff. Post Doctoral Research Associate Visiting Investigators Bahr, Michele, Ecosystems Center Podar, Mircea, WHOI Weil, Jennifer. Joslin Diabetes Center BioCurrents Research Center The Biocurrents Research Center (BRC), one of the NIH National Centers for Research Resources, has for many years been pioneering methods in the study of transmembrane currents and has hosted a variety of research pursuits. The Center provides visiting investigators access to a variety of unique technologies as well as new approaches to experimentation in the biomedical sciences. Since the early 1980s, when this Biomedical Research Technology Program (BRTP) was established at the MBL. a number of probes have been introduced. Four systems are available or being developed at the BRC. All these probe technologies are based on the principles 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 of 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 of ion transport. Most use is made of the ion-selective probes (Seris). 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, which are finding their first successful applications on biological material, are the BioKelvin Probe and the non-invasive Oxygen Probe (Serp). The BioKelvin Probe measures voltages around living tissues in air. Recently, successful measurements were made of fields derived from growing corn seedlings responding to gravity and light. Experiments will apply this new instrument to the study of skin physiology. A radically different approach is being taken to the measurements of biocurrents using the Oxygen Probe. Presently applied to molecular oxygen, such a technique offers opportunity for the study of molecular transport using redox potentials. The prototype has made measurements of the oxygen consumption of a single neuron in culture with a spatial resolution of several /jnr. We are currently developing further improvements to all our systems by incorporating super-resolution algorithms based on machine vision. 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 recently purchased Zeiss Attofluor system, all of which are finding uses in the hosted biomedical studies, as well as BRC research and development. Research by O. Shirihai. an MD/PhD student from Israel, who was at MBL as a Grass Fellow, is an example that brings together many aspects of a BRTP Center, resulting in discoveries that otherwise would not take place. While at the MBL he joined us to use the imaging capability of BRC. During that study he also applied the specialized Seris-electrodes to growing mammalian microglia, demonstrating, for the first time, a new member of the potassium/ proton ATPase family in the central nervous system. The physiological demonstration was supported by immuno-histochemical work with antibodies made available to us through another Center visiting investigator. Dr. D. Brown of Harvard and Massachusetts General Hospital. The antibodies were raised by Adam Smolka in the Carolinas. As the microglia form a principal brain reactive cell, and are implicated in neurodegenerative diseases of the CNS, we believe these discoveries will be of fundamental biomedical importance. 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 grant 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 Associate Scientist Baikie, Iain D., Associate Scientist Danuser, Gaudenz M., Visiting Scientist Hammar. Kathenne, Research Assistant Jaffe. Lionel F.. Senior Scientist McLaughlin, Jane A.. Research Assistant Porterfield, D. Marshall, Research Associate Sanger, Richard H., Senior Electronics Technician Tamse, Catherine T.. Graduate Student. University of Rhode Island Visiting Scientist and Publications This past year the Research Center hosted 25 visitors, 19 from the United States with the remaining from Canada, Israel. United Year-Round Research R45 Hecker, Barbara, Rutgers University Hinkle, Gregory, Marine Biological Laboratory Margulis, Lynn, University of Massachusetts, Amherst Moore, Michael, Woods Hole Oceanographic Institution Silver. Robert, Marine Biological Laboratory Simmons, Bill. Sandia National Laboratory Wainwright, Norman. Marine Biological Laboratory Other Dolan, Mike, Visiting Teaching Assistant Drucker, Sam, Visiting Teaching Assistant Witting, Jan. Visiting Teaching Assistant Graduate Students PhD Students Kingdom and New Zealand. Scientific publications during the year numbered 2 1 . Boston University Marine Program Facult\ Atema, Jelle, Professor of Biology, Director Dionne, Vincent. Professor of Biology, Acting Director Humes, Arthur, Professor of Biology Emeritus Kaufman. Les. Associate Professor of Biology Lobel, Phil. Associate Professor of Biology Valiela. Ivan. Professor of Biology Voigt, Rainer. Research Associate Professor Ward, Nathalie. Lecturer Staff Burns. Jennifer, Course Coordinator DiNunno. Paul. Research Assistant, Dionne Laboratory Hahn, Dorothy. Sr. Administrative Secretary Hall, Sheri. Program Manager Magee, Jennifer, Administrative Secretary Olson, Nancy, Program Assistant/Director's Secretary Proft. Heinz, Research Assistant. Lobel Laboratory Roberts, Brian, Research Technician. Valiela Laboratory Soucy. Lori, Research Assistant. Valiela Laboratory Tomasky. Gabby. Research Assistant. Valiela Laboratory Wheatley. Maryjo. Information and Development Officer Zackrison. Rebecca, Course Coordinator Postdoctoral Investigators Basil. Jenny. Atema Laboratory Cebrian, Just. Valiela Laboratory Cohen. Anne, Lobel Laboratory Delay. Rona. Dionne Laboratory Eisthen, Heather, Dionne Laboratory Grasso, Frank, Atema Laboratory Lavalli, Kari, Atema Laboratory Trott. Thomas, Atema Laboratory Visiting Facultv and Investigators Epstein, Slava. Northeastern Llniversity Hanlon. Roger, Marine Biological Laboratory Existing Batjakas, loannis Behr. Peter Dale, Jonathon Economakis, Alistair Farley, Lynda Hauxwell, Jennifer Herrold, Ruth Lindholm, James Ma, Diana McClelland, James Oliver, Steven Tamse, Armando Zhou, Qiao New Cole, Marci Kroeger. Kevin Piccillo. Bianca Sloan. Kevin Stieve, Erica Thoren. Erika Zhao, Jing Masters Students Existing Ashcraft, Susan Barry. Kevin Bell. Kimberly Demary. Kristian Ewell, Cara Filson. Jean Heiskell. Marybeth Jefferson. Shawn Keith. Lucy Kerr. Lisa Paganessi, Laura Ryan, Pamela Searcy, Brian Thompson, Sarah Timmer, Edward Valentini. Stefanie Watson, Elise Wittenberg, Kim New Atkinson. Abby Baizer, Traci R46 Annual Report Barlas, Margaret Bowen, Jennifer Cavanaugh, Joseph DeCou. Domenique Ferland, Amy Griffin, Martin Homkow, Laura Lawrence, David Smith. Spence Tober. Joanna Wright, Dana Undergraduate Students Spring 97 Balsys, Roman Bentis, Christopher Drucker, Sam Schlimmer, Lisa Singer, Emily Strongin. Daniel Wilkman, Jason Fall 97 Adams, Elizabeth Banik, Amy Bell, Richard Benjamin, Natasha Berasi, Brenda Brines, Zachary Brown, Nicole Camp. Sara Dohogne, Michelle Efros, Michelle Frenz, Christopher Furlong. Chris Harris, Jennifer Houghton, April Hubert, Jessica Jacobs, Jennafer Joyce, Kelly Kam. Adrienne Krawczyk. Jaime Levy, Ilan Lolli, Amanda Miller, Lauren Paradise, Kristen Rodriguez, Jennifer Weaver, Matthew Summer 1997 Interns Duffy, Elisabeth Elkins, Kim Heberlig, Laura Hoeppner, Susanne Javonillo. Robert Kinlan, Brian Lee, Rosalynn Markley. Jessamyn Morlock, Summer Taylor, David Voss, Daniel Whitman, Allison Laboratory of Jelle Atema 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 small untethered robots. Besides measurement and computer modeling of odor plumes and of the water currents lobsters generate to send and receive chemical signals, our research includes neurophysiology of receptor cells and anatomical studies of receptor organs and pheromone glands. Laboratory of Vincent Dionne Odors are powerful stimuli. They can focus the attention, elicit behaviors (or misbehaviors), and even resurrect forgotten memories. These actions are directed by the central nervous system, but they depend upon the initial transduction of chemical signals by olfactory receptor neurons in the nasal passages. More than just a single process appears to underlie odor transduction, and the intracellular pathways that are used are far more diverse than once thought. Hundreds of putative odor receptor molecules have been identified that work through several different second messengers to modulate the activity of various types of membrane ion channels. Our studies are being conducted with aquatic salamanders using amino acids and other soluble chemical stimuli which these animals perceive as odors. Using electrophysiological and molecular approaches, the research examines how these cellular components produce odor detection, and how odors are identified and discriminated. 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 hydrothermal vents and cold seeps. The Laboratory of Les Kaufman Current research projects in the laboratory deal with speciation and extinction dynamics of haplochromine fishes in Lake Victoria. We are studying the systematics. evolution, and conservation genetics of a species flock encompassing approximately 700 very recently evolved taxa. in the dynamic and heavily impacted landscape of northern East Africa. In the lab, we are studying evolutionary morphology, behavior, and systematics of these small, brightly colored cichlid fishes. Another area of study is developmental and skeletal plasticity in fishes; we are studying the diversity of bone tissue types in fishes, differential response to mineral and mechanical challenge, and matrophic versus environmental effects in the development of coral reef fishes. We also study the biological basis for marine reserves in Year-Round Research R47 the New England fisheries. We are involved in collaborative research with NURC. NMFS. and others on the relative impact on groundrish stocks of juvenile habitat destruction versus fishing pressure. Laborator\ of Phillip Label Fishes are the most diverse vertebrate group and provide opportunities to study many aspects of behavior, ecology, and evolution. We primarily study how fish are adapted to different habitats and the behavioral ecology of species interactions. Current research focuses on fish acoustic communication. We are also conducting a long-term study of the marine biology of Johnston Atoll, Central Pacific Ocean. Johnston Atoll has been occupied continuously by the military since the 1930s and proved a unique opportunity for assessing the biological impacts of island industrialization and effects on reefs. Johnston Atoll is the site of the US Army's chemical weapons demilitarization facility, JACADS. Laboratory of Ivan Valiela A focus of our work is the link between land use on watersheds and consequences in the receiving estuarine ecosystems. The work examines how landscape use and urbanization increase nutrient loading to groundwater and streams. Nutrients in groundwater are transported to the sea, and, after biogeochemical transformation, enter coastal waters. There, increased nutrients bring about a series of changes on the ecological components. To understand the coupling of land use and consequences to receiving waters, we study the processes involved, assess ecological consequences, and define opportunities for coastal management. A second long-term research topic is the structure and function of salt marsh ecosystems, including the processes of predution, herbivory, decomposition, and nutrient cycles. Calcium Imaging Laboratory This laboratory investigates the roles of calcium patterns in development. Our main tool uses the aequorins, a family of luminescent proteins ultimately obtained from a jellyfish and long studied by Dr. Osamu Shimomura at the MBL. Aequorins can either be microinjected into cells or transgenically expressed without disturbing function or development. The patterns of luminescence that are emitted by aequorinated cells reveals changing patterns and levels of free calcium with the cell or its progeny. Much of what we know about the role of calcium in development has been obtained with the aequorins. The four systems under present or planned investigation are the Drosophila egg (in collaboration with Carl Hashimoto at Yale), the zehrafish egg, the fucoid egg (in collaboration with Dr. Ken Robinson at Purdue), and the cellular slime mold. Dictyostelium. Staff Jaffe, Lionel, Senior Scientist Creton, Robert, Research Associate Center for Advanced Studies in the Space Life Sciences at the MBL (supported by the National Aeronautics anil Space Administration) The Marine Biological Laboratory and the National Aeronautics and Space Administration have established a cooperative arrangement with the formation of the Center for Advanced Studies in the Space Life Sciences at the MBL. This Center serves as an interface between NASA and the basic science community, addressing issues of mutual interest. A series of symposia, workshops, and seminars are held at the MBL to advise NASA on a wide variety of topics in the life sciences, including cellular, molecular, developmental, plant, neuro-, and evolutionary biology. Special attention is directed at examining how gravity and its control impact on biological processes, and how variations in gravity can be used as a probe to better understand such processes. This center provides a forum for scientists to think and discuss, often for the first time, the role that gravity and other aspects of space flight may play in fundamental cellular and physiological processes. These interactions also serve to inform the community of research opportunities in the life sciences that are of interest to NASA. In addition, a newsletter will be published to disseminate this information to a wider audience. During the past year the Center sponsored two workshops at the MBL: "Genetic Regulatory Networks in Embryogenesis and Evolution" held on June 11-14. 1997, chaired By Eric Davidson and David McClay; and "Evolution: A Molecular Point of View" held on October 24-26, 1997. chaired by Mitchell Sogin. Staff Dawidowicz, Lenny, Administrator Amit, Udeni, Administrative Assistant Nixon, Jennifer, 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 wanning 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 of 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 I5 N to investigate natural processes. A mass spectrometer facility is available. Data from field and laboratory research are used to construct mathematical models of whole-system responses to change. Some of these models are combined with geographically referenced data to produce estimates of how environmental changes affect key ecosystem indexes such as net primary productivity and carbon storage throughout the world's terrestrial biosphere. K4S Annual Report 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, a fall offering, was held for the first time in 1997. Sixteen students from 11 colleges participated in the program. There are opportunities for postdoctoral fellows. Administrative Staff' Hobbie, John E., Co-Director Melillo, Jerry M, Co-Director Berthel, Dorothy J., Administrative Assistant Chandler, Marsha, Administrative Assistant, Semester in Environmental Science Donovan, Suzanne J., Executive Assistant Foreman, Kenneth H., Associate Director of Environmental Studies Program 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 Peterson, Bruce J., Senior Scientist Shaver, Gaius R., Senior Scientist Giblin, Anne E., Associate Scientist Hopkinson. Charles S., Senior Scientist Nadelhoffer, Knute J., Associate Scientist Deegan, Linda A., Associate Scientist Rastetter, Edward B., Associate Scientist Steadier. Paul A., Senior Research Specialist Neill, Christopher, Assistant Scientist Pan, Yude, Research Associate Vallino, Joseph J., Assistant Scientist Williams, Mathew, Assistant Scientist Xiao. Xiangming, Research Associate Educational Staff Appointments Currie, William, Visiting Postdoctoral Scholar, U.S. Department of Agriculture Garcia, Diana, Postdoctoral Research Associate Gough, Laura, Postdoctoral Research Associate Hartley. Anne, Postdoctoral Research Associate Herbert, Darrell A.. Postdoctoral Research Associate Holmes, Robert M., Postdoctoral Research Associate Hughes. Jeffrey E.. Postdoctoral Research Associate Jablonski. Leanne, Postdoctoral Research Associate Stieglitz, Marc, NOAA Global Climate Change Postdoctoral Fellow Tian, Hanqin, Postdoctoral Research Associate Technical Staff Bahr. Michele P., Research Assistant Bettez, Neil D., Research Assistant Bryant, David M., Research Assistant Canary. Jana D.. Research Assistant Catricala, Christina E., Research Assistant Clark, Tamara, Research Assistant Claessens, Luc, Research Assistant Dornblaser. Mark M.. Research Assistant Downs. Martha R.. Research Assistant Garritt, Robert H., Senior Research Assistant Helfrich, John V. K.. III. Senior Research Assistant Holland, Keri, Research Assistant Hooker, Bethanie, Research Assistant Kicklighter, David W.. Senior Research Assistant Kwiatkowski, Bonnie L., Research Assistant Laundre. James A., Senior Research Assistant Micks. Patricia. Research Assistant Newkirk, Kathleen M., Research Assistant Nolin. Amy L., Research Assistant Pratt. Sara, Research Assistant Regan, Kathleen M., Research Assistant Ricca. Andrea. Research Assistant Schwamb, Carol. Laboratory Assistant Slavik. Karie A.. Research Assistant Soucy, Lori, Research Assistant Thieler, Kama. Research Assistant Thomas, Suzanne, Research Assistant Tholke, Kristin S., Research Assistant Tucker, Jane, Senior Research Assistant Weston, Nat, Research Assistant Wollheim, Wilfred M., Research Assistant Consultants Bowles, Frances P., Research Systems Consultant Principal, Research Designs Bowles, Margaret C., Administrative Consultant Golden, Heidi E., Research Consultant Visiting Scientists and Scholars Kling, George, Visiting Scientist. University of Michigan, Ann Arbor Loya, Wendy, Visiting Scholar, Kansas State University Seifert. Gabriel. CIEE Intern, Technical University of Wismar, Germany Siver, Peter, Semester in Environmental Science Faculty Fellow, Connecticut College Laboratory of Aquatic Animal Medicine and Pathology The laboratory provides diagnostic, consultative research, and educational services to the institutions and scientists of the Woods Hole community concerned with marine animal health. Diseases of wild, captive, and cultured animals are investigated. Staff Abt, Donald A.. Director and The Robert R. Marshak Term Professor of Aquatic Animal Medicine and Pathology, School of Veterinary Medicine. University of Pennsylvania Bullis, Robert A., Research Associate in Microbiology. University of Pennsylvania Leibovitz. Louis, Director Emeritus McCafferty, Michelle. Histology Technician, University of Pennsylvania Moniz, Priscilla C., Administrative Assistant Smolowitz. Roxanna M., Research Associate in Pathology, University of Pennsylvania Wadman, Elizabeth A., Microbiology Technician. University of Pennsylvania Year-Round Research R49 Laboratory of Aquatic Biomedicine Work in this laboratory centers on comparative irnmunopathology and molecular biology using marine invertebrates as experimental models. Examples of current research include determining the prevalence of leukemia in Mya 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. Developmental and chemically-induced changes in gene expression and neuronal growth are also being studied in the surf clam. Spisu/a sulidissima. 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., Associate Scientist, MBL. and Chairperson, Department of Environmental and Population Health, Tufts University School of Veterinary Medicine Jessen-Eller, Kathryn. Postdoctoral Fellow Steele, Marjorie, Research Assistant Barker, Colin, Laboratory Technician Visiting Scientist Barker, Lewellys, Senior Associate Department of International Health, Johns Hopkins University School of Public Health Student Smith, Cynthia, 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. Electrophysiological-, fluorescent- tracer-, and molecular biological techniques are used to this end. As was recently discovered in this laboratory, 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. Work is aimed now at the mechanisms of growth control and at correcting cancer growth by transferring the gene for the cell-to-cell channel protein from normal cells into the cancer cells. Molecular genetic techniques are used in this endeavor. Staff Loewenstein, Werner, Senior Scientist Rose, Birgit, Senior Scientist Jillson, Tracy, Research Assistant Laboratory of Barbara 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 Conns and the structural role of y-carboxyglutamic acid in the conopeptides. This satellite laboratory relates closely to the main laboratory on the Harvard Medical School campus in Boston; the main focus of the primary laboratory is the synthesis and function ot y-carboxyglutamic acid in blood clotting proteins and the role of vitamin K. Large numbers of cone snails from Fiji have been obtained and are being maintained in the Marine Resources Center. The marine cone snail is 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 examine the biosynthetic pathway for Gla, we have studied the Conus carboxylase which converts glutamic acid to y-carboxyglutamic acid. Of the Conus species tested, C. bandamis, C. mamwreus. C. textile, and C. leopardus had high specific y-carboxylase activity. This activity has an absolute requirement for vitamin K. The Conns carboxylase has been extensively purified and its gene is being cloned. The Conus carboxylase substrates appear to contain a carboxylation recognition site on the conotoxin precursor. The Conus vitamin K-dependent carboxylase should be an excellent model for determining the mechanism of action of vitamin K in the synthesis of y-carboxyglutamic acid. Fifteen novel y-carboxyglutamic acid-containing conopeptides have been isolated from the venom of Conus textile. The amino acid sequence, amino acid composition and molecular weights of these peptides have been determined. For several peptides, the cDNA encoding the precursor conotoxin has been cloned. The three-dimensional structure of some of these Gla-containing conopeptides as well as conantokin G have been determined by 2D NMR spectroscopy. Complete resonance assignments were made from 2D 'H NMR spectra via identification of intraresidue spin systems using 'H-'H through-bond connectivities. NOESY spectra provided d aN , d NN and d, iN NOE connectivities and vicinal spin-spin coupling constants 'J H N,, 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. Staff Barbara C. Furie, Scientist Bruce Furie, Scientist Johan Stenflo, Visiting Scientist Eva Czerwiec, Postdoctoral Fellow Gail Begley. Postdoctoral Fellow Alan Rigby. Postdoctoral Fellow R50 Annual Report Laboratory of Roger Hanlon This laboratory investigates the behavior and neurobiology of cephalopods. Studies of various learning capabilities are currently being 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 function of polarization vision in cephalopods. Complimentary 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. and statoliths). as well as immunocytochemical labelling of cell- surface antigens, neurosecretory products, second messenger proteins involved with learning and memory, and intracellular transport organelles using mono- and polyclonal antibodies on squid (Loligo peulei) giant axons and Hennissenda sensory and neurosecretory neurons, both in situ and in cell culture. Toxicity studies detailing the effects of lead on Hennissenda learning and memory, feeding, and the physiology of cultured neurons are also being conducted. Additional collaborative research includes DNA fingerprinting using RAPD-PCR techniques in preparation for isogenic strain development of laboratory-reared Hennissenda and hatchery produced bay scallops (Argopectin irradians) with distinct phenotypic markers tor the rapid field identification. Systematic and taxonomic studies of nudibranch molluscs, to include molecular phylogenetics, are also of interest. Staff Hanlon, Roger, Senior Scientist Boal. Jean, Postdoctoral Fellow Shashar. Nadav. Postdoctoral Fellow Visiting Investigators Gabr, Howaida. Graduate Student, Suez Canal University, Panama Wittenberg, Kim, Graduate Student. Boston University Marine Program Laboratory of Shinya Inoue Scientists in this laboratory study the molecular mechanism and control of mitosis, cell division, ceil 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 polarization optical and video microscopy and digital image processing techniques, and exploration of their underlying theory are an integral part of the laboratory's effort. Staff Inoue. Shinya. Distinguished Scientist Knudson. Robert, Instrument Development Engineer Maccaro. Jackie, Laboratory Assistant MacNeil, Jane, Executive Assistant Laboratory of Alan M. Kuzirian Research in the laboratory explores the functional morphology and ultrastructure of various organ systems in molluscs. The program includes mariculture of the nudibranch. Hermissemhi crassiciirnis. with emphasis on developing reliable culture methods for rearing and maintaining the animal as a research resource. The process of metamorphic induction by natural and artificial inducers is being explored in an effort to understand the processes involved and as a means to increase the yield of cultured animals. Morphologic studies stress the ontogeny of neural and sensory structures associated with the photic and vestibular systems which have been used in learning and memory studies as well as the spatial and temporal occurrence of regulatory and transmitter neurochemicals. Concurrent with these morphologic studies is the development of new histologic techniques designed to facilitate the acquisition of morphologic and structural information supporting proposed physiologic processes. Collaborative research includes histochemical investigations on strontium's role in initiating calcification in molluscan embryos (shell Staff Ku/irian, Alan M., Associate Scientist Visiting Scientists Chikarmane. Hemant, Research Scientist. Aphios Corporation, Woburn MA: Assistant Scientist. MBL Clay. John R., NINDS/NIH 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 non- invasively and non-destructively cell architecture dynamically and at high resolution, we have developed a new polarized light microscope (Pol-Scope). The Pol-Scope combines microscope optics with new electro-optical components, video, and digital image processing for fast analysis of specimen birefringence over the entire viewing field. Examples of biological systems currently investigated with the Pol- Scope are: microtubule-based structures (asters, mitotic spindles, single microtubules); actin-based structures (acrosomal process, stress fibers, nerve growth cones); zona pellucida of vertebrate oocytes; and biopolymer liquid crystals. Staff Oldenbourg. Rudolf. Associate Scientist Katoh, Kaoru. Postdoctoral Research Associate Geer. Thomas. Research Assistant Knudson, Robert, Instrument Development Engineer Maccaro, Jackie, Laboratory Assistant Laboratory for Reproductive Medicine, Brown University and Woman and Infants Hospital, Providence Work in this laboratory centers on the investigation of the underlying mechanisms behind female infertility. Particular emphasis is placed on the physiology of the oocyte or 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 new Pol-Scope to analyze the Year-Round Research R51 birefringence of the preimplantation mammalian zona pellucida a structure most predictive of successful implantation. We also have used this instrument to examine meiotic spindles. An additional aim is to continue the studies on transmembrane ion transport using the non-invasive electro-physiological techniques available at the BioCurrents Research Center. Preliminary studies indicate that the calcium transport may form an accurate predictor of oocyte and embryo health. The newly developed oxygen probe also offers the possibility of looking directly at abnormalities in the mitochondria arising from accumulated mtDNA damage. Ultimately, in addition to investigating the mechanisms behind cellular aging underlying infertility, this laboratory aims to produce clinical methods for assessing preimplantation embryo viability, a development that will make a significant contribution to the health of women and children. Staff Keefe. David. Director Liu, Lin. Research Associate Pepperell, John. Visiting Investigator Sokhal. Emily. Research Assistant Trimarchi. James, Post-doc Fellow Laboratory of Sensory Physiology Members of this laboratory have conducted research on various facets of vision since 1973. Current investigations use UV/VIS light microspectrophotometry on vertebrate retinal photoreceptors for the determination of visual pigment absorbance characteristics. One aim is to arrive at a better understanding of the method of spectral tuning that forms the chemical basis of color vision. Polarized light microscopic techniques are used to measure linear dichroism and linear birefringence aimed at revealing structure-function relationships and biophysical mechanisms. An area of interest is polarization discrimination, the mechanisms that could account for the ability of some fish species to detect the direction of polarization of light collected by their eyes. As a recent development, investigations are carried out on sickling in fish red blood cells due to hemoglobin polymerization, once again making extensive use of polarized light microscopic techniques. Staff Harosi, Ferenc I., Senior Scientist, MBL, and Boston University School of Medicine Novales Flamarique. I., Postdoctoral Fellow Visiting Scientists Van Keuren, Jeffrey R-, Postdoctoral Fellow, Woods Hole Oceanographic Institution Hunt von Herbing. I.. Assistant Professor. University of Maine Laboratory of Osamu Shimomura Biochemical mechanisms involved in the bioluminescence of various luminescent organisms are investigated. Based on the results obtained, various improved forms of bioluminescent and chemiluminescent 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, telemampulation of living cells and tissues, and modeling of decision processes. A cornerstone of the laboratory's analytical efforts is high performance computational processing and analysis of video light microscopy images and modeling. With luminescent, fluorescent, and absorptive probes, both empirical observation and computational modeling of cellular, biochemical, and biophysical processes permit interpretation and mapping of space-time patterns of intracellular chemical reactions and calcium signaling in living cells. A variety of in vitro biochemical, biophysical, and immunological methods are used. In addition to fundamental biological studies, the staff designs and fabricates optical hardware, and designs software for large video image data processing, analysis, and modeling. Staff Silver, Robert. Associate Scientist Luders, Bruce, Research Assistant Interns Hurwitz. Layne R., REU Intern, Adelphi University Sheikh, Sarah I., REU Intern, University of Edinburgh & Oxford University Strongm. Daniel E.. REU Intern, Boston University R52 Annual Report Visiting Scientistx Alt, Maxim. MikroPhotonische Universtitiit von CZI, Germany Reeves. Anthony. Cornell University Searby. Nancy. NASA Ames Research Center Stromboli. Emilio, Stazione de Napoli. Italy The Marine Resources Center The Marine Resources Center (MRC) is one of the world's most advanced facilities for maintaining and culturing aquatic organisms essential to advanced biological, biomedical, and ecological research. Service and education also play an important and complimentary role in the modem. 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. These programs will enhance and build upon the MRC's existing research activities by the University of Pennsylvania's Laboratory of Aquatic Animal Medicine and Pathology (LAAMP) and in the Laboratory of Roger Hanlon. 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, the Administrative Division, and the Laboratory for Aquatic Animal Medicine and Pathology. 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. The MRC also hosts several courses: the annual AQUAVETS courses sponsored by LAAMP. and an aquaculture course, the theme of which changes according to regional and national interests. Staff Hanlon. Roger. Director and Senior Scientist Kuzirian, Alan, Associate Scientist Boal, Jean. Postdoctoral Fellow Shashar, Nadav, Postdoctoral Fellow Visiting Investigators Baker. Robert, New York University Gilland, Edwin, Postdoctoral Fellow Adamo, Shelly, Dalhousie University, Canada Spotte, Stephen, University of Connecticut Wittenberg, Kim, Graduate Student. Boston University Marine Program Gabr, Howaida, Graduate Student, Suez Canal University, Panama Honors Friday Evening Lectures June 20 June 27 July 4 July 11 July 18 July 24, 25 August 1 August 8 August 15 Roger Y. Tsien. Howard Hughes Medical Institute, University of California, San Diego "Molecular Spies Reveal Cell Signals in Living Color" Charles F. Stevens, The Salk Institute for Biological Studies "Synapses and Memory" (Lang Lecture) Marianne Bronner-Fraser, California Institute of Technology "Development of the Neural Crest" Clara Franzini-Armstrong, University of Pennsylvania "How Muscle Contraction is Turned On" Jerry M. Melillo, Marine Biological Laboratory "Ecological Research and Global Environmental Policy: New Challenges for an Essential Partnership" Semir Zeki, University College London "The Functions of the Visual Brain" and "The Autonomy of the Visual Areas" (Forbes Lectures) Eric Wieschaus. Princeton University "Zygotic Transcription and the Control of Embryonic Development in Drosophila" (Glassman Lecture) Daniel Banry, NASA/Johnson Space Center "Space Flight from a Physician-Scientist's Perspective" Rudolf A. Raff, Indiana University "Development, Genes, and the Evolution of Animal Body Plans" Fellowships and Scholarships Robert Day Allen Fellowship Drs. Joseph W. and Jean M. Sanger ASCB Summer Research Award American Society for Cell Biology Frederik B. Bang Fellowship Mrs. Betsy G. Bang Frank A. Brown, Jr. Memorial Readership Dr. and Mrs. Francis D. Carlson The Jean and Katsuma Dan Fellowship Fund Dr. Howard Holtzer Dr. and Mrs. Shinya Inoue Drs. Joseph W. and Jean M. Sanger Mrs. H. Burr Stembach Bernard Davis Fund Mrs. Elizabeth M. Davis Aline D. Gross Scholarship Fund Mrs. Mona Gross Drs. Joan and Gerald Ruderman Mr. and Mrs. Alfred M. Weisberg Keffer Hartline Fellowship Fund Dr. Edward F. MacNichol, Jr. William Randolph Hearst Educational Endowment William R. Hearst Foundation Fred Karush Endowed Library Readership Dr. and Mrs. Laszlo Lorand Dr. and Mrs. Arthur M. Silverstein MBL Summer Fellowships Dr. and Mrs. Shinya Inoue R53 R54 Annual Report Charles Baker Metz and Charles William Metz Scholarship Fund Mr. Ronald H. Abel Mrs. Grace S. Metz Mountain Memorial Fund Ms. Brenda J. Bodian Dr. and Mrs. Benjamin Kaminer Dr. and Mrs. R. Walter Schlesinger James A. and Faith Miller Fellowship Fund Mr. and Mrs. David A. Miller Mr. Robert K. Schlessinger Frank Morrell Fund Dr. Jack C. Berger Ms. Elena N. Cohen Ms. Kathleen A. Dunn Dr. Leyla de Toledo Morrell Dr. Serge J.C. Pierre-Louis Rush-Presbyterian-St. Luke's Dr. Susan Stefoski Nikon Fellowship Nikon, Inc. Pfizer Scholarship Fund Pti/.er, Inc. William Townsend Porter Scholarship William Townsend Porter Foundation Ruth Sager Scholarship Fund Dr. and Mrs. Harlyn O. Halvorson Dr. and Mrs. Las/.lo Lorand Dr. Arthur B. Pardee Dr. and Mrs. David Shepro The Ann Osterhout Edison/ Theodore Miller Edison and Olga Osterhout Sears/Harold Bright Sears Endowed Scholarship Fund Ms. Elizabeth F. Brewster Mr. and Mrs. John W. Child Ms. Jane T. Claffey Mr. and Mrs. Putnam P. Flint Dr. and Mrs. Paul L. Goodrich Mr. and Mrs. Allan C. Henry Mr. and Mrs. Robert L. Loud Ms. Elaine M. Medeiros Mr. Maren Miles and Ms. Nancy Douglas Ms. Nancy L. Olsen Dr. and Mrs. Miles H. Robinson Mr. and Mrs. Christopher A. Sims Mr. and Mrs. Peter E. Sloane Dr. and Mrs. Thomas R. Stetson Ms. Marsden Williams Science Writing Fellowships Program American Society for Biochemistry & Molecular Biology American Society for Neurochemistry, Inc. American Society for Photobiology Association for Research in Vision and Ophthalmology Biophysical Society Charles A. Dana Foundation Federation of American Society for Experimental Biology Foundation for Microbiology Friendship Fund New York Times Foundation Nicholas B. Ottaway Foundation Society for Integrative and Comparative Biology The Washington Post Company The Moshe Shilo Memorial Scholarship Fund Dr. and Mrs. John J. Lee Dr. and Mrs. Richard I. Mateles The Evelyn and Melvin Spiegel Fellowship Fund Drs. Joseph W. and Jean M. Sanger Drs. Melvin and Evelyn Spiegel and the Sprague Foundation H. B. Steinbach Fellowship Mrs. H. Burr Steinbach Horace W. Stunkard Fellowship Mrs. Eunice Latham Dr. Albert J. Stunkard and Dr. Margaret Maunn The Walter L. Wilson Endowed Scholarship Fund Mrs. Irmgard Alexander (deceased) Dr. Paul N. Chervin Mrs. Marian Rigaumont Dr. Jean R. Wilson Young Scholars/Fellows Program Merck Research Laboratories Fellowships Awarded MBL Summer Research Fellows Elena Barbieri, M.Sc.. the Bernard Davis Fellow, is a researcher in the Bay Paul Center for Comparative Molecular Biology and Evolution at the MBL. She is also a fellow at (he University of Urbino, Urbino Italy. She worked with Dr. Norman Wainwright on the role of symbiotic bacteria in the accessory nidamental gland (ANG) of the squid, Loligo pealei. She measured secondary metabolites derived from these symbiotic bacteria. These metabolites may have antimicrobial activity in the capsule- like membranes of the squid eggs. This study may lead to the development of new antibiotics or potential treatments in clinical chemotherapy. Barbara C. Boyer, Ph.D., was an Erik B Fries and an MBL Associates Fellow. She is a professor in the Department of Biology at Union College, Schenectady, New York. Dr. Boyer studied the evolution of development using the turbellarian flatworms Hiiphiphina. Stvlochus and Neochildia. Mario H. Burgos. M.D.. was supported by the Frank R. Li/lie Fellowship while working with Dr. Shinya Inoue during the summer of 1497. Dr. Burgos is Founder and Director of the Institute of Histology and Embryology in Mendoza, Argentina. He used video Honors R55 microscopy and confocal fluorescence microscopy in his research on gossypol, a male anti-fertility agent produced from cottonseed. Dr. Burgos used the male gamete of the sea urchin as a model system in which to test the effects of the agent. Research on gossypol may eventually result in a birth control drug for men. Edwin DeMont, Ph.D., is an associate professor in the Biology Department at St. Francis Xavier University, Antigonish, Nova Scotia, Canada. Dr. DeMont was supported by the Esther A. and Joseph Klingenstein Fund during the summer of 1997 while he studied the biomechanics of jet-propelled swimming in the squid Loligo pectlei. Leah Devlin, Ph.D., was supported by the NASA Life Sciences Program Fellowship, the M.G.F. Fuortes Fellowship, and the Lucy B. Lemann Fellowship. She is an assistant professor of Biology at Penn State University in Abington, Pennsylvania. Dr. Devlin used a vibrating calcium-selective electrode to explore the movement of calcium in cardiac and smooth muscle. Her primary research organisms were the channeled whelk. Busycon canaliculatum. and the sea cucumber, Sclerodactyla briareus. Dr. Devlin's research is significant in assessing the effects of anti- arrhythmic and anti-hypertensive drugs, such as calcium blockers, on calcium channel activity in cardiac and smooth muscle. Gregg G. Gunderson, Ph.D., is an associate professor in the Department of Cell Biology and Anatomy at Columbia University in New York, New York. As the 1997 Nikon Fellow, Dr. Gunderson studied the dynamics and assembly of adhesion complexes during cell migration. Dr. Gunderson's research on the basic mechanism of cell motility may eventually aid clinicians' abilities to combat cancer and infectious diseases. Gwendolyn B. Howze, Ph.D., is an associate professor in the Biology Department of Texas Southern University. Dr. Howze's research at the MBL last summer was supported by the William Townsend Porter Fellowship for Minority investigators, as well as an MBL Associates Fellowship. Dr. Howze did on-line research of DNA sequence databases. She characterized proteins that play a role in the condensation of chromatin. Chromatin is the mass of material that condenses into individual distinguishable chromosomes just before a cell nucleus divides. Dr. Howze's research may lead to a better understanding of certain pathologies that are due to abnormal gene expression. Samantha B. Joye, Ph.D., is an assistant professor in the Department of Oceanography at Texas A&M University. She was supported by an MBL Associates Fellowship and worked with Dr. Ivan Valiela during the summer of 1997. Dr. Joye studied the reduction of nitrogen loading to estuaries by denitritication in fringing salt marshes. Kamran Khodakhah, Ph.D., is a post-doctoral researcher in the Department of Physiology at the University of Pennsylvania School of Medicine. His research last summer was supported by the H.B. Steinbach Fellowship, the Frederik B. Bang Fellowship Fund, and an MBL Associates Fellowship. Dr. Khodakhah used the American eel and catfish to study the regulation of liver metabolism. His research may have applications in the treatment of diabetes. J.H.E.M. (Anka) Klerkx, M.Sc., is a graduate student in the Department of Experimental Zoology at Utrecht University, Utrecht, The Netherlands. Her research last summer was sponsored by the Evelyn and Melvin Speigel Fellowship Fund, the James A. and Faith Miller Memorial Fund, and the Charles R. Crane Fellowship. She studied the mechanisms that evolved to control development in the spiralians, Chaetopterus and Nereis. Donald L. Lovett, Ph.D., is an associate professor in the Department of Biology at The College of New Jersey in Trenton, New Jersey. His research was supported by the John O. Crane Fellowship Fund, the Esther A. and Joseph Klingenstein Fund, and an MBL Associates Fellowship. Dr. Lovett studied the effects of methyl farnesoate on the cellular mechanisms of the green crab, Carcinus maenas. This research has potential economic applications in crustacean aquaculture and biomedical applications in the use of crustacean chitin, which is being examined tor biomedical uses. Mark Q. Martindale. Ph.D., was supported by the Erik B. Fries Endowed Fellowship and an MBL Associates Fellowship. He is an assistant professor of Organismal Biology and Anatomy at the University of Chicago, Illinois. Dr. Martindale continued his research on the evolution of embryonic development. He studied the ctenophore, Mnemiopsis leidi, and the flatworms, Hoploplana, Neochilidia, and Stylochus. His research could lead to a better understanding of birth defects. Allen Mensmger. Ph.D., is a research instructor in the Department of Otolaryngology at the Washington University School of Medicine, St. Louis, Missouri. His research was supported by a NASA Life Sciences Program Fellowship and the Esther A. and Joseph Klingenstein Fund. To understand nerve regeneration more fully. Dr. Mensinger studied the physiology of the toadfish, Opsanus tau. Charles G. Minkoff, B.A., is a graduate student in the Department of Molecular Cancer Biology at Duke University Medical Center in Durham, North Carolina. His work last summer was supported by the Frank A. Brown, Jr. Memorial Readership. Mr. Minkoff studied the effects of cholesterol-lowering drugs on the cell cycle in the surf clam, Spisula so/idissima. Takehito Saito, Ph.D., is a professor in the Institute of Biological Sciences at the University of Tsukuba, Tsukuba, Japan. The Herbert W. Rand Fellowship supported Dr. Saito' s research on the circadian rhythm of the horseshoe crab (Limulus po/yphemus) eye. Dr. Saito hopes to gain a better understanding of how circadian clocks influence vision. Alastair Simpson, B.S., is a graduate student in the Protist Research Laboratory at the University of Sydney, Sydney, Australia. His work last summer on the evolutionary relationships among protozoa was supported by the Bernard Davis Fund. By illustrating the relationships among structurally primitive protists, Mr. Simpson hopes to gain insight into the evolution of cells and a better understanding of microbial biodiversity in sediments. Peter A. Siver, Ph.D., is a professor in the Department of Botany and Chair of the Environmental Studies Program at Connecticut College. His work was supported by the Lucy B. Lemann Fellowship. Dr. Siver studied the remains of golden algae (Chrysoph\'tes) archived in lake sediments to understand more fully the effects of environmental stresses on aquatic resources. Timothy P. Spann, Ph.D., is a research associate in the Department of Cell and Molecular Biology at Northwestern University Medical School, Chicago, Illinois. He worked with Dr. Robert Goldman as a NASA Life Sciences Program Fellow and a Frederik B. Bang Fellow. Dr. Spann studied the organization of the surf clam (Spisula) nucleus as a model system to understand more fully the relationship between nuclear architecture and nuclear function. His research could lead to new methods of regulating cell growth and gene expression. Walter Steffen, Ph.D., is an assistant professor at the Institute of Biochemistry and Molecular Cell Biology in Vienna, Austria. He was a Herbert W. Rand Fellow and an MBL Associates Fellow. Dr. Steffen's research focused on the transport of membranous organelles by motor proteins in the frog, Xenopus laevi. Megumi Takahashi, M.D., Ph.D., is a psychiatrist at the Kanagawa Psychiatric Center in Yokohama, Japan. His research was supported by the Stephen W. Kuffler Fellowship and the Ann E. Kammer Memorial Fellowship Fund. Dr. Takahashi used the squid as a model for understanding the basic mechanisms of neurodegenerative diseases. R56 Annual Report Phong T. Tran. B.A.. the Ruben Duy Allen Fellow and a Lucy B. Leinann Fellow, is a graduate student in the Department of Biology at the University of North Carolina. Chapel Hill. Last summer Mr. Tran continued his research using the digital polarization microscope to obtain images of microtubules within the spindles during mitosis and kinetochore fibers during chromosome movement. He used lung cells from the newt, Taricha t>ranulosa. Matt Wachowiak. Ph.D.. is a research associate in the Department of Molecular and Cell Biology at the University of California, Berkeley, California. His research on how the brain processes odors was supported by the Esther A. and Joseph Klingenstein Fund as well as an MBL Research Fellowship. Dr. Wachowiak studied how neurons in the brain of the Florida spiny lobster (Panulirus argus) process, code, and identify odors. E. Jennifer Weil, M.D., is a research fellow at the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution at the Marine Biological Laboratory. Dr. Weil's research was supported by a NASA Life Sciences Program Fellowship. She investigated the evolution of the systems that control blood pressure and contribute to heart disease and other pathological states. She used the killifish. Fundulus hetemc/inis. as a model. Her research could lead to a better understanding of diseases like congestive heart failure, nephrotic syndrome, and cirrhosis of the liver. Grass Fellows Janet L. Casagrand. Ph.D., University of Colorado. Project: Pressure-sensitive auditory input to the mauthner cells in the goldfish: origin, response properties, and connectivity. John Gray. Ph.D., Queen's University, Canada. Project: Neural circuitry underlying a novel motor pattern expressed during metamorphosis of the hawkmoth Mandiica sexta. Matthew Halstead, University of Auckland, New Zealand. Project: Electrophysiology of the electrosensory midbrain of the little skate Raja erinacea to biologically realistic stimuli. John Layne, Duke University Marine Laboratory. Project: Coordination of optokinesis and locomotion during course control in the fiddler crab, Vca piigilator. Mark Levandowski. Ph.D., Brown University. Project: Chimeric analysis of a-bungarotoxin binding sequences in nicotimc acetylcholine receptors. Quoc Thang Nguyen, Ph.D.. University of California. Irvine. Project: Neurotransmitter synthesis in mRNA-injected Xenopus oocytes. Eleanora Palma. Ph.D.. Regina Elina Cancer Research Institute. Italy. Project: Functional expression of neuronal nAChRs subunits in the lower vertebrate using Xenopus oocytes. Nadav Shashar. Ph.D., University of Maryland, Baltimore County. Project: Polarization sensitivity in cephalopods. Hiroshi Tokumaru, Ph.D.. Duke University Medical Center. Project: The role of synapin/complexin in transmitter release at the squid giant synapse. Martina Wicklein, Ph.D.. University of Arizona. Project: Motion sensitive neurons in the visual system of the tiddler crab. MBL Science Writing Fellowships Program Fellows Marc Airhart. Assistant Producer, Earth & Sky Radio Series Rita Baron-Faust. Producer. "Report on Medicine" Steven Benowitz, Senior Editor, The Scientist Lynne Cherry, Children's Book Author Carol Ezzell, Science Editor. Journal of NIH Research (Ms.) Ronny G. Frishman. Managing Editor. INQUIRY John Fleischman. Freelance Marguerite Holloway. Contributing Editor, Scientific American Meredith Small. Freelance Terra Ziporyn. Freelance Program Directors Robert D. Goldman. Northwestern University Boyce Rensberger. The Washington Post Hands-On Laboratory Course Directors Rex Chisholm. Northwestern University Robert Palazzo, University of Kansas Hands-On Laboratory' Course Instructors Shuo Ma, Northwestern University Brad Schnackenberg. University of Kansas Wendy Wolf, Northwestern University SPINES Summer Program in Neuroscience Ethics and Survival SPINES is a month-long program directed by Joe L. Martinez. Jr., and James Townsel. The program is supported by grants from NIMH administered by the American Psychological Association and the Association of Neuroscience Departments and Programs. SPINES offers an introduction to the opportunities available at the MBL and in the field of neuroscience in general. Fellows are taught responsible conduct in research and other survival skills such as scientific writing, poster construction, presentations, grant mechanisms, and how to seek a postdoctoral or job position. Predoctoral Adwoa Aduonum-McKinney Marcus McFarren Jonathan Reasor Dani Smith Kenira Thompson Desiree Villarreal Nicole Wicha Postdoctoral Garv O. Gaufo Honors R57 Scholarships Awarded Aline D. Gross Scholarship Fund Brown, Elizabeth A., Medical College of Virginia American Society for Cell Biology Minorities Affairs Committee Chitwood. Randy. University of Texas. San Antonio DePass, Anthony. University of Massachusetts. Amherst McKnight, Spontaneous. University of Arizona Norman. Eric, University of Pittsburgh Quintero. Omar. Duke University Medical Center Reese. Eric. University of California Arthur Klorfein Scholarship Fund Cooper, Brian, National Institute of Medical Research, London. UK Damen, Wim. University of Munich (LMU). Germany Melfi. Raftaella. Universita Delgi Studi di Palermo. Italy Biology Club of the City of New York Scholarships Fund Yamaguchi. Ayako. Columbia University Burroughs Wellcome Fund Biology of Parasitism Course Brouwer, Kimberly, Johns Hopkins School of Public Health Camargo, Maristela, Federal University of Minas Gerais. Brazil Gantt. Soren. New York University Medical Center Gleeson, Michelle. University of Technology. Sydney. Australia Hensmann. Meike. Yale School of Public Health Jiang. David, Johns Hopkins University Lingnau, Andreas, Washington University Medical School Mair, Gunnar, Queen's University of Belfast, UK Marsh. Antoinette. University of California, Davis Ouko. Lillian. University of Pennsylvania Treutiger, Carl Johan. Karolinska Institute, Sweden Wille, Ulrike, University of Tubingen. Germany Burroughs Wellcome Fund Molecular Mycology Course Buchanan. Kent. University of Oklahoma Health Sciences Center Clarke. Enda E.. Royal Postgraduate Medical School. London. UK Doering, Tamara. Cornell Medical School Hopfer. Roy, University of North Carolina Hospitals Mathews, Herbert, Loyola University of Chicago Zhang. Mason, University of Nevada Caswell Grave Scholarship Fund Evans, Kelly L., Queen's University. Canada Osborne, Leslie. University of California, Berkeley Thongmee. Acharawan. University of North Texas C. Lalor Burdick Fellowship Fund Melfi, Raffaella, Universita Delgi Studi di Palermo, Italy Daniel S. Grosch Endowed Scholarship Fund Johnson. Hope. Stanford University Vasconcelos, Crisogono. Universidade Federal Fluminense. Brazil Edwin Grant Conklin Scholarship Fund Wenuganen. S., Bogor Agricultural University, Indonesia Frank R. Lillie Scholarship Fund Davis, Gregory. University of Chicago de Sa, Virginia R., University of California. San Francisco Stone. Alexandra. Ohio State University Valster. Aline, University of Massachusetts Wang, Gang, University of Iowa Gary Nathan Calkins Memorial Scholarship Fund Giuliano. Paola. Stazione Zoologica. Italy Herbert W. Rand Scholarship Fund Giuliano, Paola. Stazione Zoologica. Italy Kaufmann. Christoph. Massachusetts General Hospital, Boston Ladurner, Peter P., University of Innsbruck, Austria Lerchner, Walter. National Institute for Medical Research, London, UK Takke. Christina, University of Koln, Germany Howard A. Schneiderman Endowed Scholarship Fund Hess, Samuel, Cornell University Howard Hughes Medical Institute Educational Program Scholarship Funding Aanstad. Pia, University of Newcastle upon Tyne, UK Bacci, Alberto. University of Milano. Italy Bayliss. Richard, University of Cambridge, UK Faure. Jean-Emmanuel, University of California Gasser, Paul, Arizona State University Gries. Gundula, University of Pennsylvania Hess, Samuel, Cornell University Holzmann, Maria, University of Geneva. Switzerland Karthikeyan. G., Tata Institute of Fundamental Research, India Nelson, Craig, Harvard University Nilsson, Helen, University of Goteborg, Sweden Ozoren, Nesrin. University of Pennsylvania Polnaii. Dorit, Northeastern University White, Kathryn. Scripps Institute of Oceanography Zurek, Ludek. University of Alberta Jacques Loeb Founder's Scholarship Fund Ketelaar, Tijs J., Wageningen Agricultural University. The Netherlands R58 Annual Report Marjorie Roloff Stetten Scholarship Fund Krause. Sabine, Max-Planck-Instiliit fur Molekulare Genetik, Germany Massachusetts Space Grant Consortium Blank. Carrine, University of California, Berkeley Gaidos, Eric, Massachusetts Institute of Technology Merck and Company, Inc. Shoda, Lisl, Washington State University Treutiger, Carl Johan, Karolinska Institute, Sweden Triplett, Elisabeth. Cornell University Villegas. Eric, University of Pennsylvania Wille, Ulrike, University of Tubingen. Germany Moshe Shilo Memorial Scholarship Fund Banin. Ehud. Tel Aviv University, Israel Mountain Memorial Fund Bayhss, Richard, University of Cambridge, UK Bell, George, University of Arizona Broome. Jill, University of North Carolina, Chapel Hill Diggins, John, Providence College Ozoren, Nesrin, University of Pennsylvania Ream. Rachael A., Stanford University. Hopkins Marine Station Phillip H. Presley Memorial Scholarships, Carl Zeiss, Inc. Bauer. Eric, University of Texas, Austin Bonham, Ben, University of California, San Francisco Broome, Jill, University of North Carolina, Chapel Hill Hughes. Deborah. Scripps Institution of Oceanography Jaspers. Elke, Universilat Oldenburg. Germany Kilroy, Christopher, University of North Carolina, Wilmington Ream, Rachael A., Stanford University. Hopkins Marine Station Pioneers Fund Barghuthy. Fikry, The Hebrew University of Jerusalem, Israel Evans, Kelly L., Queen's University. Canada Planetary Biology Internships Johnson. Hope, Stanford University Vasconcelos, Crisogono, Universidade Federal Fluminense. Brazil Post-Course Research Awards Dale L. Beach, University of North Carolina, Chapel Hill, Physiology Julie Canman, University of North Carolina. Chapel Hill, Physiology Gregory Davis, University of Chicago, Embryology Anthony DePass. University of Massachusetts, Amherst, Physiology Gundula E. Gries, University of Pennsylvania. Physiology Xiang Dong (Edward) Guo, Columbia University, Physiology Kaoru Katoh, University of Tsukuha, Japan, Physiology Malleus Kelelaar, Wageningen Agricultural University. The Netherlands, Physiology Peter Ladurner. University of Innsbruck, Austria, Embryology Susan Laessig, University of Maryland, Baltimore, Neural Syslems & Behavior Laura Linz, Louisiana Slale University. Physiology S. O. Mast Founders' Scholarship Fund Meyer. Axel. State University of New York Society of General Physiologists' Scholarships Gregory K. Davis. University of Chicago Timothy E. Holy, Princeton University Justin S. Koble, Children's Hospital of Pennsylvania Surdna Foundation Banin, Ehud, Tel Aviv Universily, Israel Jaspers, Elke, Universitat Oldenburg. Germany Krause. Sabine, Max-Planek-Inslilul ftir Molekulare Genelik. Germany Wenuganen. S., Bogor Agricultural University, Indonesia Zurek. Ludek. University of Alberta, Canada Walter L. Wilson Endowed Scholarship Fund Katoh, Kaoru, Marine Biological Laboratory Nilsson, Helen, University of Goteborg, Sweden William F. and Irene C. Diller Scholarship Fund Melfi, Raffaella, Universita Degli Studi di Palermo, Italy Stone, Alexandra, Ohio Slate University William Morton Wheeler Family Founder's Scholarship Fund Wenuganen, S.. Bogor Agricultural University. Indonesia William Randolph Hearst Educational Endowment Scholarships Kim. Warren. Yale University School of Medicine Nelson, Craig. Harvard University Shelton, Marilee, Universily of North Carolina, Chapel Hill William Townsend Porter Scholarship Fund DePass. Anthony, Universily of Massachuselts, Amherst McKnight. Spontaneous, University of Arizona Norman, Eric, University of Pittsburgh Qumtero, Omar. Duke University Medical Center Paul Maddox. University of North Carolina, Chapel Hill. Physiology Lynne Merchant, University of California, San Diego. Neural Syslems & Behavior Helen Nilsson, University of Goteborg, Sweden, Physiology Yasushi Satoh, Universily of Tokyo, Japan, Physiology Elaine Seaver, University of Texas. Auslin. Embryology Sen Song, Brandeis University. Neural Systems & Behavior Aline Valster, Universily of Massachuselts. Amherst. Physiology Cris Vasconcelos, ETH Zentrug, Zurich. Switzerland. Microbial Diversity Gang Wang, University of Iowa, Physiology S, Wenuganen, Bogor Agricultural University, Indonesia, Microbial Diversity 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. James D. Ebert, The Johns Hopkins 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, Neil Jacobs, Hale and Don- Chair of the Science Council, Ronald L. Calabrese. Emory University* Class of 1998 Norman Bernstein, Diane and Norman Bernstein Foundation. Inc. John R. Lakian, The Fort Hill Group, Inc. Joan V. Ruderman, Harvard Medical School Sheldon J. Segal, The Population Council William T. Speck, Columbia-Presbyterian Medical Center Alfred Zeien, The Gillette Company Class of 1999 Mary-Ellen Cunningham, Grosse Pointe Farms, MI Neil Jacobs, Hale and Dorr Darcy Brisbane Kelley, Columbia University Laurie J. Landeau. Marinetics, Inc. Burton J. Lee, III, Edgartown, MA Robert E. Mainer, The Boston Company Frank Press, Carnegie Institution of Washington. DC Christopher M. Weld, Sullivan & Worcester, Boston Class of 2000 Alexander W. Clowes, University of Washington School of Medicine Story C. Landis, Case Western Reserve University Irwin B. Levitan. Brandeis University G. William Miller. G. William Miller & Co.. Inc., Washington. DC *Ex officiti Class of 2001 Porter Anderson, North Miami Beach, FL Frederick Bay, Josephine Bay Paul and C. Michael Paul Foundation, Inc. Martha W. Cox, Hobe Sound, FL Mary J. Greer, New York. NY William C. Steere. Jr., Pfizer Inc. Gerald Weissmann, New York University School of Medicine Honorary Trustees William T. Golden, New York, NY Ellen R. Grass. The Grass Foundation Trustees Emeriti Edward A. Adelberg. Yale University, New Haven, CT John B. Buck. Sykesville, MD Seymour S. Cohen, Woods Hole, MA Arthur L. Colwin, Key Biscayne, FL Laura Hunter Colwin. Key Biscayne, FL Donald Eugene Copeland, Woods Hole, MA Sears Crowell. Indiana University, Bloomington, IN Alexander T. Daignault. Falmouth, MA Teru Hayashi. Woods Hole. MA Ruth Hubbard, Cambridge, MA Lewis Kleinholz, Reed College, Portland. OR Maurice E. Krahl, Tucson. AZ Charles B. Metz, Miami. FL (deceased) Keith R. Porter, University of Pennsylvania, Philadelphia, PA (deceased) C. Ladd Prosser, University of Illinois, Urbana, IL W. D. Russell-Hunter, Syracuse University, Syracuse, NY Mary Sears, Woods Hole, MA (deceased) David Shepro, Boston University, Boston, MA D. Thomas Trigg, Wellesley. MA Walter S. Vincent. Woods Hole. MA George Wald. Cambridge, MA (deceased) R59 R60 Annual Report Science Council Ronald L. Calabrese. Chair Donald Abt Clay Armstrong Robert Barlow, Jr. (from 8/97) Kerry S. Bloom John Burris,* Vincent E. Dionne (from 8/97) John Dowling Barbara Ehrlich Bruce J. Peterson (1998) Mitch Sogin (1998) Ann E. Stuart (ending 8/97) Executive Committee of the Board of Trustees Sheldon J. Segal. Chair Frederick Bay, Co-vice Chair Mary J. Greer, Co-vice Chair John E. Burris* Ronald L. Calabrese Mary B. Conrad Mary-Ellen Cunningham Robert Mainur Joan V. Ruderman Gerald Weissmunn Standing Committees of the Board of Trustees Development Mary-Ellen Cunningham, Chair Porter W. Anderson Robert Barlow Frederick Bay Mary B. Conrad Martha Cox James Ebert Neil Jacobs John Lakian Burton Lee Irwin Levitan G. William Miller William Speck William Steere Christopher Weld Facilities and Capital Equipment Joan Ruderman, Chair Porter W. Anderson Lawrence Cohen Neal Cornell Story Landis Irwin Levitan Frank Press Christopher M. Weld Finance and Investment Robert Mainer, Chair Norman Bernstein Alexander Clowes Mary B. Conrad Donald DeHart Neil Jacobs Darcy Kelley John Lakian Laurie Landeau Werner LoewenMem Robert Man/. G. William Miller Ronald P. O'Hanley Irving Rabb Alfred Zeien Nominating Gerald Weissmann. Chair Ronald Calabrese Alexander Clowes Martha Cox Mary-Ellen Cunningham Mary Greer Story Landis Tom Pollard Sheldon Segal William Steere Standing Committees of the Corporation and Science Council Buildings and Grounds Lawrence B. Cohen, Chair Barbara C. Boyer Alfred B. Chaet Richard Cutler* William R. Eckberg Barry Fleet' Ferenc Harosi Joe Hayes* Bruce J. Peterson Kenvon S. Tweedell *Ex officio Trustees and Committees R61 Education John E. Dowling. Chair Elaine L. Bearer Vincent E. Dionne Paul V. Dunlap Rachel D. Fink Roger T. Hanlon Holger W. Jannasch George M. Langford Dorianne Mehane* Michael E. Mendelsohn John D. Rummel* Steven J. Zottoli Fellowships Thoru Pederson. Chair Kathleen Dunlap Barbara E. Ehrlich Anne E. Giblin Jose Lemos Carol L. Reinisch John D. Rummel* Housing, Food Service, and Child Care Kerry S. Bloom Carole L. Browne LouAnn King* Robert P. Malchow Darrell R. Stokes Ann E. Stuart Janis C. Weeks MBL/WHOI Library Joint Advisory Committee David Shepro, Chair, MBL Judy Ashmore, MBL* Cabell Davis, WHOI David Dow, NMFS John Hobbie, MBL Colleen Hurter, WHOI* Mark Kurz, WHOI Cathy Norton, MBL* Monica Riley, MBL Jim Robb, USGS Peter J. S. Smith, MBL Bruce Warren, WHOI Research Services and Space Hans Laufer. Chair Peter B. Armstrong Neal W. Cornell Richard Cutler* Kenneth H. Foreman Louis M. Ken- David Landowne Andy Mattox* Jerry M. Melillo Merle Mizell Peter J. S. Smith Paul A. Steudler Ivan Valiela Discovery: The Campaign for Science at the Marine Biological Laboratory Steering Committee Frederick Bay, Campaign Chair William T. Golden, Honorary Campaign Chair Ellen R. Grass, Honorary Campaign Chair Alexander Clowes, Campaign Vice-chair Martha W. Cox, Campaign Vice-chair G. William Miller, Campaign Vice-chair Gerald Weissmann. Campaign Vice-chair Porter Anderson Robert B. Barlow, Jr. Norman Bernstein Jewel Plummer Cobb Mary B. Conrad Mary-Ellen Cunningham John E. Dowling James D. Ebert Gerald D. Fischbach Robert D. Goldman Mary J. Greer M. Howard Jacobson Laurie J. Landeau George M. Langford Burton J. Lee, III Robert A. Prendergast David Shepro William T. Speck William C. Steere. Jr. Christopher M. Weld. Esq. Alfred M. Zeien *Ex officio Administrative Support Staff 1 Biological Bulletin Greenberg, Michael J., Editor-in-Chief Clapp, Pamela L., Managing Editor Burns, Patricia DeBenedictis, Lisa M. Gibson, Victoria R. Pennmgton, Susan M. Schachinger, Carol H. Financial Sen'ices Office Roddy, Timothy, Chief Financial Officer Bowman, Richard, Controller Ghetti, Pamela M.. Controller Afonso, Janis Cornette, Ruth Dwyer, Patricia E. Hopkins, Ann E. Iwaszko, Roxanne M. ; Lancaster, Cindy Pacheco, Anthony F. 2 Palmer, Pamela 2 Poravas, Maria Ranzinger. Laura Sprague. Patricia A. Stark. Judy Slock Room Schorer, Timothy M., Supervisor Cameron, Alicia A. : Nelson, Beth A. O'Connor-Lough, Susan Robinson, Mary M. : Director's Office Burris, John E., Director and Chief Executive Officer Burrhus, I. Elaine Donovan. Marcia H. MacNeil, Jane L. External Affairs Carotenuto, Frank C., Director Black, Nancy O. Faxon, Wendy P. Martin, Theresa H. Martinez, Mario R. 2 Maxwell, Thanh L. : Patch-Wing, Dolores Quigley, Barbara A. Scibek, John C. Shaw, Kathleen L. Wessling, Gail M. Wicklund, Eileen R Associates Program Bohr, Kendall B. Pratt, Jennifer A. : Communications Office Clapp, Pamela, L. Director Clowes, Sarah W. : Dykstra, Margaret L. : Furfey, Susan C. 2 Kenna, Laura M. Liles, Beth R. Pratt, Sara Williams, Sara V. Purchasing Hall Jr., Lionel E., Supervisor Mancini, Mary Nelson, Beth A. Shamon, Lynne R. 2 Stone, Janice G. 2 1 Including persons who joined or left the staff during 1997. 2 Summer or temporary. Housing and Conferences King. LouAnn D.. Director Barry, Maureen J. Johnson, Frances N. Hanlon. Arlene K. 2 Switchboard Baker. Ida M. Dunn-Fall. Martha F. 2 Ridley. Alberta W. R62 Administrative Support Staff R63 Human Resources Goux, Susan P., Director Donovan, Marcia H. Drange, Stacey B. Marine Resources Center Administrative Staff Hanlon, Roger T., Director Moniz, Priscilla Aquatic Resources Department Enos. Jr., Edward G., Superintendent Bourque, Ryan M. : Chappell, P. Dreux 2 DeGiorgis, Joseph A. 2 Erlingsson, Erik C. 2 Freeman, Darren M. 2 Grossman. William M. Klimm III. Henry W. Luther, Herbert Mansfield, Darren P. 2 Parent, Scott M. 2 Schneider. Peter J. : Sexton, Andrew W. Sullivan, Daniel A. Tassinari, Eugene MRC Life Support System Mebane. William N.. Systems Operator Hanley, Janice S. Kuzirian, Alan Stukey, Jetley M. Till, Geoffrey A. MBUWHOl Librae Norton, Catherine N., Director Ashmore. Judith A. Connelly. Michelle F.A. Costa, Marguerite E. Cullen, Cynthia M, 2 Deveer, Joseph M. Duda. Laurel E. Farrar, Stephen R. L. Jackson, James R, Medeiros, Melissa Monahan. A. Jean Moniz. Kimberly L. Nelson, Heidi Pratson, Patricia F. Riley, Jacqueline Ulbrich, Ciona 2 Zuwallack, Barbara Zuwallack, Raymond Zuwallack. Ronald L. Copy Center Mountford, Rebecca J., Supervisor Abisla, Richard L. : Adams, Cathryn L. : Clark, Tamara L. 2 Johnson, Courtney M. 2 Kefeauver. Lee LaPlante, Robert F Mancini. Mary E. Warner, Kathleen 2 Information Systems Division Smith, Adrian P., Assistant Director Ennis, Douglas E. 2 Gage, Timothy J. 2 Mahoney, Timothy P. Moynihan, James V. Mountford, Rebecca J. Remsen, David P. Renna. Denis J. Space, David B. Swasey. Anne E. 2 Safety Sen'ices Mattox, Andrew H., Environmental, Health, and Safety Manager Kelly, Niamh O. : O'Neill. Maureen D. 2 Sen'ice. Projects, and Facilities Cutler, Richard D., Director Enos, Joyce B. Apparatus Baptiste. Michael G. Barnes. Franklin D. Haskins, William A. Building Sen'ices and Grounds Hayes. Joseph H., Superintendent Anderson, Lewis B. Atwood, Paul R. Baker, Harrison S. Barnes. Susan M. Berrios, Jessica L. 2 Boucher. Richard L. Brereton, Richard S. 2 Callahan, John J. Cameron, Lawrence M. 2 Collins, Paul J. Cowan, Matthew B. 2 Cutler, Matthew D. 2 Dole. Adam J. 2 Dorris. John J. Fernandez, Peter R. : Gibbons, Roberto G. Gonsalves, Nelson Hanmgan, Catherine Harrington, James D. Illgen. Robert F. Kennette. Kirsten E. 2 Lawrence, Adam G. 2 Lawson, Christina C. 2 Leary, Jason C. : Luther, Herbert Lynch. Henry L. Maccaro, Jackie McNamara, Noreen M. Massey Eric 2 Rattacasa. Frank D. Sholkovitz. Nathan 2 R64 Annual Report Schrontz. Mathieu D. : Silva, Cynthia C. Wessling, Kellen A. 2 Varao, John : Ware, Lynn M. Plant Operations and Maintenance Fleet. Barry M., Superintendent Barnes, John S. Blunt, Hugh F. Bourgoin. Lee E. Cadose. James W. Carini, Robert J. Carroll, James R. Fish Jr., David L. Fuglister. Charles K. Gonsalves, Jr., Walter W. Hathaway, Peter J. Henderson, Jon R. Justason, C. Scott Lochhead, William M. McAdams 111, Herbert M. McHugh, Michael O. Mills, Stephen A. Olive, Jr.. Charles W. Schoepf, Claude Serrano, Robert A. Shepherd, Denise M. Toner, Michael Machine Shop Sylvia, Frank E. Wetzel, Ernest D. : Photolab Nelson, Linda M., Supervisor Clark, Tamara L. : Colder, Robert J. Hong, Theresa H. : Richmond. Hazel E. 2 Research Atlniinixtnition ami Educational Programs Rummel, John D., Director Barry, Kevin 2 Chandler, Marsha J. Hamel, Carol C. Hunt, Sharon L. Huffer, Linda Kaufmann, Sandra J. Mebane, Dorianne C. Moynihan, Brenda L. 2 Central Microscopy Facility and General Use Rooms Kerr, Louis M., Supervisor DeProto, Jamin E. : Lavalli, Kari Lee 2 Peterson, Martha B. Soucy, Lori A. : Josephine Bci\ Paul Center for Comparative^ Molecular Biology and Evolution Administrative Staff Amit. Udeni Journal of Menihrane Biology Loewenstein. Werner R., Editor Cicora, Judith M. 2 Fay. Catherine H. Howard, Linda L. Lynch. Kathleen F. NASA Center for Advanced Studies in the Space Life Sciences Administrative Staff Dawidowicz, Eliezar A., Administrator Amit. Udeni P. Nixon, Jennifer L. Satellite/Periwinkle Children 's Programs Robinson. April 2 Robinson. Paulina H. 2 Brown. Shannon K. : Browne. Jennifer L. 2 Douglas, Alicia D. 2 Gallant, Carolyn A. 2 Griffin, Courtney A. 2 Lee, Annette M. 2 Martinez. Adria E. 2 Robinson, Jayma L. 2 Robinson, Milton G. 2 Simpson, Christopher F. 2 Strout, Kerry L. 2 Ecosystems Center Administrative Staff Berthel. Dorothy J. Donovan, Suzanne J. Nunez. Guillermo Seifert, Mary Ann Members of the Corporation 1 Life Members Acheson, George H., 25 Quissett Avenue, Woods Hole. MA 02543 Adelberg. Edward A., Lincoln Tower Apt. 802. 2400 Presidential Way, West Palm Beach. FL 33401 Afzelius, Bjorn, University of Stockholm. Wenner-Gven Institute. Department of Ultrastructure Research. Stockholm. SWEDEN Amatniek, Ernest, 1112 Northwest 5th Avenue. Gainesville. FL 32601 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, Dept. of Physiology, Box 1116, MR4, Charlottesville. VA 22903 Bernht'imer, Alan W., New York University Medical Center. Dept. of Microbiology, 550 First Avenue, New York, NY 10016 Bertholf, Lloyd M., Westminster Village. #2114. 2025 E. Lincoln Street. Bloommgton. IL 61701-5995 Bosch, Herman F., Box 617. Woods Hole, MA 02543 Buck, John B., 7200 Third Avenue, #C-020, Sykesville, MD 21784 Burbanck, Madeline P., Box 15134. Atlanta, GA 30333 Burbanck, William IX, P.O. Box 15134. Atlanta. GA 30333 Carlson, Francis D., Johns Hopkins University, Biophysics Dept.. Jenkins Hall, N. Charles Street, Baltimore, MD 212 IS Clark, Arnold M., 53 Wilson Road, Woods Hole, MA 02543 Clark, James M., 210 Emerald Lane, Palm Beach. FL 33480 Cohen, Seymour S., 10 Carrot Hill Road, Woods Hole. MA 02543 Colwin, Arthur L., 320 Woodcrest Road, Key Biscayne, FL 33149- 1322 Colwin, Laura Hunter, 320 Woodcrest Road, Key Biscayne, FL 33149 Cooperstein, Sherwin J., University of Connecticut, School of Medicine, Department of Anatomy, Farmington, CT 06030-3405 Copeland, D. Eugene, 4 1 Fern Lane, Woods Hole. MA 02543 Corliss, John O., P.O. Box 2729, Bala Cynwyd. PA 19004-21 16 Costello, Helen M., Carolina Meadows, Villa 137, Chapel Hill, NC 27514-8512 Crouse, Helen, Rte. 3. Box 213. Hayesville. NC 28904 Dudley, Patricia L., 3200 Alki Avenue SW, #401, Seattle, WA 98116 Edwards, Charles, 2244 Harbour Court Drive. Longboat Key, FL 34228 Elliott, Gerald F., The Open University Research Unit, Foxcombe Hall, Berkeley Road. Boars Hill, Oxford OX1 5HR, UK Failla, Patricia M., 2149 Loblolly Lane, Johns Island, SC 29455 Ferguson, James K. W., 56 Clarkehaven Street. Thornhill. Ontario L4J 2B4. Canada Glusman, Murray, New York State Psychiatric Institute. 722 W. 168 Street, Unit #70, New York, NY 10032 Goldman, David E., 140 Ter Heun Drive, Rm 212. Falmouth, MA 02540 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., 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 0455 1 Hayashi, Teru, 15 Gardiner Road, Woods Hole. MA 02543-1 113 Hisaw , Frederick L., 1 765 SW Tamarack Street. Apt II. McMinnville, OR 97128-7416 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 Hurwitz, Charles, Stratum VA Medical Center, Research Service, Albany, NY 12208 DeHaan, Robert L., Emory University School of Medicine. Department of Anatomy and Cell Biology. 1648 Pierce Drive, Rm. 108, Atlanta, GA 30322 Including action of the 1997 Annual Meeting. Katz, George, Merck. Sharp and Dohme, Fundamental & Experimental Research Laboratory, P.O. Box 2000. Rahway, NJ 07065 Kingsbury, John M., Cornell University. Dept. of Plant Biology, Plant Science Building, Ithaca, NY 14853 R65 R66 Annual Report Kleinholz, Lewis, Reed College. Department of Biology, 3203 SE Woodstock Blvd.. Portland. OR 97202 Laderman, Ezra, Yale University. New Haven. CT 06520 LaMarche, Paul H., Eastern Maine Medical Center, 489 State Street. Bangor. ME 04401 Lauffer, Max A., Penn State University Medical Center. Dept. of Biophysics & Physiology. Hershey, PA 17033 LeFevre, Paul G., 15 Agassis Road, Woods Hole, MA 02543 Levine, Rachmiel, City of Hope Medical Center. Shapiro Building, Duarte, CA 91010 (deceased) Lochhead, John H., 49 Woodlawn Road, London SW6 6PS. UK Loevvus, Frank A., Washington State University. Institute of Biological Chemistry. Pullman, WA 99164 Loftfield, Robert B., University of New Mexico, School of Medicine, Albuquerque. NM Malkiel, Saul, Allergic Diseases Inc., 130 Lincoln Street. Worcester. MA 01 609 Marsh, Julian B., Medical College of Pennsylvania. Dept. of Physiology & Biochemistry, Philadelphia, PA 19129 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 Moscona, A. Aaron, University of Chicago, Dept. of Molecular Genetics & Cell Biology, 920 East 58 Street, 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 Prosser, C. Ladd, University of Illinois. Dept. of Physiology, 524 Bun-ill Hall. Urhana, IL 61X01 Prvtz. Margaret McDonald, address unknown Ratnt'r, Sarah, Public Health Research Institute. Department of Biochemistry, 455 First Avenue, New York, NY 10016 Renn, Charles E., address unknown Reynolds, George T., Department of Physics, Princeton University, Jadwin Hall, Princeton, NJ 08544 Rice, Robert V., 30 Burnham Drive. Falmouth. MA 02540 Rockstein, Morris, 600 Biltmore Way. Apartment 805, Coral Gables, FL 33134 Ronkin, Raphael R., 3212 McKinley Street, NW. Washington. DC 20015-1635 Sanders, Howard L., Woods Hole Oceanographic Institute, Woods Hole, MA 02543 Sato, Hidemi, Professor Emeritus. Nagova Llniversity, 3-24-101. Oakinishi Machi. Toba Mie 517-0023. Japan Saz, Arthur K., Georgetown University Medical School. Department of Immunology. Washington, DC 20007 Schlesinger, R. Walter, 7 Langley Road, Falmouth, MA 02540-1809 Scott, Allan C., Colby College. Waterville. ME 04901 Silverstein, Arthur M., John Hopkins University, Institute of the History of Medicine, 1900 E. Monument Street, Baltimore, MD 21205 Sjodin, Raymond A., University of Maryland. Department of Biophysics, Baltimore, MD 21201 Smith, Paul F., P.O. Box 264, Woods Hole. MA 02543-0264 Sonnenblick, Benjamin P., 515A Hentage Hill Village, Southbury, CT 06488 (deceased) Speer, John W., 293 West Main Road, Portsmouth, RI 02871 Sperelakis, Nicholas, University of Cincinnati, Dept. of Physiol./ Biophysics, 231 Bethesda Avenue, Cincinnati, OH 45267-0576 Spiegel, Evelyn, Dartmouth College, Dept. of Biological Sciences, 204 Oilman, Hanover, NH 03755 Spiegel, Melvin, Dartmouth College, Dept. of Biological Sciences, 204 Oilman, Hanover. NH 03755 Steinhardt, Jacinto, 1508 Spruce Street, Berkeley. CA 94709 Stephens, Grover C., University of California, School of Biological Sciences, Dept. of Ecology & Evolutionary/Biology, Irvine. CA 92717 Strehler, Bernard L., 2310 N. Laguna Circle Drive, Agoura. CA 91301-2884 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 Taylor, Robert E., 339 Gifford Street. Apt. 303. Falmouth. MA 02540 Thorndike, VV. Nicholas, Wellington Management Company, 200 State Street, Boston. MA 02109 Trager, William, The Rockefeller University. 1230 York Avenue, New York. NY 10021-6399 Trinkaus, J. Philip, Yale University, Dept. of Biology, New Haven, CT 065 1 1 Villee, Claude A., 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 Wilber, Charles G., Colorado State University, Department of Biology, Forensic Science Laboratory, Fort Collins, CO 80523 Zweifach, Benjamin W., 8811 Nottingham Place, La Jolla, CA 92037 (deceased) Members Abt, Donald A., Marine Biological Laboratory. Laboratory of Aquatic Animal Medicine & Pathology, Woods Hole, MA 02543 Adams, James A., 3481 Paces Ferry Road, Tallahassee, FL 32308 Adelman, William J., 160 Locust Street, Falmouth, MA 02540 Alkon, Daniel L.. NIH Lab. of Adaptive Systems. 36 Convent Drive. MSC 4124. 36/4A21. Bethesda, MD 20892-4124 Allen. Garland F., Washington University. Dept. of Biology, Box I 137, One Brookings Drive, St. Louis, MO 63130-4899 Allen. Nina S., No. Carolina State University, Department of Botany, Box 7612, Raleigh, NC 27695 Alliegro, Mark C., Louisiana State University Medical Center, Dept. of Cell Biology & Anatomy, 1901 Perdido Street, New Orleans, LA 701 12 Members of the Corporation R67 Anderson, Everett, Harvard Medical School, Dept. of Cell Biology, 24(1 Longwood Avenue. Boston. MA 021 15-6092 Anderson, John M., 1 10 Roat Street, Ithaca, NY 14850 Anderson, Porter W., 100 Bayview Drive, Apt. 2224. North Miami Beach, FL 33160 Armett-Kihel, Christine, University of Massachusetts, Boston, Dean of Science Faculty, Boston, MA 02125 Armstrong, Clay M., University of Pennsylvania School of Medicine. B701 Richards Bldg., Department of Physiology, 3700 Hamilton Walk. Philadelphia, PA 19104-6085 Armstrong, Ellen Prosser, 57 Millfield Street, Woods Hole. MA 02543 Armstrong, Peter B., University of California, Davis, Dept. of Molec. & Cell. Biology, Davis, CA 95616-8755 Arnold, William A., Oak Ridge National Laboratory. Biology Division. 102 Balsalm Road, Oak Ridge. TN 37830 Ashton. Robert W., Bay Foundation. 17 West 94th Street, New York. NY 10025 Atema, Jelle, Boston University Marine Program. Marine Biological Laboratory, Woods Hole. MA 02543 Baccetti, Baccio, University of Sienna, Institute of Zoology, 53100 Siena, Italy Baker, Robert G., New York University Medical Center, Dept. of Physiol. & Biophysics, 550 First Avenue, New York, NY 10016 Baldwin, Thomas O., Texas A & M University, Department of Biochemistry and Biophysics, College Station, TX 77843 Baltimore. David, California Institute of Technology, 256-80, Pasadena, CA 9 1 1 25 Barlow, Robert B., SUNY Health Science Center. Dept. of Physiology. 750 East Adams St., Weiskotten Hall. Syracuse. NY 13210 Barry, Daniel T., 2415 Fairwind Drive, Houston. TX 77062-4756 Barry, Susan R., Mount Holyoke College, Dept. of Biological Sciences, So. Hadley. MA 01075 Bass, Andrew H., Cornell University. Dept. of Neurobiology & Behavior, Seely Mudd Hall. Ithaca. NY 14853 Battelle, Barbara-Anne, University of Florida. Whitney Laboratory, 9505 Ocean Shore Boulevard, St. Augustine. FL 32086 Bay, Frederick, Bay Foundation, 17 W. 94th Street, First Floor, New- York, NY 10025-7116 Baylor, Martha B., P.O. Box 93, Woods Hole, MA 02543 Bearer, Elaine L., Brown University, Div. of Biology & Medicine. Dept. of Pathology, Box G, Providence, RI 02912 Beatty, John M., University of Minnesota, Dept. of Ecology & Behavioral, Biology. 1445 Gortner, St. Paul, MN 55108 Beauge, Luis Alberto, Institute M. & M. Ferreyra, Dept. 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. Dept. of Cell Biology & Anatomy. Edmonton. Alberta T6G 2H7, Canada Bell, Eugene, 305 Commonwealth Avenue. Boston. MA 021 15 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. Dept. of Neuroscience. 1300 Morris Park Avenue, Bronx. NY 10461 Bennett, Miriam F., Colby College. Department of Biology. Waterville, ME 04901 Berg, Carl .)., P.O. Box 681, Kilauea, Kauai, HI 96754-0681 Berlin. Suzanne T., 5 Highland Street, Gloucester, MA 01930 Bernstein, Norman, Diane and Norman Bernstein Foundation. 5301 Wisconsin Ave.. 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 02 1 1 5 Bishop, Stephen H., Iowa State University. Dept. of Zoology, Ames, IA 50010 Blaustein, Mordecai P., University of Maryland, School of Medicine, Department of Physiology. Baltimore. MD 21201 Blennemann, Dieter, 1117 E. Putnam Avenue. Apt. #174, Riverside, CT 06878-1333 Bloom, George S.. University of Texas Southwestern, Medical Center, Cell Bio. & Neuroscience Dept., 5323 Harry Hines Blvd.. Dallas, TX 75235-9039 Bloom, Kerry S., University of North Carolina at Chapel Hill, Department of Biology, 623 Fordham Hall, CB #3280. Chapel Hill. NC 27599 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 Blvd., West. Bronx, NY 10468 Borst, David W., Illinois State University, Department of Biological Sciences, Normal, IL 61790 Bowles, Francis P., Marine Biological Laboratory, The Ecosystems Center. Woods Hole, MA 02543 Boyer, Barbara C., Union College, Biology Department, Schenectady. NY 12308 Brandhorst, Bruce P., Simon Eraser University, Inst. of Molec. Biol./Biochem. Barnaby. B.C. V5A 1S6, Canada Brinley, F. J., NINCDS/NIH, Neurological Disorders Program, Rm. 812 Federal Building. Bethesda, MD 20892 Bronner-Fraser, Marianne, California Institute of Technology. Division of Biology 139-74, Pasadena, CA 91 125 Brown. Stephen C., SUNY. Dept. of Biological Sciences. Albany. NY 12222 Brown, William L., BankBoston. 100 Federal Street, 01-23-11, Boston. MA 02106-2016 Browne, Carole L., Wake Forest University. Dept. of Biology, Box 7325, Winston-Salem, NC 27109 Browne, Robert A., Wake Forest University, Dept. of Biology. Box 7325. Winston-Salem, NC 27109 Bucklin, Anne C., University of New Hampshire, Ocean Process Analysis Lab. 142 Morse Hall. Durham. NH 03824 Bullis, Robert A., Marine Biological Laboratory, Laboratory of Aquatic Animal Medicine, Woods Hole, MA 02543 Burger, Max M., Friedrich Miescher Institute, P.O. Box 2543, CH 4002 Basel. Switzerland Burgess, David R., University of Pittsburgh, Dept. of Biological Sciences. 234 Langley. Pittsburgh. PA 15260 Burgos, Mario, 1HEM Medical School, UNC Conicet. Casilla de Correo 56, Mendoza. 5500, 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 Calahrese, Ronald L., Emory University, Department of Biology. 1510 Clifton Road. Atlanta, GA 30322 R68 Annual Report Callaway, Joseph C., New York Medical College, Dept. of Physiology, Basic Sciences Bldg., Valhalla, NY 10595 Cameron, R. Andrew, California Institute of Technology, Division of Biology 156-29. Pasadena. CA 9112? 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 Carew, Thomas J., Yale University, Department of Psychology, P.O. Box 11A, Yale Station, New Haven, CT 06520 Cariello, Lucio, Stazione Zoologica Villa Comunale, 80121 Naples. Italy Case, James F., University of California. Santa Barbara, Marine Science Institute, Santa Barbara. CA 93106 Cassidy, J. D., Providence College. Priory of St. Thomas Aquinas, Providence, RI 02918-0001 Cavanaugh, Colleen M., Harvard University. Biological Laboratories, 16 Divinity Avenue, Cambridge. MA 02138 Chaet, Alfred B., University of West Florida, Dept. of Cell & Molec. Biol.. 1 1000 University Parkway, Pensacola, FL 32514 Chambers, Edward L., University of Miami School of Medicine. Dept. of Physiology & Biophys., P.O. Box 016430, Miami, FL 33101 Chang, Donald C., Hong Kong University of Science and Technology, Department of Biology, Clear Water Bay, Row loon. Hong Kong Chappell, Richard L., Hunter College. CUNY. Dept. of Biological Sciences, Box 210, 695 Park Avenue, New York, NY 10021 Chikarmane, Hemant M., 12 Middle Street, Reading, MA 01867 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 0651 1 Clark, Eloise E., Bowling Green State University. Biological Sciences Department. Bowling Green, OH 43403 Clark, Hays, 26 Deer Park Drive, Greenwich, CT 06830 Clark, Wallis H., 12705 NW 1 12th Avenue. Alachua, FL 32615 Claude, Philippa, University of Wisconsin. Dept Zoology, Zoology Research Building 125. 1 1 17 W Johnson St., Madison, WI 53706 Clay, John R., NIH-NINDS, Building 36, Room 2-CO2, Bethesda, MD 20892 Clowes, Alexander W., University of Washington, School of Medicine, Dept. of Surgery, Box 356410, Seattle, WA 98195-6410 Clutter, Mary, 2555 Pennsylvania Avenue, N.W., Apt. 611, Washington, DC 20037-1646 Cobb, Jewel Plummer, California State University, Office of the President, 5151 University Drive, 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, Dept. of Physiology, 333 Cedar Street, New Haven, CT 06520 Cohen, Maynard M., Rush Medical College, Dept. of Neurological Sciences. 600 South Paulina, Chicago, IL 60612 Cohen, William D., Hunter College. Dept. of Biological Sciences, 695 Park Avenue, Box 79. New York, NY 10021 Coleman, Annette W., Brown University, Div. of Biology and Medicine, Providence, RI 02912 Colinvaux, Paul, Smithsonian Tropical Research Institute, Unit 0948, Apo AA 34002-0948, USA Collier, Jack R., P.O. Box 139. 3431 Highway, #107, Effie, LA 71331 Collier, Marjorie McCann, P.O Box 139, 3431 Highway 107. Effie, LA 71331 Collin, Carlos, NIH, Dept. of LAS, NINDS, Bldg. 36. 36 Convent Drive, Room B306, Bethesda. MD 20892-4124 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 Cornwall, Melvin C., Boston University, School of Medicine, Dept. of Physiology L714, Boston, MA 021 18 Corson, D. Wesley, Storm Eye Institute, Room 537, 171 Ashley Avenue. Charleston. SC 29425 Corwin, Jeffrey T., University of Virginia, School of Medicine, Dept. of Otolaryngology, HNS and Neuroscience, 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 Institution, Biology Department. Woods Hole, MA 02543 Crane, Sylvia E., 438 Wendover Drive. Princeton, NJ 08540 Cremer-Bartels, Gertrud Universities Augenklinik. 44 Munster, Germany Crow, Terry J., University of Texas Medical School. Dept. of Neurobiol. & Anatomy, Houston, TX 77225 Crowell, Sears, Indiana University. Department of Biology. Bloomington, IN 47405 Crowther, Robert J., Shriners Burns Institute Research Center. One Kendall Square. Building 1400. Cambridge, MA 02139 Cunningham, Mary-Ellen, 62 Cloverly Road, Grosse Pointe Farms, MI 48236-3313 Cutler, Richard D., Marine Biological Laboratory, Woods Hole, MA 02543 Daignault, Alexander T., Edgewood, 575 Osgood Street. North Andover. MA 01875 Davidson. Eric H., CA Institute of Technology. Division of Biology, 156-29. 1201 E. California Blvd.. Pasadena, CA 91 125 Daw, Nigel W., 5 Old Pawson Road, Branford. CT 06405 De Weer. Paul J., University of Pennsylvania, B400 Richards Bldg., 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 B., Johns Hopkins University, School of Medicine, Kennedy-Krieger Inst, 707 North Broadway, Baltimore, MD 21205 Del'hillips, 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 Ctr., Charlottesville, VA 22908 Dettbarn, Wolf-Dietrich, Vanderbilt University, School of Medicine, Department of Pharmacology, Nashville, TN 37232 Dionne, Vincent E., Boston University Marine Program. Marine Biological Laboratory. Woods Hole, MA 02543 Dixon, Keith E., Flinders University, School of Biological Sciences, Bedford Park. 5042, SO. Australia Dow ling. John E., Harvard University, Biological Laboratories, 16 Divinity Street, Cambridge, MA 02138 I >i ;i|> .HI, Pierre, Montreal General Hospital. Dept. of Neurology. 1650 Cedar Avenue. Montreal H3G 1A4, Canada DuKuis. Arthur Brooks, John B. Pierce Foundation Lab., 290 Congress Avenue. New Haven, CT 06519 Members of the Corporation R69 Duncan, Thomas K., Nichols College, Environmental Sciences Dept.. Dudley, MA 01571 Dunham, Philip B., Syracuse University, Department of Biology, Syracuse, NY 13244 Dunlap, Paul V., University of Maryland Biotechnology Institute, Center of Marine Biotechnology, Columbus Center, Suite 236, 701 East Pratt Street, Baltimore. MD 21202 Ehert, James D., Johns Hopkins University, Dept. of Biology, Homewood. 3400 No. Charles Street, Baltimore. MD 21218-2685 Eckberg, William R., Howard University. Department of Biology, P.O. Box 887, Admin. Bldg., Washington, DC 20059 Edds, Kenneth T., R&D Systems, Inc., Hematology Division, 614 McKinley Place, NE, Minneapolis, MN 55413 Eder, Howard A., Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Edstrom, Joan, 53 Two Ponds Road, Falmouth. MA 02540 Egyud, Laszlo G., Cell Research Corporation. P.O. Box 67209, Chestnut Hill, MA 02167-0209 Ehrlich, Barbara E., Yale University Medical School, B207 SHM. New Haven, CT Eisen, Arthur Z., Washington University, Division of Dermatology. St. Louis, MO 63110 Eisen, Herman N., Massachusetts Institute of Technology, Center for Cancer Research, El 7- 128, 77 Massachusetts Ave.. Cambridge, MA 02139-4307 Elder, Hugh Young, University of Glasgow, Institute of Physiology. Glasgow G12 8QQ, Scotland, UK Englund, Paul T., Johns Hopkins Medical School, Dept. of Biological Chemistry. 725 N. Wolfe Street, Baltimore. MD 21205 Epel, David, Stanford University, Hopkins Marine Station. Ocean View Blvd., Pacific Grove, CA 93950 Epstein, Herman T., 18 Lawrence Farm Road, Woods Hole, MA 02543 Epstein, Ray L., 1602 W. Olympia Street, Hemando. FL 34442 Farb, David H.. Boston University School of Med., Dept. of Pharmacology L603. 80 East Concord Street, Boston, MA 021 18 Farmanfarmaian, A., Rutgers University. Dept. of Biological Sciences. Nelson Biology Lab. POB 1059. Piscataway, NJ 08855 Feldman, Susan C., University of Medicine & Dentistry, New Jersey Medical School, 100 Bergen Street, Newark, NJ 07103 Festoff, Barry William, VA Medical Center, Neurology Service (151). 4801 Linwood Blvd., Kansas City. MO 64128 Fink, Rachel D., Mount Holyoke College, Dept. 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., Harvard Medical School, Neurobiology Department, 220 Longwood Avenue, Boston. MA 021 15 Fishman, Harvey M., University of Texas Medical Branch, Dept. of Physiology & Biophys., 301 University Blvd.. Galveston, TX 77555-0641 Flanagan, Dennis, 12 Gay Street, New York. NY 10014 Fluck, Richard Allen, Franklin & Marshall College, Department of Biology, Box 3003, Lancaster, PA 17604-3003 Foreman, Kenneth H., Marine Biological Laboratory. The Ecosystems Center, Woods Hole, MA 02543 Fox, Thomas Oren, Harvard Medical School, Division of Medical Sciences, 260 Longwood Avenue, Boston, MA 02115 Franzini-Armstrong, Clara, University of Pennsylvania, School of Medicine, 330 S. 46th Street, Philadelphia, PA 19143 Frazier, Donald T., LIniversity of Kentucky Medical Center, Dept. of Physiology & Biophysics, Lexington, KY 40536 French, Robert J., University of Calgary, Health Sciences Centre, Alberta. T2N 4N1, Canada Fulton, Chandler M., Brandeis University. Department of Biology, Waltham, MA 02254 Furie, Barbara C., Beth Israel Deaconess Medical Center, B1DMC Cancer Center. Kirstein 1, 330 Brookline Avenue, Boston. MA (12215 Furie, Bruce, Beth Israel Deaconess Medical Center. B1DMC Cancer Center. Kirstein 1, 330 Brookline Avenue, Boston, MA 02215 Furshpan, Edwin J., Harvard Medical School. Department of Neurophysiology, 220 Longwood Avenue, Boston, MA 02115 Fulrelle, Robert P., Northeastern University, College of Computer Science, 360 Huntington Avenue, Boston, MA 021 15 Gabriel, Mordecai L., Brooklyn College, Department of Biology, 2900 Bedford Avenue, Brooklyn, NY 11210 Gadsby, David C., The Rockefeller University, Laboratory of Cardiac Physiol., 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., 180 North Michigan Avenue. Suite 2401, Chicago, IL 60601 Gall, Joseph G., Carnegie Institution, 115 W. University Parkway. Baltimore. MD 21210 Garber, Sarah S., Medical College of Pennsylvania, Dept. 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, AT & T Bell Labs. Dept. of Biophysics, Rm. 1C464. 600 Mountain Avenue. Murray Hill, NJ 07974 German, James L., The New York Blood Center, Lab. 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 Giblin, Anne E., Marine Biological Laboratory, The Ecosystems Center. Woods Hole. MA 02543 Gibson, A. Jane, Cornell University. Dept. of Biochemistry, Biotech. Building, Ithaca, NY 14850 Gifford, Prosser, 540 N Street, SW, Apt. #S-903. Washington, DC 20024-4557 Gilbert, Daniel L., NIH/NINDS, Biophys. Sec.. BNP, Bldg. 36. Rm 5A-27, Bethesda. MD 20892 Giudice, Giovanni, L'niversita di Palermo. Dipartimento di Biologia, Cellulare e Dello Sviluppo. 1-90123 Palermo, Italy Giuditta, Antonio, University of Naples, Dept. of Gen. Physiology, Via Mezzocannone 8. Naples. 80134. 80134 Italy Glynn, Paul, P.O. Box 6083, Brunswick, ME 0401 1-6083 Golden, William T., Golden Family Trust, Room 4201. 42nd Floor, 40 Wall Street, New York, NY 10005 Goldman, Robert D., Northwestern University Medical School, Dept. of Cell & Molecular Biology, 303 E. Chicago Avenue, Chicago. IL 60611-3008 Goldsmith, Paul K., National Institutes of Health. Bldg. 10. Room 9C-101, Bethesda, MD 20892 Goldsmith, Timothy H., Yale University, Department of Biology, New Haven. CT 06510 Goldstein, Moise H., Johns Hopkins University. ECE Department, Barton Hall, Baltimore, MD 21218 Goodman, Lesley Jean, Queen Mary College, Dept. of Biological Sciences. Mile End Road. London El 4NS, England. UK Gould, Robert Michael, Institute for Basic Research in R70 Annual Report Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314-6399 Govind, C. K., Scarborough College, Life Sciences Division, 1265 Military Trail. West Hill, Ontario, MIC 1A4 Canada Grace, Dick, Doreen Grace Fund, The Brain Center, Promontory Point, New Seabury, MA 02649 Graf, Werner M., College of France, 1 1 Place Marcelin Berthelot, 75231 Paris Cedex 05, France Grant, Philip, National Institutes of Health, NINDS. BN, DIR, Neurochemistry, Bldg. 36, Rm. 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 & Coastal Studies, Box 231, New Brunswick, NJ 08903 Graubard, Katherine G., University of Washington, Dept. of Zoology, NJ-15. Box 351800, Seattle, WA 98195-1800 Greenberg, Everett Peter, University of Iowa, College of Medicine, Dept. of Microbiology, Iowa City, IA 52242 Greenberg, Michael J., University of Florida, The Whitney Laboratory, 9505 Ocean Shore Boulevard, St. Augustine, FL 32086-8623 Greer, Mary J., 176 West 87 Street, #12A, New York, NY 10024 Griffin, Donald R., Harvard University. Concord Field Station, Old Causeway Road. Bedford. MA 01730 Gross, Paul R., 53 Two Ponds Road, Falmouth, MA 02540 Grossman, Albert, New York University Medical Center, 550 First Avenue, New York, NY 10016 Grossman, Lawrence, Johns Hopkins University, Dept. of Biochemistry. 615 North Wolfe Street, Baltimore, MD 21205 Gruner, John A., Cephalon, Inc., 145 Brandywine Parkway, W. Chester. PA 19380-4245 Gunning, A. Robert, P.O. Box 165, Falmouth, MA 02541 Gwilliam, G. F., 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, Dept. of Biochemistry and Molecular Genetics, 1918 University Blvd.. Birmingham, AL 35294 Hall, Linda M., SUNY at Buffalo, Dept. of Biochem. Pharmacol., 329 Hochstetter Hall, Buffalo, NY 14260-3850 Hall, Zach W., NINDS/NHS, Office of the Director, Bldg. 31-8A52, Rockville Pike, Bethesda, MD 20892-2540 Halvorson, Harlyn O., Policy Center for Marine Biosciences & Technology, UMass Boston, 100 Morrissey Blvd., Boston, MA 02125-3393 Haneji, Tatsuji, Kyushu Dental College, Dept. of Anatomy, 2-6-1, Manazuru, Kokurakita-Ku, Kitakyushu 803, Japan Hanlon, Roger T., Marine Biological Laboratory, Marine Resources Center, 7 MBL Street, Woods Hole, MA 02543-1015 Harosi, Ferenc, Marine Biological Laboratory, Laboratory of Sensory Physiology, Woods Hole, MA 02543 Harrigan, June F., 7415 Makaa Place, Honolulu, HI 96825 Harrington, Glenn W., Weber State University, Dept. of Microbiology, Ogden, UT 84408 Harrison, Stephen C., Harvard University, Dept. of Molecular & Cell Biology, 7 Divinity Avenue, Cambridge, MA 02138 Haselkorn, Robert, University of Chicago, Dept. of Molecular Genetics & Cell Biology, Chicago, IL 60637 Hastings, J. Woodland, Harvard University, The Biological Laboratories, 16 Divinity Street, Cambridge, MA 02138-2020 Haydon-Baillie, Wensley G., Porton Int.. 2 Lowndes Place, London SW1 X8D, England, UK Hayes, Raymond L., Howard University, College of Medicine, 520 W Street. NW, Washington. DC 20059 Heck, Diane E., EOHS1. Dept. of Pharmacology and Toxicol., 681 Frelinghuysen Road, Piscataway, NJ 08855 Henry, Jonathan Joseph, University of Illinois, Dept. of Cell & Struct. Biol., 505 South Goodwin Avenue, Urbana, IL 61801 Hepler, Peter K., University of Massachusetts, Department of Biology, Morrill III, Amherst, MA 01003 Herndon, Walter R., University of Tennessee, Department of Botany, Km.xville, TN 37996-1100 Herskovits, Theodore T., Fordham University, Dept. of Chemistry, John Muleahy Hall, Rm. 638, Bronx, NY 10458 Hiatt, Howard H., Brigham and Women's Hospital, Department of Medicine, 75 Francis Street, Boston, MA 021 15 Highstein, Stephen M., Washington University School of Medicine. Dept. of Otolaryngology, Box 8115, 4566 Scott Avenue, St. Louis, MO 63110 Hildebrand, John G., University of Arizona, ARL Div. of Neurobiology, 603 Gould-Simpson Sci. Bldg., Tucson, AZ 85721 Hill, Richard W., Michigan State University. Department of Zoology. East Lansing, MI 48824 Hill, Susan. Michigan State University. Department of Zoology, East Lansing, MI 48824 Hillis, Llewellya W., Smithsonian Tropical Research Institute, Unit 0948 APO, AA 34002-0948 USA Hinegardner, Ralph T., University of California, Division of Natural Sciences, Santa Cruz, CA 95064 Hinsch, Gertrude W., University of South Florida. Department of Biology, Tampa, FL 33620 Hinsch, Jan, Leica, Inc., 110 Commerce Drive, Allendale. NJ 07401 Hobble, John E., Marine Biological Laboratory, The Ecosystems Center, Woods Hole, MA 02543 Hodge, Alan J., 3843 Mt. Blackburn Avenue. San Diego, CA 92111 Hoffman, Joseph F., Yale University School of Medicine, Dept. of Cellular and. Molecular Physiology, New Haven, CT 06520 Holz, George G., Massachusetts General Hospital, Lab. of Molec. Endocrinology. Wellman 320, 50 Blossom St., Boston, MA 021 14 Houk, James C., Northwestern University Medical School, 303 E. Chicago Ave.. Ward 5-315. Chicago, IL 6061 1-3008 Hoy, Ronald R., Cornell University, Section of Neurobiology & Behavior, 215 Mudd Hall, Ithaca, NY 14853 Huang, Alice S., California Institute of Technology, Mail Code 1-9, Pasadena, CA 9 1 1 25 Hufnagel-Zackroff, Linda A., University of Rhode Island, Department of Microbiology, Kingston, RI 02881 Hummon, William D., Ohio University, Dept. of Biological Sciences, Athens. OH 45701 Humphreys, Susie H., Food and Drug Administration. HFS-308. 200 C Street, SW. Washington, DC 20204-0001 Humphreys, Thomas D., University of Hawaii, Kewalo Marine Lab., 41 Ahui Street, Honolulu, HI 96813 Hunt, Richard T., ICRF, Clare Hall Laboratories, South Minims Potter's Bar, Herb EN6-3LD, England, UK Hunter, Robert D., Oakland University. Dept. of Biological Sciences, Rochester, MI 48309-4401 Huxley, Hugh E., Brandeis University, Rosenstiel Center. Biology Department. Waltham, MA 02154 Ilan, Joseph, Case Western Reserve University, School of Medicine, Department of Anatomy, Cleveland, OH 44106 Ingoglia, Nicholas A., New Jersey Medical School. Dept. of Pharmacology/Physiology, 185 South Orange Avenue, Newark. NJ 07103 Members of the Corporation R71 Inoue, Saduyki, McGill University, Dept. of Anatomy, 3640 University Street, Montreal. PQ H3A 2B2. Canada Inoue, Shinya, Marine Biological Laboratory, 7 MBL Street. Woods Hole, MA 02543 Isselbacher, Kurt J., Massachusetts General Hospital, Cancer Center, Charlestown, MA 02129 Issidorides, Marietta Radovic, University of Athens, Department of Psychiatry, Monis Petraki 8, Athens, 140, Greece Izzard, Colin S., SUNY. Albany. Dept. of Biological Sciences, 1400 Washington Avenue, Albany, NY 12222 Jacobs, Neil, Hale & Dorr, 60 State Street, Boston, MA 02109 Jaffe, Laurinda A., University of Connecticut Health Center, Dept. of Physiology, Farmmgton Avenue, Farmington, CT 06032 Jaffe, Lionel, Marine Biological Laboratory. Woods Hole. MA 02543 Jannasch, Holger W., Woods Hole Oceanographic Institution, Department of Biology, Woods Hole, MA 02543 Jeffery, William R., Penn State University, Dept. of Biology, 208 Mueller Lab, University Park, PA 16802 Johnston, Daniel, Baylor College of Medicine, Division of Neuroscience, 1 Baylor Plaza. Houston. TX 77030 Josephson, Robert K., University of California. Irvine, School of Biological Science, Dept, of Psychobiology, Irvine, CA 92697 Kaczmarek, Leonard K., Yale University School of Medicine, Dept. 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 of Biological Sciences, South Hadley, MA 01075 Kaminer, Benjamin, Boston University Medical School, Physiology Dept., 80 East Concord Street, Boston, MA 02118 Kaneshiro, Edna S., University of Cincinnati, Biological Sciences Department, JL 006, Cincinnati. OH 45221-0006 Kaplan, Ehud, Mt. Sinai School of Medicine. Dept. of Ophthalmology, 1 Gustave Levy Place, Box 1183, New York, NY 10029 Karakashian, Stephen J., Apartment 16-F. 165 West 91st Street. New York. NY 10024 Karlin, Arthur, Columbia University, Ctr. for Molecular Recognition, 630 West 168th St., Rm. 11-401. New York, NY 10032 Keller, Hartmut Ernst, Carl Zeiss, Inc.. One Zeiss Drive. Thorn wood, NY 10594 Kelley, Darcy B., Columbia University, Dept. of Biological Sciences, 911 Fairchild, Mailcode 2432, New York. NY 10027 Kelly, Robert E., 5 Little Harbor Road, Woods Hole, MA 02543 Kemp, Norman E., University of Michigan. Department of Biology. Ann Arbor. MI 48109 Kendall, John P., Faneuil Hall Associates. 176 Federal Street, 2nd Fir, 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, Dept. of Physiol. & Biophysics, 1300 Morris Park Avenue. Bronx. NY 10461 Khodakhah, Kamran, University of Pennsylvania, Dept. of Physiology, Philadalphia, PA 19104 Kiehart, Daniel P., Duke University Medical Center, Dept. of Cell Biol., Box 3709, 307 Nanaline Duke Bldg., Durham, NC 2771(1 Kl< inlc Id. David, University of California, San Diego. Physics Department, 0319. 9500 Gilman Drive. La Jolla, CA 92093 Klessen, Rainer, Carl Zeiss, Inc., 1 Zeiss Drive, Thornwood, NY 10594 Klotz, Irving M., Northwestern University. Department of Chemistry. Evanston, IL 60201 Knudson, Robert A., Marine Biological Laboratory. Instrument Development Laboratory, Woods Hole, MA 02543 Koide, Samuel S., The Rockefeller University, The Population Council, 123(1 York Avenue, New York, NY 10021 Kornberg, Hans, 134 Sewall Ave., #2, Brookline, MA 02146 Kosower, Edward M., Tel-Aviv University, Dept. of Chemistry, Ramat-Aviv, Tel Aviv. 69978, Israel Krahl, Maurice E., 2783 W. Casas Circle. Tucson, AZ 85741 Krane, Stephen M., Massachusetts General Hospital, Arthritis Unit, Fruit Street, Boston, MA 021 14 Krauss, Robert, P.O. Box 291, Demon. MD 21629 Kravitz, Edward A., Harvard Medical School, Dept. of Neurobiology, 220 Longwood Avenue, Boston, MA 021 15 Kriebel, Mahlon E., SUNY Health Science Center, Dept. of Physiology, Syracuse, NY 13210 Kristan, William B., University of California, San Diego, Dept. of Biology. B-0357. La Jolla, CA 92093-0357 Kropinski, Andrew M., Queen's University, Dept. of Microbiol./ Immunology. Kingston. Ontario K7L 3N6, Canada Kuffler, Damien P., Insitute of Neurobiology, 201 Blvd. del Valle, San Juan, PR 00901 Kuhns, William J., Hospital tor Sick Children, Biochemistry Research, 555 University Ave., Toronto. Ontario. M5G 1X8 Canada Kunkel, Joseph G., University of Massachusetts, Dept. of Biology. Amherst. MA 01003 Kusano, Kiyoshi, National Institutes of Health, Bldg. 36, Room 4D- 20, Bethesda, MD 20892 Kuzirian. Alan M., Marine Biological Laboratory, Woods Hole, MA 02543-1015 Laderman, Aimlee D., Yale University, School of Forestry & Environmental Studies, 370 Prospect Street. New Haven, CT 06511 Landeau, Laurie J., Listowel, Inc., 2 Park Avenue, Suite 1525. New York. NY 10016 Landis, Dennis M.D., University Hospital of Cleveland, Dept. of Neurology, 1 1 100 Euclid Ave., Cleveland, OH 44106 Landis, Story C., National Institutes of Health, Bldg. 36, Room 5A05. 36 Convent Drive, Bethesda, MD 20892-4150 Landowne, David, University of Miami Medical School, Dept. of Physiology. PO Box 016430, Miami. FL 33101 Langford, George M., Dartmouth College. Dept. of Biological Sciences, 6044 Gilman Laboratory, Hanover, NH 03755 Laskin. Jeffrey, University of Medicine & Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854 Lasser-Ross, Nechama, New York Medical College. Dept. 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. Dept. of Molec. & Cell Biol., U-125, 75 North Eagleville Rd., Storrs, CT 06269-3125 Lazarow, Paul B., Mt. Sinai School of Medicine, Dept. of Cell Biol. & Anatomy. Box 1007, 5th Ave. & 100th St.. New York, NY 10029 R72 Annual Report Lazarus, Maurice, Federated Department Stores, Sears Crescent. City Hall Plaza. Boston. MA 02108 Leadbetter, Edward R., University of Connecticut. Dept. of Molec. & Cell Biology. U-131. Storrs, CT 06266-2131 Lederberg, Joshua, The Rockefeller University, 1230 York Avenue. New York. NY 10021 Lee, John J., City College of CUNY, Department of Biology, Convent Avenue & 138th Street. New York. NY 10031 Lehy, Donald B., 35 Willow Field Drive, No. Fulmouth, MA 02556 Leibovitz, Louis, 3 Kettle Hole Road. Falmouth. MA 02540 Leighton, Joseph, Aeron Biotechnology. Inc.. 1933 Davis Street #310. San Leandro, CA 94577 Ltighton, Stephen B., National Institutes of Health. Bldg. 13. 3W13. Bethesda, MD 20892 Lemos, Jose Ramon, University of Massachusetts Medical Center. Worcester Foundation Campus, 222 Maple Avenue, Shrewsbury. MA 01 545-2737 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, 111 H2. 4150 Clement Street, San Francisco. CA 94121 Levine, Michael, University of California, Molecular & Cell Biology, 401 Barker Hall #3204. Berkeley. CA 92093-3204 Levine, Richard B., Univ. of Arizona. ARL, Div. of Neurobiology. Room 611, Gould-Simpson Bldg.. Tucson, AZ 85721 Levinthal, Francoise, Columbia University, Dept. of Biological Sciences, Broadway & 1 16th Street. New York. NY 10026 Levitan, Herbert, National Science Foundation. 4201 Wilson Boulevard, Room 835, Arlington, VA 22230 Levitan, Irwin B., Brandeis University, Volen Center for Complex Sys., 415 South Street. Waltham. MA 02254 Linck, Richard W., University of Minnesota, School of Medicine. Cell Biology & Neuroanatomy Dept.. 4-135 Jackson Hall, 321 Church St.. Minneapolis. MN 55455 Lipicky, Raymond J., FDA/CDER/ODEI/ HFD-1 10, 5600 Fishers Lane, Rockville, MD 20857 Lisman, John E., 199 Coolidge Avenue, #902. Watertown, MA 02172-1572 Liuzzi. Anthony, 320 Beacon Street. Boston, MA 021 16 Llinas, Rodolfo R., NYU Medical Center, Dept. of Physiology & Neurosc., 550 First Ave, Rm 442. New York. NY 10016 Lobel, Phillip S., Boston University Marine Program. Marine Biological Laboratory. Woods Hole, MA 02543 Loew, Franklin M., Medical Foods, Inc.. 5 Cambridge Ctr. Ste. 8, Cambridge, MA 02142 Loewenstein, Birgit Rose, Marine Biological Laboratory. Woods Hole, MA 02543 Loewenstein, Werner R., Marine Biological Laboratory, Woods Hole, MA 02543 London. Irving M., Johnson & Johnson. Harvard-MIT Division, E- 25-551, Cambridge. MA 02139 Longo, Frank J., University of Iowa, Department of Anatomy, Iowa City. IA 52442 I 01 .UK], Laszlo, Northwestern University Medical School, CMS Biology. Searle 4-555. 303 East Chicago Avenue, Chicago. IL 6061 I-300X Luckenbill, Louise M., Ohio University, Dept. of Biological Sciences, Irvine Hall. Athens, OH 45701 Macagno, Eduardo R., Columbia University, Department of Biosciences, 1003B Fairchild. New York. NY 10027 MacNichol, Edward E., Boston University School of Medicine, Dept. of Physiol.. HO E. Concord Street, Boston, MA 021 IS Maglott-Dufh'eld, Donna R., American Type Culture Collection. 12301 Parklawn Dnve. Rockville. MD 20852-1776 Maienschein, Jane Ann, Arizona State University. Department of Philosophy. P.O. Box 872004. Tempe, AZ 85287-2004 Mainer, Robert E., The Boston Company. Inc.. One Boston Place, OBP-15-D, Boston. MA 02108 Malbon, Craig C., SUNY, Stony Brook. Univ. Medical Center. Pharmacology-HSC, Stony Brook. NY 11794-8651 Manalis, Richard S., Indiana-Purdue University, Dept. of Biological Science, 2101 Coliseum Blvd. E.. Fort Wayne, IN 46805 Mangum, Charlotte P., College of William and Mary, Department of Biology, Williamsburg. VA 23187-8795 Manz, Robert D., 304 Adams Street, Milton, MA 02186 Margulis, Lynn, University of Massachusetts, Amherst, Dept. of Geosciences, Morrill Science Center, Box 35820. Amherst, MA 01003-5X20 Marinucci, Andrew C., 102 Nancy Drive, Mercerville. NJ 08619 Martinez, Joe L., University of Texas, San Antonio, 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, Hospital of University of Pennsylvania, 106 Dulles. 3400 Spruce Street, Philadephia, PA 19104-4283 Mauzerall, David, Rockefeller University, 1230 York Avenue, New York. NY 10021 McCann, Erances V., Dartmouth Medical School, Department of Physiology. Lebanon. NH 03756 McLaughlin, Jane A., Marine Biological Laboratory, Woods Hole, MA 02543 McMahon, Robert F., University of Texas, Arlington, Department of Biology. Box 19498. Arlington. TX 76019 Meedel, Thomas, Rhode Island College, Biology Dept., 600 Mt. Pleasant Avenue, Providence, RI 02908 Meinertzhagen, Ian A., Dalhousie University, Dept. Psychology, Halifax. NS B3H 4J1. Canada Meiss, Dennis E., Immunodiagnostic Laboratories. 488 McCormick Street. San Leandro, CA 94577 Melilln, Jerry M., Marine Biological Laboratory, The Ecosystems Center. Woods Hole, MA 02543 Mellon. DeEorest, 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., Harvard Medical School, Cardiovascular Division, 75 Francis Street, Boston, MA 021 15 Merriman, Melanie Pratt, 751 1 Beachview Dr.. N. Bay Village, FL 33141 Meselson, Matthew, Harvard University, Fairchild Biochem. Building. 7 Divinity Avenue. Cambridge, MA 02138 Metuzals, Janis, University of Ottawa. Dept. of Pathology & Lab. Med.. 451 Smyth Rd.. Ottawa. Ontario. KIH 8M5 Canada Miledi, Ricardo, University of California. Irvine. Dept. of Psychobiology. 2205 Bio. Sci. II. Irvine, CA 92697-4550 Milkman, Roger D., University of Iowa. Dept. of Biological Sciences, 138 Biology Building. Iowa City. IA 52242-1324 Miller, Andrew L., Hong Kong University. Dept. of Biology. Clearwater Bay. Kowloon, Hong Kong Mills. Robert, 10315 44th Avenue, W. 12 H Street. Brandenton. FL 34210 Misevic, Gradimir, University Hospital of Basel, Dept. ot Research, Mehelstr. 20, CH-4031 Basel, Switzerland Mitchell, Ralph, Harvard University, Division of Applied Sciences, 29 Oxford Street, Cambridge, MA 02138 Members of (he Corporation R73 Mivakawa, Hiroyoshi, Tokyo College of Pharmacy. Lab. of Cellular Neurobiology, 1432-1 Horinouchi, Hachiouji. Tokyo 192-03, Japan Miyamoto, David M., Drew University, Department of Biology, Madison, NJ 0794(1 Mizell, Merle, Tulane University, Dept. of Cell & Molecular Biology, New Orleans, LA 701 18 Moore, John W., Duke University Medical Center, Department of Neurobiology, Box 3209, Durham, NC 27710 Moreira, Jorge E., NIH/NICHD, Dept. of Cell and Molec. Biol., Bethesda, MD 20852 Morin, James G., University of California, Department of Biology. Los Angeles. CA 90024 Morrell, Leyla de Toledo, Rush-Presbyterian-St. Lukes Medical Center, 1653 West Congress Parkway, Chicago. [L 60612 Morse, M. Patricia, Northeastern University, Marine Science Center, Nahant, MA 01908 Morse, Stephen $., DARPA/DSO, 3701 N. Fairfax Drive. Arlington, VA 22203-1714 Mote, Michael I., Temple University, Department of Biology, Philadelphia, PA 19122 Muller, Kenneth J., University of Miami Medical School. Dept. of Physiol./Biophysics. RMSB-5092 1600 NW 10th Avenue, Miami, FL 33136 Murray, Andrew W., University of California, Dept. of Physiology. Box 0444. 513 Parnassus Avenue, San Francisco, CA 94143-0444 Nabrit, S. M., 686 Beckwith Street, SW. Atlanta. GA 30314 Nadelhoffer, Knute J., Marine Biological Laboratory, The Ecosystems Center, Woods Hole, MA 02543 Naka, Ken-ichi, 2-9-2 Tatumi Higashi. Okazaki. 444, Japan Nakajima, Yasuko, University of Illinois, College of Medicine, Anat. & Cell Biol. Dept.. M/C 512, Chicago, IL 60612 Narahashi, Toshio, Northwestern University Medical School. Dept. of Pharmacology. 303 E. Chicago Avenue. Chicago. IL 6061 1 Nasi, Enrico, Boston University School of Medicine, Dept. of Physiology, R-406, 80 E. Concord Street, Boston, MA 021 18 Neill, Christopher, Marine Biological Laboratory, The Ecosystems Center, Woods Hole. MA 02543 Nelson, Leonard, Medical College of Ohio, Department of Physiology, CS 10008, Toledo, OH 43699 Nelson, Margaret C., Cornell University, Section of Neurobiology, and Behavior, Ithaca, NY 14850 Nicholls, John G., Biocenter, Klingelbergstrasse 70, 4056 Basel, Switzerland Nickerson, Peter A., SUNY, Buffalo, Dept. 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, Dept. of Anatomy & Cell Biol., Atlanta. GA 30322 Northcutt, R. Glenn, University of California, San Diego, Neuroscience 0201, 9500 Gilman Drive, La Jolla, CA 92093-0201 Norton, Catherine N., Marine Biological Laboratory, Woods Hole, MA 02543 Nusbaum, Michael P., University of Pennsylvania School of Medicine, Dept. of Neuroscience, 215 Stemmler Hall. Philadelphia, PA 19104-6074 O'Herron, Jonathan, Lazard Freres & Co.. 30 Rockefeller Plaza. 59th Fir, 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, Buffalo. Dept. of Biophysical Sciences, 224 Cary Hall, Buffalo, NY 14214 Oklenhoiirg, Rudolf, Marine Biological Laboratory. 7 MBL Street, Woods Hole, MA 02543 Olds, James L., American Association of Anatomists, 9650 Rockville Pike, Ste. 4514, Bethesda, MD 20814-3998 Olins, Ada L., University of Tennessee, Oak Ridge, Grad. School of Biomed. Sci.. Biology Div. ORNL-POB 2009, Oak Ridge. TN 37831-8077 Olins, Donald E., University of Tennessee, Oak Ridge, Grad. School of Biomed. Sci.. Biology Division ORNL-POB 2009. Oak Ridge. TN 37831-8077 Oschman, James L., 31 Whittier Street, Dover, NH 03820 Palazzo, Robert E., University of Kansas, Dept. of Physiol. & Cell Biol., Haworth Hall, Lawrence, KS 66045 Palmer, John D., University of Massachusetts, Department of Zoology, 221 Morrill Science Center, Amherst, MA 01003 Pant, Harish C., NINCDS/NIH. Lab. of Neurochemistry, Building 36, Room 4D20. Bethesda. MD 20892 Pappas, George D., University of Illinois. College of Medicine, Department of Anatomy. Chicago. IL 606 1 2 Pardee, Arthur B., Dana-Farber Cancer Institute, D810, 44 Binney Street, Boston. MA 02115 Pardy, Rosevelt L., University of Nebraska, School of Life Sciences, Lincoln, NE 68588 Parmentier, James L., 175 S. Great Road, Lincoln, MA 01773-41 12 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, The Ecosystems Center. Woods Hole. MA 02543 Pethig, Ronald, University College of No. Wales. School of Electronic Eng. Sci., Bangor, Gwynedd. LL 57 IUT. UK Pfohl, Ronald J., Miami University. Department of Zoology, Oxford, OH 45056 Pierce, Sidney K., University of Maryland, Department of Zoology. College Park. MD 20742 Pleasure, David E., Children's Hospital, Neurology Research, 5th Floor. Ambramson Building. Philadelphia, PA 19 104 Poindexter, Jeanne S., Barnard College, Columbia University, 3009 Broadway, New York, NY 10027-6598 Pollard, Harvey B., NIH/NIDDKD, Lab. of Cell Biol. & Genetics. Bldg. 8. Rm. 401. Bethesda. MD 20892 Pollard, Thomas D., Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037 Porter, Beverly H., 5542 Windysun Ct., Columbia, MD 21045 Porter, Mary E., University of Minnesota Medical School, Dept. of Cell Biol. & Neuroanatomy, 4-135 Jackson Hall. 321 Church St. 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, UK Powers, Maureen K., Vanderbilt University, Dept. of Psychology, 301 Arts & Sci. Psych. Bldg, Nashville. TN 37240 Prendergast, Robert A., Wilmer Institute. Johns Hopkins Hospital, 457 Wilmer-Woods Building, 600 N. Wolfe Street, Baltimore. MD 21287-9142 Price, Carl A., Rutgers University, Waksman Inst. of Microbiology. P.O. Box 759, Piscataway, NJ 08855-0759 R74 Annual Report Prior, David J.. Northern Arizona University, Arts and Sci. Dean's Office. Box 5621. Flagstaff. AZ 86011 Prusch, Robert D., Gonzaga University, Department of Life Sciences, Spokane, WA 99258 Purves, Dale, Duke University Medical Center, Dept. of Neurobiology, B\ 3209, 1011 Bryan Res. Bldg., Durham. NC 27710 Quigley, James P., SUNY Health Sciences Center, Dept. of Pathology, BHS Tower 9, Rm. 140, Stony Brook, NY 11794-8691 Rottenl'usser. Rudi, Carl Zeiss. Inc.. Marine Biological Laboratory. Woods Hole. MA 02543 Rowland, Lewis P., Neurological Institute, 710 West 168th Street, New York, NY 10032 Ruderman, Joan V., Harvard Medical School, Dept. of Cell Biology. 240 Longwood Avenue, Boston, MA 021 15 Rummel, John D., Marine Biological Laboratory. Woods Hole. MA 02543 Rushl'orth, Norman B., Case Western Reserve University, Department of Biology. Cleveland, OH 441 Oh Russell-Hunter, W. D., 711 Howard Street, Easton, MD 21601-3934 Rabb, Irving W., 1010 Memorial Drive, Cambridge, MA 02138 Rabin, Harvey, P.O. Box 4022, Wilmington, DE 19807 Rabinowitz, Michael B., Marine Biological Laboratory, 7 MBL St.. Woods Hole. MA 02543 Rafferty, Nancy S., Marine Biological Laboratory, 7 MBL, Woods Hole, MA 02543 Rakowski, Robert F., UHS/The Chicago Medical School, Dept. of Physiol. & Biophysics. 3333 Greenbay Road, N. Chicago, IL 60064 Ramon, Fidel, UNAM-CU, Div. Est. Posgrado E Invest., Facultad de Medicina, 04510, D.F., Mexico Ranzi, Silvio, Sez. Zoologia Scienze Naturali. Dip. Di Biologia, Via Celoria, 26, 20133 Milano, Italy 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. Dept. of Biological Sciences, Rochester. Ml 48309-44(11 Reese, Thomas S., NIH. Bldg. 36. Room 2A21, 9000 Rockville Pike. Bethesda. MD 20892 Reinisch, Carol L., Marine Biological Laboratory, 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, Woods Hole, MA 02543 Ripps. Harris, University of Illinois at Chicago, Dept. of Ophthal/ Vis. Sci., 1855 West Taylor Street. Chicago. IL 60612 Ritchie, J. M., Yale University School of Medicine. Dept. of Pharmacology, 333 Cedar Street. New Haven, CT 06510 Rome, Lawrence C., University of Pennsylvania, Dept. of Biology, Philadelphia, PA 19104 Rosenbaum, Joel L., University of Pennsylvania School of Medicine, c/o Brian Salzberg, Dept. of Physiology. Philadelphia, PA 19104 Rosenbluth, Jack, New York University School of Medicine, Dept. of Physiology, RF 714. 40(1 East 34th Street, New York, NY 10016 Rosenbluth, Raja. Simon Fraser University, Inst. of Molec. Biology & Biochem.. Burnaby, BC, Canada, V5A 1S6 Rosenfield, Allan, Columbia University School of Public Health. 600 West 168th Street, New York. NY 10032-3702 Rosenkranz, Herbert S., University of Pittsburgh. Dept. of Environ. & Occup. Hlth.. 260 Kappa Drive, Pittsburgh. PA 15238 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 Ross, William N., New York Medical College, Department of Physiology, Valhalla, NY 10595 Roth, Jay S., P.O. Box 692. Woods Hole. MA 02543-0692 Saffo, Mary Beth, Arizona State University, Life Sci. Dept., MC 2352. P.O. Box 37100, Phoenix. AZ 85069-7100 s.il.iin.i, Guy, University of Pittsburgh. Department of Physiology. Pittsburgh. PA 15261 Salmon, Edward D., University of North Carolina, Dept. of Biology. Wilson Hall. CB 3280, Chapel Hill, NC 27599 Salyers, Abigail. University of Illinois, Dept. of Microbiology. 407 S. Goodwin Avenue, Urbana. IL 61801 Salzberg, Brian M., University of Pennsylvania School of Medicine. Dept. of Neuroscience. 215 Stemmler Hall. Philadelphia. PA 19104-6074 Sanger, Jean M., University of Pennsylvania School of Medicine. Dept. of Anatomy, 36th and Hamilton Walk, Philadelphia, PA 19104 Sanger, Joseph W., University of Pennsylvania Medical Center, Dept. of Cell and Devel. Biol.. 36th and Hamilton Walk. Philadelphia. PA 19104-6058 Saunders, John W., 1 18 Metoxit Road. P.O. Box 3381, Waquoit. MA 02536 Schachman, Howard K., LIniversity of California, Berkeley, Molecular & Cell Biology Dept., 229 Stanley Hall. #3206. Berkeley. CA 94720-3206 Schatten, Gerald P., University of Wisconsin. 1117 W. Johnson Street. Madison. WI 53706 Schatten, Heide, University of Wisconsin. Department of Zoology, Madison, WI 53706 Schmeer, Arlene C., Mercenene Cancer Research Institute. 790 Prospect Street. New Haven. CT 06511 Schuel, Herbert, SUNY, Buffalo, Dept. of Anatomy/Cell Biology, Buffalo, NY 14214 Schwartz, James H., New York State Psychiatric Institute. Research Annex, 722 West 168lh St.. 7th floor. New York. NY 10032 Schwartz, Lawrence, University of Massachusetts. Amherst, Department of Biology, Morrill Science Center. Amherst, MA 01003 Schweitzer, A. Nicola, Brigham & Women's Hospital, Immunology Division. Dept. of Pathology. 221 Longwood Ave., LMRC 521. Boston. MA 021 15 Segal, Sheldon J., The Population Council. One Dag Hammarskjold Plaza. New York. NY 10036 Shanklin, Douglas R., University of Tennessee, Dept. of Pathology. Rm. 576, 800 Madison Avenue. Memphis, TN 381 17 Shashoua, Victor E., Harvard Medical School. Ralph Lowell Labs. McLean Hospital, 115 Mill St.. Belmont. MA 02178 Shaver, Gains R., Marine Biological Laboratory. The Ecosystems Center, Woods Hole. MA 02543 Shaver, John R., Michigan State University, Dept. of Zoology. East Lansing, MI 48824 Sheet/, Michael P., Duke University Medical Center, Dept. of Cell Biology. Bx 3709, 388 Nanaline Duke Bldg., Durham. NC 27710 Shepro, David, Boston University, CAS Biology, 5 Cummington Street, Boston, MA 02215 Members of the Corporation R75 Shimomura, Osamu, Marine Biological Laboratory. Woods Hole, MA 02543 Shipley, Alan M., P.O. Box 2036, Sandwich. MA 02563 Silver, Robert B., Marine Biological Laboratory, Woods Hole, MA 02543 Siwicki, Kathleen K., Swarthmore College, Biology Department, 500 College Avenue, Swarthmore, PA 19081-1397 Skinner, Dorothy M., Oak Ridge National Laboratory, Biology Division, P.O. Box 2009, Oak Ridge. TN 3783 I Slohoda. Roger D., Dartmouth College, Dept. of Biological Sciences, 6044 Oilman Laboratory, Hanover. NH 03755 Sluder, Greenfield, University of Massachusetts Medical Center, Worcester Foundation Campus, 222 Maple Avenue. Shrewsbury, MA 01545 Smith. Peter J. S., Mamie Biological Laboratory, Woods Hole, MA 02543 Smith, Stephen J., Stanford University School of Medicine, Dept. of Cell. & Molec. Phys.. Beckman Center, Stanford. CA 94305-5426 Smolowitz, Roxanna S., Marine Biological Laboratory, Laboratory of Aquatic Animal Medicine, Woods Hole, MA 02543 Sogin, Mitchell L., Marine Biological Laboratory. Woods Hole. MA 02543 Sorenson, Martha M., Cidade Universitaria-UFRJ, Dept. Bioquimica Medica-ICB, 21941-590 Rio de Janerio, Brazil Speck, William T., Columbia-Presbyterian Medical Center. 161 Fort Washington Avenue. 14th Floor. Room 1470, New York, NY 10032-3784 Spector, Abraham, Columbia University. Dept. of Ophthalmology, 630 West 168th Street, New York. NY 10032 Speksnijder, Johanna E., University of Groningen. Dept. of Genetics. Kerklaan 30, 9751 NN Haren, The Netherlands Spray, David C., Albert Einstein College of Medicine, Dept. of Neurosci., 1300 Morris Park Avenue, Bronx, NY 10461 Spring, Kenneth R., National Institutes of Health, Building 10, Room 6N260, 10 Center Drive, MSC 1603. Bethesda. MD 20892- 1603 Steele, John H., Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Steinacker, Antoinette, University of Puerto Rico, Medical Sciences, Institute of Neurobiology, 201 Boulevard Del Valle, San Juan. PR 00901 Steinberg, Malcolm, Princeton University, Dept. of Molecular Biology. M-18 Moffett Laboratory, Princeton. NJ 08544-1014 Stemmer, Andreas C., Institut fiir Robotik, ETH-Sentrum, 8092 Zurich. Switzerland Stenflo, Julian. University of Lund, Dept. of Clinical Chemistry, Malmo General Hospital, S-205 02 Malmo, Sweden Stetten, Jane Lazarow, 4701 Willard Ave #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 Rd., NE. Atlanta, GA 30322-1 100 Stommel, Elijah W., Dartmouth-Hitchcock Medical Center, Neurology Dept.. Lebanon. NH 03756 Stracher, Alfred, SUNY Health Science Center, Dept. of Biochemistry, 450 Clarkson Avenue, Brooklyn. NY 1 1203 Strumwasser, Felix, 39 Fox Run Drive. Hatchville, MA 02536 Stuart, Ann E., University of North Carolina, Department of Physiology, Medical Res. Bldg. 206H. Chapel Hill, NC 27599- 7545 Sugimori, Mutsuyuki, New York University Medical Center, Dept. of Physiology & Neuroscience, Rm 442, 550 First Avenue, New York, NY 10016 Summers, William C., Western Washington University. Huxley Coll. of Environ. Stud.. Bellmgham. WA 98225 Suprenant, Kathy A., University of Kansas, Dept. of Physiol. & Cell Biol., 4010 Haworth Hall, Lawrence. KS 66045 Sweet, Frederick, Washington University, School of Medicine, Dept. of OB & GYN, Box 8064, St. Louis, MO 631 10 Swenson, Katherine 1., Duke University Medical Center, Dept. of Molec. Cancer Biology, Box 3686, Durham. NC 27710 Sydlik, Mary Anne, Hope College, Peale Science Center, 35 East 12th St., PO Box 9000, Holland, MI 49422 Szent-Gyorgyi, Andrew, Brandeis University, Department of Biology, Bassine 244. 415 South Street, Waltham, MA 02254 Tabares, Lucia, University of Seville School of Medicine, Dept. of Physiology, Avda. Sanchez Pizjuan. 4, Seville 41009, SPAIN Tamm, Sidney L., Boston University. 725 Commonwealth Avenue, Boston, MA 02215 Tanzer, Marvin L., University of Connecticut School of Dental Medicine. Dept. of Biostructure & Funct.. Farmington, CT 06030- 3705 Tasaki. Ichiji, NIMH/NIH, Laboratory of Neurobiology, Building 36, Room 2B-16, Bethesda, MD 20892 Taylor, D. Lansing, Carnegie Mellon University, Ctr. for Flurorescence Res.. 4400 Fifth Avenue, Pittsburgh, PA 15213 Taylor, Edwin W., University of Chicago. Dept. of Mol. Gen. & Cell Bio.. 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 19104 Telzer, Bruce, Pomona College. Dept. of Biol., Thille Bldg., 175 W. 6th Street. Claremont. CA 9171 1 Townsel, James G., Meharry Medical College. Dept. of Physiology, 1005 D. B. 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 0221 1 Troll, Walter, NYU Medical Center. 550 First Avenue. New York, NY 10016 Troxler, Robert F., Boston University School of Medicine, Dept. of Biochem., 80 East Concord Street, Boston, MA 02118 Tucker, Edward B., Baruch College, CUNY, Dept. 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, Dept. of Biological Sciences, Notre Dame. IN 46656 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, Dept. of Anatomy & Neurobio., Winston-Salem, NC 27157 Ueno, Hiroshi, Kyoto University, AGR Chemistry Faculty, 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. Cell Biol.. 222 Maple Avenue, Shrewsbury, MA 01545 R76 Annual Report Valois, John J., 420 Woods Hole Road, Woods Hole. MA 02543 Van Dover. Cindy Lee, University of Alaska, P.O. Box 757220, Fairbanks. AK 99775 Van Holde, Kensal E., Oregon State University, Biochemistry & Biophysics Dept.. Corvallis, OR 97331-7503 Vogl, Thomas P., Environmental Research Institute of Michigan, 1101 Wilson Boulevard, Arlington. VA 22209 Wainwright, Norman R., Marine Biological Laboratory. Woods Hole. MA 02543 Waksman, Byron, NYU Medical Center, Department of Pathology, 550 First Avenue, New York. NY 1 00 Id Wall, Betty, 9 George Street, Woods Hole, MA 02543 Wang, Hsien-Yu, SUNY, Stony Brook, Univ. Medical Center, Physiology & Biophysics-HSC, Stony Brook, NY 11794-8633 Wangh, Lawrence J., Brandeis University, Dept. of Biology, 415 South Street. Waltham. MA 02254 Warner, Robert C., University of California. Irvine. Molecular Bio. & Biochemistry, Irvine. CA 92717 Warren, Leonard, Wistar Institute. 36th and Spruce Streets. Philadelphia. PA 19104 Waterbury, John B., Woods Hole Oceanographic Institution. Dept. 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. M., 426 Woods Hole Road. Woods Hole, MA 02543 Weber, Anne Marie, University of Pennsylvania School of Medicine, Dept. of Biochem. & Biophysics, Philadelphia, PA 19066 Weeks, Janis C., Institute for Neuroscience, University of Oregon, Box 1254, Eugene, OR 97403-1254 Weidner, Earl, Louisiana State University, Dept. of Zoology & Physiology. Baton Rouge. LA 70803 Weiss, Alice Sara, 105 University Blvd. West, Silver Spring, MD 20901 Weiss, Dieter, University of Rostock, Biology. 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, Dept. of Med/Div. Rheumatology. 550 First Avenue. New York. NY 10016 Westerfield, R. Monte, University of Oregon, Institute of Neuroscience, Eugene. OR 97403 Whittaker, J. Richard, University of New Brunswick. Dept. of Biology. BS 4511. Fredericton. NB E3B 6E1. Canada Wilkens, Lon A., University of Missouri. St. Louis, Dept. of Biology, 8001 Natural Bridge Road. St. Louis. MO 63121-4499 Wilson, Darcy B., San Diego Regional Cancer Center. 3099 Science Park Road. San Diego. CA 92121 Wilson, T. Hastings, Harvard Medical School, Department of Physiology. 25 Shattuck Street, Boston, MA 021 15 Witkovsky, Paul, NYU Medical Center, Department ot Ophthalmology. 550 First Avenue, New York, NY 10016 Wittenberg, Beatrice, Albert Einstein College of Medicine, Dept. of Physiol. & Biophysics, Bronx, NY 10461 Wittenberg, Jonathan B., Albert Einstein College of Medicine. Dept. of Physiol. & Biophysics, Bronx. NY 10461 Wolken, Jerome J., Caniegie Mellon LIniversity. Dept. of Biological Sciences. 440 Fifth Avenue, Pittsburgh, PA 15213 \\onderlin, William F., West Virginia University, Pharmacology & Toxicology Dept., Morgantown. WV 26506 Worden, Mary Kate, University of Virginia, Dept. of Molecular Physics and Biological Physics, P.O. Box 10011, Charlottesville. VA 22906 Worgul, Basil V., Columbia University, Department of Ophthalmology, 630 West 168 Street, New York, NY 10032 Wu, Chau Hsiung, Northwestern University Medical School, Dept. ul Pharmacology (S215). 303 E. Chicago Avenue. Chicago, II. 60611-3008 Wj'ttenbach, Charles R., University of Kansas, Biological Sciences Dept., 2045 Haworth Hall, Lawrence, KS 66045-2 lOh Yeh, Jay Z., Northwestern University, Medical School. Department of Pharmacology, Chicago. IL 6061 1 Zacks, Sumner I., 65 Saconesset Road. Falmouth, MA 02540-1851 Zigman, Seymour, University of Rochester Medical School, Ophthalmol. Research. Box 314. 601 Elmwood Avenue, Rochester, NY 14640 Zigmond. Michael J., University of Pittsburgh. Dept. of Neuroscience. 570 Crawford Hall, Pittsburgh. PA 15260 Zimmerberg. Joshua J., LCMB. NICHD, NIH. Building 10. Room IODI4. 10 Center Drive, MSC 1855. Bethesda. MD 20892-1855 Zottoli, Steven J., Williams College. Dept. of Biology. Williamstown. MA 01267 Zucker, Robert S., University of California, Neurobiology Div.. Molecular & Cellular Biol. Dept., Berkeley, CA 94720 /nUiii. R. Suzanne. Albert Einstein College of Medicine, Dept. of Neurosci.. 1410 Pelham Parkway South, Bronx, NY 10461 MEL Associates Executive Board Julie S. Child, President Ruth Ann Laster. Vice President Priscilla Roslansky. Secretary Hanna Hastings, Treasurer Mary Ulbrich, Membership Chair Duncan Aspinwall Barbara Atwood Kitty Brown Seymour Cohen Molly Cornell Elizabeth Farnham Michael Fenlon Megan Jones Alice Knowles Rebecca Lash Barbara Little Jack Pearce Joan Pearlman Deborah Senft John Valois Kensal Van Holde Sustaining Associate Josephine B. Crane Foundation George Frederick Jewett Foundation Mr. Edward F. MacNichol. Jr. Plymouth Savings Bank Mr. and Mrs. William A. Putnam. Ill Members of the Corporation R77 Supporting Associate Mrs. George H. A. Clowes Dr. and Mrs. James D. Ebert Dr. and Mrs. Prosser Gifford Drs. Alfred and Joan Goldberg Mr. and Mrs. Lon Hocker Drs. Luigi and Elaine Mastroianni Dr. and Mrs. William M. McDermott Dr. and Mrs. Courtland D. 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Zinn MBL Associates Gift Shop Volunteers Marion Adelberg Marianne Angell Barbara Atwood Harriet Bernheimer Gloria Borgese Jennie Brown Elizabeth Buck Jewel Cobb Janet Daniels Carol De Young Fran Eastman Alma Ebert Pat Ferguson Becky Glazebrook Muriel Gould Rose Grant Edie Grosch Bobbie Grossman Pat Hancox Hanna Hastings Sally Karush Alice Knowles Barbara Little Sally Loessel Winnie Mackey Miriam Mauzerall Mary Mavor Elizabeth Mosley Jane McCormack Polly Miles Florence Mixter Lorraine Mizell Eleanor Nace Bertha Person Liz Price Julie Rankin Jean Ripps Arlene Rogers Lilyan Saunders Merilyn Shepro Cynthia Smith Peggy Smith Louise Specht Jane Stetten Elaine Troll Natalie Trousof Mary Ulbrich Barbara Van Holde Mabel Whelpley Barbara Whitehead Clare Wilber MBL Summer Tour Guides Kevin Barry John Buck Sears Crowell Barbara Little Giselle Magnusson Kathy Mullin Julie Rankin Lola Robertson Priscilla Roslansky Erin Smyth Mary Ulbrich John Valois Certificate of Organization Articles of Amendment Bylaws Certificate of Organization Articles of Amendment (On File in the Office of the Secretary of the Commonwealth) No. 3170 We, Alpheus Hyatt. President. William Stanford Stevens. Treasurer, and William T. Sedgwick. Edward G. Gardiner. Susan Mims and Charles Sedgwick Minot being a majority of the Trustees of the Marine Biological Laboratory in compli- ance with the requirements of the fourth section of chapter one hundred and fifteen of the Public Statutes do hereby certify that the following is a true copy of the agreement of association to constitute said Corporation, with the names of the subscribers thereto: We, whose names are hereto subscribed, do, by this agreement, associate ourselves with the intention to constitute a Corporation according to the provisions of the one hundred and fifteenth chapter of the Public Statutes of the Commonwealth of Massachusetts, and the Acts in amendment thereof and in addition thereto. The name by which the Corporation shall be known is THE MARINE BIOLOGICAL LABORATORY The purpose for which the Corporation is constituted is to establish and maintain a laboratory or station for scientific study and investigations, and a school for instruction in biology and natural history. The place within which the Corporation is established or located is the city of Boston within said Commonwealth. The amount of its capital stock is none In Witness Whereof, we have hereunto set our hands, this twenty seventh day of February in the year eighteen hundred and eighty-eight, Alpheus Hyatt. Samuel Mills. William T. Sedgwick, Edward G. Gardiner, Charles Sedgwick Minot. Wil- liam G. Farlow. William Stanford Stevens, Anna D. Phillips, Susan Mims, B. H. Van Vleck. That the first meeting of the subscribers to said agreement was held on the thirteenth day of March in the year eighteen hundred and eighty-eight. In Witness Whereof, we have hereunto signed our names, this thirteenth day of March in the year eighteen hundred and eighty-eight, Alpheus Hyatt. President, William Stanford Stevens, Treasurer. Edward G. Gardiner, William T. Sedgwick. Susan Mims. Charles Sedgwick Minot. (Approved on March 20, 1888 as follows: I hereby certify that it appears upon an examination of the within written certificate and the records of the corporation duly submitted to my inspection, that the requirements of sections one, two and three of chapter one hundred and fifteen, and sections eighteen, twenty and twenty-one of chapter one hundred and six, of the Public Statutes, have been complied with and I hereby approve said certificate this twentieth day of March A.D. eighteen hundred and eighty-eight. Charles Endicott Commissioner of Corporations) (On File in the Office of the Secretary of the Commonwealth) We, James D. Ebert, President, and David Shepro, Clerk of the Marine Biological Laboratory, located at Woods Hole. Massachusetts 02543. do hereby certify that the following amendment to the Articles of Organization of the Corporation was duly adopted at a meeting held on August 15. 1975, as adjourned to August 29, 1975, by vote of 444 members, being at least two-thirds of its members legally qualified to vote in the meeting of the corporation: Voted: That the Certificate of Organization of this corporation be and it hereby is amended by the addition of the following provisions: "No Officer, Trustee or Corporate Member of the corporation shall be personally liable for the payment or satisfaction of any obligation or liabilities incurred as a result of. or otherwise in connection with, any commitments, agreements, activi- ties or affairs of the corporation. "Except as otherwise specifically provided by the Bylaws of the corporation, meetings of the Corporate Members of the corporation may be held anywhere in the United States. "The Trustees of the corporation may make, amend or repeal the Bylaws of the corporation in whole or in part, except with respect to any provisions thereof which shall by law, this Certificate or the bylaws of the corporation, require action by the Corporate Members." The foregoing amendment will become effective when these articles of amend- ment are filed in accordance with Chapter 180, Section 7 of the General Laws unless these articles specify, in accordance with the vote adopting the amendment, a later effective date not more than thirty days after such filing, in which event the amendment will become effective on such later date. In Witness whereof and Under the Penalties of Perjury, we have hereto signed our names this 2nd day of September, in the year 1975. James D. Ebert, President: David Shepro, Clerk. (Approved on October 24. 1975. as follows: I hereby approve the within articles of amendment and. the filing fee in the amount of $10 having been paid, said articles are deemed to have been filed with me this 24th day of October. 1975. Paul Guzzi Secretary of the Commonwealth) Bylaws (Revised August 7, 1992 and December 10. 1992) ARTICLE I THE CORPORATION A. Name and Purpose. The name of the Corporation shall be The Marine Biological Laboratory. The Corporation's purpose shall be to establish and mam- R79 R80 Annual Report tarn a laboratory or station lor scientific study and investigation and a school for instruction in biology and natural history. B. Nondiscrimination. The Corporation shall not discriminate on the basis of age, religion, color, race, national or ethnic origin, sex or sexual preference in its policies on employment and administration or in its educational and other programs. ARTICLE II MEMBERSHIP A. Members The Members of the Corporation ("Members") shall consist of persons elected by the Board of Trustees (the "Board"), upon such terms and conditions and in accordance with such procedures, not inconsistent with law or Ihese Bylaws, as may be determined hy the Board. At any regular or special meeting of the Board, the Board may elect new Members. Members shall have no voting or other rights with respect to the Corporation or its activities except as specified in these Bylaws, and any Member may vote at any meeting ol the Members in person only and not by proxy. Members shall serve until their death or resignation unless earlier removed with or without cause by the affirmative vote of two-thirds of the Trustees then in office. Any Member who has retired from his or her home institution may. upon written request to the Corporation, be designated a Life Member. Life Members shall not have the right to vote and shall not be assessed for dues. B. Meetings. The annual meeting of the Members shall be held on the Friday following the first Tuesday in August of each year, at the Laboratory of the Corporation in Woods Hole. Massachusetts, at 9:30 a.m. The Chairperson of the Board shall preside at meetings of the Corporation. If no annual meeting is held in accordance with the foregoing provision, a special meeting may be held in lieu thereof with the same effect as the annual meeting, and in such case all references in these Bylaws, except in this Article II. B., to the annual meeting of the Members shall be deemed to refer to such special meeting. Members shall transact business as may properly come before the meeting. Special meetings of the Members may be called by the Chairperson or the Trustees, and shall be called by the Clerk, or in the case of the death, absence, incapacity or refusal by the Clerk, by any other officer, upon written application of Members representing at least ten percent of the smallest quorum of Members required for a vote upon any matter at the annual meeting of the Members, to be held at such time and place as may be designated C. Quiiriiin. One hundred (100) Members shall constitute a quorum at any meeting. Except as otherwise required by law or these Bylaws, the affirmative vote of a majority of the Members voting in person al a meeting attended by a quorum shall constitute action on behalf of the Members. D. Notice of Meetings. Notice of any annual meeting or special meeting of Members, if necessary, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting at least 15 days before such meeting to each Member at his or her address as shown on the records of the Corporation. E. Wavier of Notice. Whenever notice of a meeting is required to be given a Member, under any provision of the Articles or Organization or Bylaws of (In- corporation, a written waiver thereof, executed before or after the Meeting by such Member, or his or her duly authorized attorney, shall be deemed equivalent to such notice. F. Adjournments. Any meeting of the Members may he adjourned to any other time and place by the vote of a majority of those Members present at the mcciiii!. 1 . whether or not such Members constitute a quorum, or by any officer entitled to preside at or to act as Clerk of such meeting, if no Member is present or repre- sented. It shall not be necessary to notify any Members of any adjournment unless no Member is present or represented at the meeting which is adjourned, in which case, notice of the adjournment shall be given in accordance with Article II. D. Any business which could have been transacted at any meeting of the Members as originally called may be transacted at an adjournment thereol. ARTICLE III ASSOCIATES OF THE CORPORATION Associates of the Corporation. The Associates of the Marine Biological Labora- tory shall he an unincorporated group of persons (including associations and corporations) interested in the l.ah.ii.itory and shall be organized and operated under the general supervision and authority of the Trustees. The Associates of the Marine Biological Laboratory shall have no voting rights ARTICLE IV BOARD OF TRUSTEES A. I'un-ers. The Board of Trustees shall have the control and management of the affairs of the Corporation. The Trustees shall elect a Chairperson of the Board who skill serve until his or her successor is elected and qualified. They shall annually elect a President of the Corporation. They shall annually elect a Vice Chairperson of the Board who shall be Vice Chairperson of the meetings of the Corporation. They shall annually elect a Treasurer. They shall annually elect a Clerk, who shall be a resident of Massachusetts. They shall elect Trustees-at-Large as specified in this Article IV. They shall appoint a Director of the Laboratory for a term not to exceed five years, provided the term shall not exceed one year if the candidate has attained the age of 65 years prior to the date of the appointment. They shall choose such other officers and agents as they shall think best. They may fix the compensation of all officers and agents of the Corporation and may remove them at any time. They may fill vacancies occurring in any of the offices. The Board shall have the power to choose an Executive Committee from their own number as provided in Article V, and to delegate to such Committee such ol their own powers as they may deem expedient in addition to those powers conferred by Article V. They shall, from time to time, elect Members to the Corporation upon such terms and conditions as they shall have determined, not inconsistent with law or these Bylaws. B. Composition and Election. (1) The Board shall include 24 Trustees elected by the Board as provided below: (a) At least six Trustees ("Corporate Trustees") shall be Members who are scientists, and the other Trustees ("Trustees-at-Large") shall be individuals who need not be Members or otherwise affiliated with the Corporation. (h) The 24 elected Trustees shall be divided into four classes of six Trust- ees each, with one class to be elected each year to serve for a term of four years, and with each such class to include at least one Corporate Trustee. Such classes of Trustees shall be designated by the year of expiration of their respective terms. (2) The Board shall also include the Chief Executive Officer, Treasurer and the Chairperson of the Science Council, who shall be ex officio voting members of the Board. (.1) Although Members or Trustees may recommend individuals for nomina- tion as Trustees, nominations for Trustee elections shall be made by the Nomi- nating Committee in its sole discretion. The Board may also elect Trustees who have not been nominated hy the Nominating Committee. C Eligibility. A Corporate Trustee or a Trustee-at-Large who has been elected to an initial tour-year term or remaining portion thereof, of which he/she has served at least two years, shall be eligible for re-election to a second four-year term, but shall be ineligible for re-election to any subsequent term until one year has elapsed after he/she has last served as a Trustee. D. Removal. Any Trustee may be removed from office at any time with or without cause, by vote of a majority of the Members entitled to vote in the election of Trustees; or for cause, by vote of two-thirds of the Trustees then in office. A Trustee may be removed for cause only if notice of such action shall have been given to all of the Trustees or Members entitled to vote, as the case may be. prior in the meeting at which such action is to be taken and if the Trustee to be so removed shall have been given reasonable notice and opportunity to be heard betore the body proposing to remove him or her. E. Vacancies. Any vacancy in the Board may be filled by vote of a majority of the remaining Trustees present at a meeting of Trustees at which a quorum is present. Any vacancy in the Board resulting from the resignation or removal of a Corporate Trustee shall be filled by a Member who is a scientist. F. Meetings. Meetings of the Board shall be held from time to time, not less frequently than twice annually, as determined by the Board. Special meetings of Trustees may be called by the Chairperson, or by any seven Trustees, to be held at such time and place as may be designated. The Chairperson of the Board, when present, shall preside over all meetings of the Trustees. Written notice shall be sent to a Trustee's usual or last known place of residence at least two weeks before the meeting. Notice of a meeting need not be given to any Trustee if a written waiver of notice executed by such Trustee before or after the meeting is filed with the records of the meeting, or if such Trustee shall attend the meeting without protesting prior thereto or at its commencement the lack of notice given to him or her. G. Quorum niiii Action hy Trustees. A majority of all Trustees then in office shall constitute a quorum. Any meeting of Trustees may be adjourned by vote of a majority of Trustees present, whether or not a quorum is present, and the meeting may be held as adjourned without further notice. When a quorum is present at any meeting of the Trustees, a majority of the Trustees present and voting (excluding abstentions) shall decide any question, including the election of officers, unless otherwise required by law. the Articles of Organization or these Bylaws. H 7'nins/ers of Interests in Luinl. There shall be no transfer of title nor long- term lease ot real property held by the Corporation without prior approval of not less than two-thirds of the Trustees. Such real property transactions shall be finally acted upon at a meeting of the Board only if presented and discussed at a prior meeting of the Board. Either meeting may be a special meeting and no less than lour weeks shall elapse between the two meetings. Any property acquired by the Bylaws of the Corporation R81 Corporalion after December 1. 19X9 may be sold, any mortgage or pledge of real property (regardless of when acquired) to secure borrowings by the Corporation may be granted, and any transfer of title or interest in real property pursuant to the foreclosure or endorsement of any such mortgage or pledge of real property may be effected by any holder of a mortgage or pledge of real property of the Corporation, with the prior approval of not less than two-thirds of the Trustees (other than any Trustee or Trustees with a direct or indirect financial interest in the transaction being considered for approval) who are present at a regular or special meeting of the Board at which there is a quorum. ARTICLE V COMMITTEES A. Executive Committee. There shall be an Executive Committee of the Board of Trustees which shall consist of not more than eleven (II) Trustees, including ex officio Trustees, elected by the Board. The Chairperson of the Board shall act as Chairperson of the Executive Commit- tee and the Vice Chairperson as Vice Chairperson. The Executive Committee shall meet at such times and places and upon such notice and appoint such subcommittees as the Committee shall determine. The Executive Committee shall have and may exercise all the powers of the Board during the intervals between meetings of the Board except those powers specifically withheld, from time to time, by vote of the Board or by law. The Executive Committee may also appoint such committees, including persons who are not Trustees, as it may, from time to time, approve to make recommendations with respect to matters to be acted upon by the Executive Committee or the Board. The Executive Committee shall keep appropriate minutes of its meetings, which shall be reported to the Board. Any actions taken by the Executive Committee shall also be reported to the Board. B. Nominating Committee. There shall be a Nominating Committee which shall consist of not fewer than four nor more than six Trustees appointed by the Board in a manner which shall reflect the balance between Corporate Trustees and Trustees-at-Large on the Board. The Nominating Committee shall nominate persons for election as Corporate Trustees and Trustees-at-Large, Chairperson of the Board, Vice Chairperson of the Board. President, Treasurer, Clerk. Director of the Laboratory and such other officers, if any, as needed, in accordance with the requirements of these Bylaws. The Nominating Committee shall also be responsible for overseeing the training of new Trustees. The Chairperson of the Board of Trustees shall appoint the Chairperson of the Nominating Committee. The Chairperson of the Science Council shall be an ex officio voting member of the Nominating Committee. C. Science Council. There shall be a Science Council (the 'Council") which shall consist of Members of the Corporation elected to the Council by vote of the Members of the Corporation, and which shall advise the Board with respect to matters concerning the Corporation's mission, its scientific and instructional endeavors, and the appointment and promotions of persons or committees with responsibility for matters requiring scientific expertise. Unless otherwise approved by a majority of the members of the Council, the Chairperson of the Council shall be elected annually by the Council. The chief executive officer of the Corporation shall be an ?.v officio voting member of the Council. D. Board of Overseers. There shall be a Board of Overseers which shall consist of not fewer than five nor more than eight scientists who have expertise concerning matters with which the Corporation is involved. Members of the Board of Over- seers may or may not be Members of the Corporation and may be appointed by the Board of Trustees on the basis of recommendations submitted from scientists and scientific organizations or societies. The Board of Overseers shall be available to review and offer recommendations to the officers. Trustees and Science Council regarding scientific activities conducted or proposed by the Corporation and shall meet from time to time, not less frequently than annually, as determined by the Board of Trustees. E. Board Committees Generally. The Trustees may elect or appoint one or more other committees (including, but not limited to. an Investment Committee, a Development Committee, an Audit Committee, a Facilities and Capital Equipment Committee and a Long-Range Planning Committee) and may delegate to any such committee or committees any or all of their powers, except those which by law. the Articles of Organization or these Bylaws the Trustees are prohibited from delegating; provided that any committee to which the powers of the Trustees are delegated shall consist solely of Trustees. The members of any such committee shall have such tenure and duties as the Trustees shall determine. The Investment Committee, which shall oversee the management of the Corporation's endowment funds and marketable securities shall include as e.\ officio members, the Chairper- son 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 ot the Investment Committee to consist of Trustees. Except as otherwise provided by these Bylaws or determined by the Trustees, any such committee may make rules for the conduct of its business, but, unless otherwise provided by the Trustees or in such rules, its business shall be conducted as nearly as possible in the same manner as is provided by these Bylaws for the Trustees. F. Actions Without a Meeting. Any action required or permitted to be taken at any meeting of the Executive Committee or any other committee elected by the Trustees may be taken without a meeting if all members of such committees consent to the action in writing and such written consents are riled 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 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 Chairperson and one or more Vice Presidents, Assistant Treasurers or Assistant Clerks. Any two or more offices may be held by the same person. The Chairperson and Vice Chairperson of the Board shall be elected by and from the Trustees, but other officers of the Corporation need 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 Organiza- tion 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 Mem- bers and thereafter, until his or her successor is chosen and qualified. C. Resignation. Any officer may resign by delivering his or her written resigna- tion 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 exer- cise 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 other- wise voted by the Trustees, the chief executive officer of the Corporation. The Director shall, subject to the direction of the Trustees, have general supervision of the Laboratory and control of the business of the Corporation. 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 Director. 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 R82 Annual Report 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 direc- tion of the Trustees, have general charge of the financial affairs of the Corporation, including its long-range financial planning, and shall cause to be kept accurate books of account. The Treasurer shall prepare a yearly report on the financial status of the Corporation to be delivered at the annual meeting. The Treasurer shall also prepare or oversee all filings required by the Commonwealth of Massa- chusetts, 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 Common- wealth 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 lime, prescribe In the absence of the Clerk and an Assistant Clerk from any meeting, a tempo- rary Clerk shall be appointed at the meeting. M. Other Powers ami Duties. Each officer shall have in addition to the duties and powers specifically set forth in these Bylaws, such duties and powers as are customarily 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, indemnity 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] im- posed 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 de- fending 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 indemni- fied 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 m the Article VI11. 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 deter- mined 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 Corporation 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, dii) "fines" shall be deemed to include any excise tax plan pursuant to ERISA; and (iv) actions taken or omitted by a person with respect to an employee benefit plan in the performance of such person's duties for a purpose reasonably believed by such person to be in the interest of the participants and beneficiaries of the plan shall be deemed to be for a purpose which is in the best interests of the Corporatton. 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 Corpora- tion'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 dis- posed 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 Commonwealth of Massachusetts. ARTICLE \-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 onginal. or attested copies, of the Articles of Organization. Bylaws and records of all meetings of the Members shall be kept in Massachusetts 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. Bylaws of the Corporation R83 E. Arliclt f \ c/ Orxtint:titn>n. All references in these Bylaws to the Articles of Organization shall be deemed to refer to the Articles of Organi/ation of ihe Corporation, 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 individually or as a director, member or officer of such corporation, firm, association or partnership in such a party or is so interested shall be disclosed to or shall have been known by the Board of Trustees or a majority of such Members thereof as shall be present at a meeting of the Board of Trustees at which action upon any such contract or transaction shall be taken; any Trustee may be counted in determining the existence of a quorum and may vote at any meeting of the Board of Trustees for the purpose of authorizing any such contract or transaction with like force and effect as if he/she were not so interested, or were not a director, member or officer of such other corporation, firm, association or part- nership, provided that any vote with respect to such contract or transaction must be adopted by a majority of the Trustees then in office who have no interest in such contract or transaction- THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Associate Editors JAMES A. BLAKE, ENSR Marine & Coastal Center, Woods Hole Louis E. BURNETT, Grice Marine Biological Laboratory, College of Charleston WILLIAM D. COHEN, Hunter College, City University of New York CHARLES D. DERBY, Georgia State University SHINYA INOUE, Marine Biological Laboratory RUDOLF A. RAFH, Indiana University NOV 9 1998 Editorial Board PETER B. ARMSTRONG, University of California, Davis ANDREW R. CAMERON, California Institute of Technology THOMAS H. DIETZ, Louisiana State University RICHARD B EMLET, Oregon Institute of Marine Biology, University of Oregon DAVID EPEL, Hopkins Marine Station, Stanford Univer- sity DAPHNE GAIL FAUTIN, University of Kansas WILLIAM F. GILLY, Hopkins Marine Station, Stanford University ROGER T. HANLON, Marine Biological Laboratory GREGORY HINKLE, University of Massachusetts, Dart- mouth MAKOTO KOBAYASHI, Hiroshima University of Eco- nomics MICHAEL LABARBERA, University of Chicago DONAL T. MANAHAN, University of Southern California MARGARET McFALL-NGAi, Kewalo Marine Laboratory. University of Hawaii MARK W. MILLER, Institute of Neurobiology, University of Puerto Rico TATSUO MOTOKAWA, Tokyo Institute of Technology YOSHITAKA NAGAHAMA, National Institute for Basic Biology, Japan SHERRY D. PAINTER, Marine Biomedical Institute, Uni- versity of Texas Medical Branch K. RANGA RAO, University of West Florida BARUCH RINKEVICH, Israel Oceanographic & Limno- logical Research Ltd. RICHARD STRATHMANN, Friday Harbor Laboratories, University of Washington STEVEN VOGEL, Duke University J. HERBERT WAITE, University of Delaware SARAH ANN WOODIN, University of South Carolina RICHARD K. ZIMMER-FAUST, University of California, Los Angeles Editor MICHAEL J GREENBERG, The Whitney Laboratory, University of Florida Managing Editor: PAMELA L. CLAPP, Marine Biological Laboratory OCTOBER, 1998 Printed and Issued by LANCASTER PRESS, Inc. 3575 HEMPLAND ROAD LANCASTER, PA Cover The bobtail squid Euprymna scolopes is a benthic nocturnal predator endemic to the Hawaiian archi- pelago. Each individual of this species like the adult (2-3 cm) resting on the sandy substrate in the upper right photograph entertains a colony of bioluminescent bacterial symbionts ( Vibrio fi- scheri) which it houses in a specialized light or- gan. The light produced by these bacteria, having been diffused into the environment, is used in anti- predatory behavior. Each day at dawn, a fraction of the bacterial colony is vented into the ambient seawater a form of population control. If the light organ of an adult squid is exposed at dawn by ventral dis- section of the mantle, freshly released bacteria- rich exudate can be seen emerging from the right lobe of the organ (bottom photograph). In this is- sue. Spencer V. Nyholm and Margaret J. McFall- Ngai exploit this regulatory behavior; they sample the exudate, study its contents the cellular com- ponents of the light organ, both animal and bacte- rial and explore the mechanisms that sustain this stable, dynamic mutualism. Newly hatched E. scolopes are typically 2 to 3 mm long (on sand pebbles, upper left photograph). Such hatchling squid obtain their symbionts from the environment and undergo a series of develop- mental changes that are induced by V. fischeri. Once the symbiosis is established, the association is maintained throughout the life history of the host. Fortunately, the host and its bacterial symbi- onts can be reared and cultured separately under laboratory conditions; thus, the effects of the mu- tualistic bacteria on the development of animal tissues can be readily studied. In the December issue of the Bulletin, Mary K. Montgomery and McFall-Ngai will describe the final developmental stages of the light organ system. CONTENTS SYMBIOSIS Nyholm, Spencer V., and Margaret J. McFall-Ngai Sampling the light organ microenvironment of Eu- prymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri CELL BIOLOGY AND DEVELOPMENT Rinkevich, Baruch, Irving L. Weissman, and An- thony W. DeTomaso Transplantation of Fu/HC-incompatible zooids in Bo- tryllux schlosseri results in chimerism 98 Yamada, Katsuyuki, and Koshin Mihashi Temperature-independent period immediately after fertilization in sea urchin eggs 107 Abdu, Uri, Peter Takac, Hans Laufer, and Amir Sagi Effect of methyl farnesoate on late larval development and metamorphosis in the prawn Macrobrachium ro- senbergii (Decapoda, Palaemonidae): a juvenoid-like effect? 112 ECOLOGY AND EVOLUTION Bavestrello, Giorgio, Umberto Benatti, Barbara Calcinai, Riccardo Cattaneo-Vietti, Carlo Cerrano, Anna Favre, Marco Giovine, Serena Lanza, Roberto Pronzato, and Michele Sara Body polanty and mineral selectivity in the de- mosponge Chondrosia reniformis 120 Wendt, Dean E. Effect of larval swimming duration on growth and reproduction of Bugula neritina (Bryozoa) under field conditions 126 PHYSIOLOGY Ellers, Olaf, Amy S. Johnson, and Philip E. Moberg Structural strengthening of urchin skeletons by collag- enous sutural ligaments 136 Thorington, Glyne U., and David A. Hessinger Efferent mechanisms of discharging cnidae: II. A ne- matocyst release response in the sea anemone tentacle 145 NEUROBIOLOGY AND BEHAVIOR Herberholz, Jens, and Barbara Schmitz Role of mechanosensory stimuli in intraspecific ago- nistic encounters of the snapping shrimp (Alpheus liet- erochaelis) . 156 RESEARCH NOTE Tankersley, Richard A., Maria G. Wieber, Marco A. Sigala, and Kristen A. Kachurak Migratory behavior of ovigerous blue crabs Calli- nectes sapidus: evidence for selective tidal-stream transport 168 SHORT REPORTS FROM THE 1998 GENERAL SCIENTIFIC MEETINGS OF THE MARINE BIOLOGICAL LABORATORY FEATURED ARTICLES Atema, Jelle Introduction. Tracking turbulence: processing the bi- modal signals that define an odor plume .... 179 Weaver, Matthew, and Jelle Atema Hydrodynamic coupling of lobster antennule motion to oscillatory water flow 180 Guenther, Carla M., and Jelle Atema Distribution of setae on the Homarus americanus lat- eral antennule flagellum 182 SENSORY BIOLOGY Mead, Kristina S. The biomechanics of odorant access to aesthetascs in the grass shrimp, Palaemonetes vulgaris 184 Quinn, Elizabeth, Kristen Paradise, and Jelle Atema Juvenile Limulus polypliemus generate two water cur- rents that contact one proven and one putative chemo- receptor organ 185 Shashar, Nadav, Ferenc I. Harosi, Anastazia T. Ba- naszak, and Roger T. Hanlon UV radiation blocking compounds in the eye of the cuttlefish Sepia officinalis 187 Ruta, Vanessa J., Frederick A. Dodge, and Robert B. Barlow Efferent modulation of physiological properties of the Limulus lateral eye 189 Edds- Walton, P. L., and R. R. Fay Directional auditory responses in the descending octa- val nucleus of the toadfish (Opxanus tan) 191 BEHAVIOR AND NEUROBIOLOGY King, Jane Roche, and Hans Straka Effects of vestibular nerve lesions on orientation turn- ing in the leopard frog. Rana pipiens 193 Mensinger, Allen I .. and Max DefTenbaugh Prototype rechargeable tag for acoustical neural telem- etry 194 Billack, Blase, Jeffrey D. Laskin, Prudence T. Heck, Walter Troll, Michael A. Gallo, and Diane E. Heck Alterations in cholinergic signaling modulate contrac- tion of isolated sea urchin tube feet: potential role of nitric oxide 196 Hoskin, Francis C. G., and John E. Walker A closer look at the natural substrate for a nerve-gas hydrolyzing enzyme in squid nerve 197 Kuzirian, Alan M., Herman T. Epstein, Thomas J. Nelson, and Nancy S. Rafferty Lead, learning, and calexcitin in Hermissenda 198 Tamse, Catherine T., Katherine Hammar, D. Mar- shall Porterfleld, and Peter J. S. Smith Transmembrane calcium flux in Pb : * -exposed Aplysia neurons 201 Malchow, Robert Paul, Michael P. Verzi, and Peter J. S. Smith Extracellular pH gradients measured from isolated ret- inal cells 203 Andersen, Kristen A., and Robert Paul Malchow Fluorometric analysis of intracellular sodium concen- trations in isolated retinal horizontal cells 204 Jessen-Eller, Kathryn, Marjorie Steele, Carol Rein- isch, and Nicholas Spitzer Blockade of ryanodine receptors stimulates neurite outgrowth in embryos of Spisula snlidissima 206 CELL AND DEVELOPMENTAL BIOLOGY Porterfleld, D. M., J. R. Trimarchi, D. L. Keefe, and P. J. S. Smith Characterization of oxygen and calcium fluxes from early mouse embryos and oocytes 208 Silver, Robert B., Leslie A. King, and Alyssa F. Wise Calcium regulatory endomembranes of the prophase mitotic apparatus of sand dollar cells contain enzyme activities that produce leukotriene B 4 but not 1,4,5- inositol triphosphate 209 Lee, Kyeng G., Nishal Mohan, Zoya Koroleva, Li- Fang Huang, and William D. Cohen Fluorescence localization of cytoskeletal proteins in fibrin-trapped cells 211 Goda, Makoto, Shinya Inoue, and Robert Knudson Oocyte maturation in Chaetopterus pergamentaceous observed with centrifuge polarizing microscope ... 212 Miyake, Katsuya, and Paul L. McNeil A little shell to live in: evidence that the fertilization envelope can prevent mechanically induced damage of the developing sea urchin embryo 214 Kuhns, William J., Xavier Fernandez-Busquets, Max M. Burger, Michael Ho, and Eva Turley Hyaluronic acid-receptor binding demonstrated by synthetic adhesive proteoglycan peptide constructs and by cell receptors on the marine sponge Microciona prolifera 216 Kubke, M. F., E. Gilland, and R. Baker Lipophilic dye labeling distinguishes segregated cen- tral components of the eighth cranial nerve in embry- onic chicken 218 Straka, Hans, Edwin Gilland, and Robert Baker Rhombomeric organization of brainstem motor neu- rons in larval frogs 220 ANIMAL HUSBANDRY AND DISEASE Hanley, Janice S., Nadav Shashar, Roxanna Smolo- witz, Robert A. Bullis, William N. Mebane, Howaida R. Gabr, and Roger T. Hanlon Modified laboratory culture techniques for the Euro- pean cuttlefish Sepia officinulis 223 Maxwell, Michael R., William K. Macy, Shobu Odate, and Roger T. Hanlon Evidence for multiple spawning by squids (Loligo pealei ) in captivity 225 Weidner, Earl, and Teresa King In vivo and in vitro growth of nerve parasite from Lophius americanus 227 O'Neill, Maureen D., Heather M. Wesp, Allen F. Mensinger, and Roger T. Hanlon Initial baseline blood chemistry of the oyster toadfish, Opsanus tau 228 Smolowitz, Roxanna, Elizabeth Wadman, and H. M. Chikarmane Pseudomonas putida infections of the oyster toadfish ( Opsanus tau ) 229 ECOLOGY Schmitt, Catherine, Nathaniel Weston, and Charles Hopkinson Preliminary evaluation of sedimentation rates and spe- cies distribution in Plum Island Estuary, Massachu- setts 232 Griffin, Martin P. A., Marci L. Cole, Kevin D. Kroeger, and Just Cebrian Dependence of herbivory on autotrophic nitrogen con- tent and on net primary production across ecosystems 233 Rogers, Jennifer, Jennifer Harris, and Ivan Valiela Interaction of nitrogen supply, sea level rise, and ele- vation on species form and composition of salt marsh plants 235 Sweeney, Jennifer, Linda Deegan, and Robert Garritt Population size and site fidelity of Fundulus heterocli- tus in a macrotidul saltmarsh creek 238 KJrkpatrick, John, Ken Foreman, and Ivan Valiela Dissolved inorganic nitrogen flux and mineralization in Waquoit Bay sediments as measured by core incu- bations 240 Graham, Suzanne, Jessica Davis, Linda Deegan, Just Cebrian, Jeff Hughes, and Jennifer Hauxwell Effect of eelgrass (Zostera marina) density on the feeding efficiency of mummichog ( Fundulus hetero- clitus) . 241 Legra, Jessica C., Roselle E. Safran, and Ivan Valiela Costello, John H., and Rebecca Coverdale Lead concentration as an indicator of contamination Planktonic feeding and evolutionary significance of history in estuarine sediments 243 the lobate body plan within the Ctenophora 247 Safran, Roselle E., Jessica C. Legra, and Ivan Valiela Effects of nitrogen loading on eelgrass seed coat abun- AHSTRACTS dance, C to N ratios, and 6'^N in sediments of Waquoit Bay 245 Papers listed by title only 249 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. Subscription per year (six issues, two volumes): $195 tor libraries; $95 for individuals. Subscription per volume (three issues): $97.50 for libraries; $50 for individuals. 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Reprints normally will be delivered about 2 to 3 months after the issue date. Authors (or delegates for foreign authors) will receive page proofs of articles shortly before publication. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). Other than these charges for au- thors' alterations. The Biological Bulletin does not have page charges. Reference: fi/o/. Hull. 195: 89-97. (October, 1998) Sampling the Light-Organ Microenvironment of Euprymna scolopes: Description of a Population of Host Cells in Association With the Bacterial Symbiont Vibrio fischeri SPENCER V. NYHOLM AND MARGARET J. MCFALL-NGAI* Pacific Biomedical Research Center, University of Hawaii, 41 Ahni Street, Honolulu, Hawaii 96813 Abstract. The symbiosis between the squid Enpryinna scolopes and the luminous bacterium Vibrio fischeri has a pronounced diel rhythm, one component of which is the venting of the contents of the light organ into the surrounding seawater each day at dawn. In this study, we explored the use of this behavior to sample the microenvi- ronment of the light-organ crypts. Intact crypt contents, which emerge from the lateral pores of the organ as a thick paste-like exudate. were collected from anesthetized host animals that had been exposed to a light cue. Micros- copy revealed that the expelled material is composed of a conspicuous population of host cells in association with the bacterial symbionts, all of which are embedded in a dense acellular matrix that strongly resembles the bacte- ria-based biofilms described in other systems. Assays of the viability of expelled crypt cells revealed no dead bac- terial symbionts and a mixture of live and dead host cells. Analyses of the ultrastructure, biochemistry, and phago- cytic activity of a subset of the host cell population sug- gested that some of these cells are macrophage-like mol- luscan hemocytes. Introduction The microenvironment surrounding extracellular bacte- rial symbionts that associate with animal hosts is the dy- namic zone of interchange between the partners. In a number of associations, most notably the cow rumen (Flint, 1997) and the termite hindgut (Breznak, 1982; Received 30 April 1998; accepted 22 July 1998. * To whom correspondence should be addressed. E-mail: mcfallng hawaii.edu Breznak and Brune, 1994), the characterization of this interface has provided insight into the basic nature of the symbiosis, including aspects of nutrient exchange, host immune response, and the control of symbiont number. In such consortial associations, the contribution of any given symbiont species to the dynamics of the whole has been difficult to assess. Thus, the precise mechanisms by which the environment is created and maintained have not been determined. The symbiosis between the Hawaiian bobtail squid Eu- pryinna scolopes and its bioluminescent bacterial partner Vibrio fischeri provides a research system complementary to the more prevalent consortial symbioses (Ruby, 1996; McFall-Ngai and Ruby, 1998). Because both partners are cultivable outside of the symbiosis, and the bacterial sym- biont can be genetically manipulated, this two-species association can be used to study the underlying biochemi- cal and molecular contributions of each partner to the dynamics of the symbiosis. The microenvironment surrounding the bacterial sym- bionts in this symbiosis can now be readily analyzed (Graf and Ruby, 1998). The squid houses its extracellular sym- bionts in the epithelial crypts of a conspicuous bilobed light organ located in the center of its mantle cavity (McFall-Ngai and Montgomery, 1990). Lee and Ruby (1994) and Boettcher et al. ( 1996) showed that the symbi- osis is characterized by a pronounced diel rhythm, one aspect of which is the daily venting of 90% of the bacterial culture into the surrounding seawater. At dawn, each day, the animal expels the bacteria-containing crypt material into the mantle cavity through lateral pores on either side of the light organ (see Fig. 1 ). This daily venting appears to have a variety of consequences for the symbiosis. Lee 89 90 S. V. NYHOLM AND M. J. McFALL-NGAI and Ruby ( 1994) provided evidence that, in addition to the obvious function of controlling symbiont number, venting increases population densities of V. fischeri in the ambient seawater, which is essential for the horizontal transmis- sion of the symbiosis between generations; specifically, they showed that the natural seawater is only infective to newly hatched squids when it is sampled from environ- ments with large populations of adults. Under laboratory conditions, Graf and Ruby ( 1998) exploited this diel vent- ing behavior of the host to show that the squid host pro- vides the bacteria in the symbiosis with amino acids. In the present study, we explore the cellular compo- nents of the vented crypt contents. Our data show that the host vents a dense assemblage of its own cells in association with the bacterial symbionts. This mixed pop- ulation of cells is embedded in a conspicuous acellulur matrix. Among the host cell population, there is a subset of cells that have a morphology, biochemistry, and phago- cytic activity suggestive of macrophage-like molluscan hemocytes. In addition, we demonstrate that the animals can be experimentally induced to vent their crypt contents at times of day other than dawn, a feature that will provide an opportunity for future studies of diel fluctuations in the microenvironment of the light organ crypts. Materials and Methods General procedures Adult E. scolopes were collected with the use of dip nets from shallow, subtidal regions surrounding Oahu, Hawaii, and were either transported to the University of Southern California, Los Angeles, and maintained in a 265-liter recirculating aquarium at 23C, or maintained in flow-through seawater aquaria at Kewalo Marine Labo- ratory, University of Hawaii, Manoa. All chemicals were obtained from Sigma Chemical Co. (St. Louis, Missouri) unless otherwise stated. Fixa- tives, embedding media, and supplies for electron mi- croscopy were purchased from Ted Pella, Inc. (Redding. California). Characterizing natural venting behavior and ohtainini; crypt contents We used the methods of Graf and Ruby ( I99S) to in- duce venting behavior and to acquire the contents of the light organ crypts for further analysis. Briefly, adult ani- mals were maintained under natural environmental light conditions of about 12 h of light and 12 h of darkness. Minutes prior to dawn, just before venting normally oc- curs (Lee and Ruby, 1994), animals were anesthetized in 2% ethanol in seawater. Under red light, we made a midventral transection of the mantle to expose the light organ. The animals were then subjected to a constant light stimulus, using a 150-W halogen light source placed several centimeters above the eyes. Either immediately or within 60 min of the onset of the stimulus, they vented their light organ contents. This variation in the timing of venting following the stimulus did not affect the factors that were characterized in this study. As they were vented, the crypt contents were collected with a 5-pl hematocrit tube fitted with a plunger. To determine whether light, and not another aspect of our procedure (i.e., anesthesia or dissection), was the necessary stimulus for the induc- tion of venting, animals were anesthetized during their natural dark period and dissected, but not given a light stimulus. The possibility that venting behavior occurs in newly hatched squid at the first dawn following hatching was tested as follows. Juvenile squid were anesthetized for 60 s in a solution containing 0.37 M MgCL and seawater (1:1). Acridine orange was added to the solution to a final concentration of 5 ng/ml. which uniformly stained the host cells. The light organs were exposed by removing the mantle tissue, animals were exposed to a light stimu- lus, and the vented contents were viewed under confocal microscopy. Preparation for electron microscopy For scanning electron microscopy (SEM), the exuded crypt contents were placed on a nitrocellulose membrane, which was then immersed and fixed for 15 min in a solu- tion of 5% formalin in filtered seawater (FSW). The sam- ples were rinsed in FSW, dehydrated in a 15%- 100% ethanol series, desiccated with hexamethyldisilazane, sputter coated with gold, and examined with a Cambridge 360 scanning electron microscope. For transmission electron microscopy (TEM), the exu- date was fixed in 4% glutaraldehyde in 0.1%' sodium cacodylate with 0.45 M NaCl, pH 7.4 (Fixative A), for 30 min. Samples were rinsed for 15 min in 0.1% sodium cacodylate with 0.45 M NaCl, pH 7.4 (Buffer A) and then postnxed in 1% osmium tetroxide in Buffer A for 20 min followed by rinsing with Buffer A for 10 min. Samples were then dehydrated through a graded series from 30% to 100% ethanol in distilled water followed by infiltration with propylene oxide. Samples were placed in a 50:50 mixture of propylene oxide and unaccelerated Spurr (Spurr, 1969) for 15 min. and then transferred first to 100% unaccelerated Spun' for 3 h, and then 100% acceler- ated Spurr for 2 h. Samples were embedded in freshly prepared accelerated Spurr at 67C for 48 h. The embed- ded exudate was sectioned (80 to 90 nm thick), stained with Reynolds lead citrate solution and 3% uranyl acetate, and viewed with a JEOL CX-100 transmission electron microscope. To determine whether the composition of vented mate- SAMPLING OF A SYMBIOTIC M1CROENVIRONMENT 91 rial was influenced by the experimental procedure by which it was obtained (i.e., from anesthetized, ventrally dissected animals), we analyzed the crypt contents in in- tact, unanesthetized animals just prior to venting. Whole adult animals were placed at dawn directly in Fixative A. The light organs were then dissected out and prepared for TEM as described previously (McFall-Ngai and Mont- gomery, 1990). Analysis of animal ami bacterial cells in the exudate The total numbers of bacterial and animal cells present in fresh exudate were determined for adult specimens \n = 3; average mantle length, 25 mm]. To quantify cultura- ble bacterial cells per microliter in the exudate, serial dilutions of fresh exudate were plated on seawater tryp- tone (SWT) agar medium and allowed to incubate at 25C overnight (Ruby and Asato, 1993). The resulting colonies were counted, giving the number of colony forming units (CPUs) in the exudate material. This plating technique detects culturable bacterial cells with greater than 95% efficiency (Ruby and Asato, 1993). In situ viability of bacterial cells in the exudate was determined by fluores- cence viability staining (BacL\ght Live/Dead Viability Assay Kit, Molecular Probes. Eugene, OR). We used two nucleic acid stains, which are provided separately and mixed immediately prior to application: SYTO-9, which is taken up by all cells and fluoresces green, and propid- ium iodide, which fluoresces red and is only taken up by dead or dying bacterial cells with damaged membranes (Lloyd and Hayes. 1995). Cells were observed by confo- cal microscopy immediately after collection, and then 2 h later to ensure that propidium iodide had penetrated dead V. fischeri. The number of animal cells was determined as follows. Fresh exudate of a known volume was placed in a Petroff- Hauser hemacytometer, and the cells were either counted under phase contrast microscopy or stained with acridine orange and counted under fluorescent microscopy. Viabil- ity of animal cells in the exudate was determined by exposing the material to trypan blue, which is excluded from healthy animal cells but taken up freely by dead and dying cells with compromised membranes. Samples were exposed to 0.1% trypan blue in FSW for 3.0 min, rinsed in FSW, and examined under light microscopy. To determine the abundance of acidic compartments (e.g., lysosomes) within the animal cells, fresh exudate was exposed to a 1 fj,M solution of LysoTracker Green (Molecular Probes, Eugene, OR) for 30 min, rinsed in FSW, and viewed under confocal microscopy. Lyso- Tracker consists of a fluorophore moiety linked to a weak base that permeates cell membranes; it concentrates in cellular compartments with low internal pH. such as lyso- somes, where it fluoresces under acidic conditions (Diwu et al., 1994). Experimental manipulation of venting behavior To determine whether crypt contents could be obtained at times other than dawn, we attempted to induce venting behavior by exposing E. scolopes adults to our experi- mental conditions (i.e., anesthetic, ventral dissection, and a light stimulus) at different times during the day. Spe- cifically, we assayed in the hours preceding [-12 h. -9 h, -6 h. -4 h. and -I h] and following [+4 h. +6 h. +8 h, and +10 h] dawn, when the light organ would presumably have either abundant or depleted crypt con- tents, respectively. In addition, to determine whether the crypt contents would be retained past dawn in the absence of the natural light cue, adult animals were maintained in the dark for up to 8 h after their natural venting time and then exposed to our experimental conditions. Results Natural venting behavior After a continuous light stimulus of 5-60 min, the re- lease of the crypt contents (Fig. 1) was preceded by a large contraction of the light organ. The exudate that resulted from this event emerged from the pores of the light organ as a thick, white, paste-like material (Fig. IB). An adult animal with a mantle length of 25 mm typically vented between 10 and 20 /jl of exudate material, which was then collected intact for further analyses. Animals that were anesthetized and ventrally dissected, but not exposed to a light stimulus, did not expel their crypt contents neither during the natural dark cycle nor after dark adaptation. Therefore, a light stimulus is required to induce venting, and the experimental conditions them- selves did not cause this behavior. Under the experimental conditions used for adults, ju- venile animals also exhibited venting behavior beginning with the first dawn after hatching. Large eukaryotic cells of a diameter similar to those seen in adults (about 10 /vm) were observed emerging from the pores leading to the juvenile crypts (Fig. 1C). Cellular constituents of the exudate Observations of exudate under light microscopy re- vealed a mixture of animal and bacterial cells. SEM of the exudate showed that the population of animal and bacterial cells is surrounded by a dense matrix (Fig. 2A- C). Animal cells in the exudate were 10 to 20 /j,m in diameter. Their membranes often had a ruffled appear- ance, and cytoplasmic blebbing was regularly observed, but these cells otherwise appeared healthy (Fig. 2D). Both SEM and TEM revealed a close association between the animal cells and bacterial cells within the exudate, with bacterial cells often adhering to the surface of the eukary- otic cells (Fig. 2C-D). TEM of the exudate matrix 92 S. V. NYHOLM AND M. J. MrFALL-NGAl Figure 1. Venting of crypt contents by Euprymna xcolnpes. (A) Ventral dissection of an adult, revealing the bilobed light organ (arrow). Scale, 5 mm. (B) Exudate emerging from one of the lateral pores of the light organ. The exudate can be seen lying across the digestive gland (yellow), which is directly dorsal to the light organ. Scale. 1 mm. (C) Animal cells (white arrow) emerging from the pore of a 48-h juvenile squid. Scale, 25 fjm. p. pore; e. exudate; dg, digestive gland. showed a mixture of participate material suggestive of cell membranes, possibly of lysed host cells (Fig. 2D). Based on counts of CPUs, an average of 2.8 x 10" ( 1 .3 X 10'', n = 3) bacterial cells were contained within the exudate of an E. scolopes adult. After venting, homog- enates of the bacteria-containing crypt epithelia of the light organ of these same animals contained, on average, 10 X bacterial cells; thus, 5% to 10% of the symbiont popu- lation remained in the hosts. This number of bacteria corresponds to estimates of symbiont retention in animals that were allowed to vent naturally (Lee and Ruby, 1994). In adult squid (average mantle length, 25 mm), the num- ber of animal cells in the exudate, as determined by light and fluorescence microscopy, averaged between 10' and 1(V total cells. Control studies with fixed, intact, unvented light organs showed animal and bacterial cells in the collection ducts leading up to and in the crypt spaces (Fig. 3). These cells were similar in appearance to cells found in freshly vented exudate samples. TEM revealed that the accompanying matrix material was also similar to that observed in exu- date (data not shown). Viabililv oj animal and bacterial cells About half the freshly vented animal cells that were stained with trypan blue were observed to exclude the dye, indicating that they were viable. The remainder took up the blue stain, indicating that they were dead or dying cells with compromised membranes (Fig. 4A). Staining patterns under confocal microscopy, with two nucleic acid stains that distinguish live and dead bacterial cells, indi- cated that the vast majority of bacteria in the exudate are viable (Fig. 4B). To ensure that these nucleic acid stains could reveal dead cells, the crypt contents were stained 2 h after being expelled. An increase in bacterial cells fluorescing red was observed 2 h after venting, so the propidium iodide revealed dying or dead V. fisclieri (data not shown). Acidic compartments of animal cells in the exudate The animal cells in the exudate were examined for the presence of acidic compartments characteristic of phago- cytic or macrophage-like cells. To this end, fresh exudate stained with LysoTracker dye was observed with both light and fluorescence microscopy. Under differential in- terference contrast (DIG) microscopy, exudate cells ap- peared to be surrounded by matrix and adherent bacterial cells (Fig. 5A). In the presence of LysoTracker, fluores- cent compartments were seen within the animal cells, whereas the nucleus and surrounding extracellular matrix, as well as bacterial cells in the mixture, showed no fluo- rescence (Fig. 5B). SAMPLING OF A SYMBIOTIC MICROENVIRONMENT 93 V < I i V Vi . ***' *l " v -$ + & ' i Figure 2. Electron micrographs of freshly collected exudate (A-C were taken with a scanning scope, and D with a transmission scope). (A) Bacterial cells embedded in a thick matrix. Scale, 3 pm. (B) Higher magnification, showing individual bacteria (arrows) surrounded by the matrix. Scale, 1 ^m. (C) The outline of animal cells surrounded by bacteria (arrows). Scale, 2 /jm. (D) Bacterial cells (arrows) adjacent to a larger animal cell. The membrane exhibits ruffling, and cytoplasmic blebbing is evident. The bacteria are surrounded by a mixture of paniculate matter that constitutes the matrix. Scale, 1 fan. m. matrix; n. nucleus; b, cytoplasmic blebbing of the host cell. Evidence of bacterial phagocytosis in light organ cells Eukaryotic cells in the crypt spaces of intact light or- gans occasionally contained intracellular bacteria (Fig. 6). Like the majority of the animal cells within the exudate, these cells averaged 10 /jm in diameter and often con- tained 3-5 bacterial cells each. The bacteria appeared to be contained within membrane-bound vacuoles of the animal cells, and many of these bacterial cells appeared to be undergoing degradation. Manipulation of venting behavior Under our experimental conditions, we were able to induce venting behavior between 1 and 12 h prior to dawn, i.e., during the dark portion of the animal's natural cycle the time period when the bacterial population of the light organ is at its highest. This behavior could not be induced, however, during the light portion of the animal's natural cycle; this is the period between 4 and 10 h after dawn, when the crypt spaces should have the lowest amount of extracellular constituents, i.e., matrix material, as well as animal and bacterial cells. Animals kept in the dark past dawn retained their crypt contents until given a light stimulus. Under these condi- tions, we could induce expulsion up to 8 h past the natural venting time. These data suggest that the behavior is not under an independent circadian rhythm, but requires a light cue each day. Discussion The opportunity to sample the microenvironment sur- rounding bacterial symbionts is rare because, in most as- sociations, the bacteria are embedded deeply within tis- sues that are difficult to access. In the present study, taking 94 S. V. NYHOLM AND M. J. McFALL-NGAI .| " W" A Ife Jr I ' : ;;.-- .-..,. -^Sif~ t - .' -9" VSK-T ' V*,'^; ';.'V I ,[>'.- /'i 1 A'tjt-,. > ifefei > : ^ f -v " Figurc 3. Exudate in the duct and anterior crypt spaces of a sectioned adult light organ. The image is a montage of light micrographs of tissue that was fixed at dawn, just before the natural venting. The typical experimental conditions that produce venting (e.g.. anesthetic and light stimulus) were not applied. Eukaryotic cells (arrows) in the crypt and duct spaces are surrounded hy bacterial cells. The sample was stained with 2% toluidine blue. Scale, 20 fim. advantage of a natural behavior of the host squid, we have described the cellular components of the microenvi- ronment of the symbiotic light organ and have determined the feasibility of sampling this microenvironment under experimental conditions. These studies have revealed that ( 1 ) the diel venting \ * Figure 4. Viability of cells in the exudale. (A) Results ot staining the exudate with ().17r trypan blue to test viability of animal cells. The exudate contains a mixture of living cells, which exclude the stain and remain a golden hue (small arrow), and dead or dying cells, which do not exclude the stain (large arrow). The matrix also interacted with the dye and appears blue. Magnification. 400-'. Scale. 10 /vm. (B) Results ol two nucleic acid fluorescent stains that distinguish viable from dead bacterial cells. The vast majority of freshly emerged bacterial cells were viable, as indicated by the green field of bacteria surrounding the animal cell. Scale, 5 ;/m. SAMPLING OF A SYMBIOTIC MICROENVIRONMENT 95 Figure 5. Stained acidic compartments within a freshly vented animal cell. (A) Differential interference microscopy; the cell is surrounded by bacteria and matrix material. Scale, 10 /jm. (B) The same cell stained with I /jM LysoTracker; note the acidic compartments, possibly lysosomes. Scale, 10 pm. of the symbiotic organ contents by the host squid provides an intact sample of the light organ crypt microenviron- ment; (2) this diel behavior, which occurs in juveniles as well as adults, is dependent upon a light cue; (3) the cellular constituents of the crypts consist of a population of viable V. fischeri cells in association with a mixture of live and dead host cells; (4) the characteristics of some of the animal cells found in the exudate suggest that these cells are molluscan phagocytic hemocytes (Cheng, 1981; Sminia, 1981; Cowden and Curtis, 1981); (5) these host and bacterial cells are embedded in a conspicuous matrix Figure 6. Bacteria within a crypt cell. A TEM of a eukaryotic cell found within the crypt spaces of a juvenile E. scolopes light organ. This cell contains several intracellular bacteria (arrows). Scale. I pm. similar in appearance to other bacteria-based biofilms; and (6) the venting of crypt contents can be experimentally induced at times in the diel cycle other than dawn. Our finding that about 90% of the symbiont culture is vented suggests strongly that this behavior is controlling the number of symbionts in the light organ (Lee and Ruby, 1994; present study). Research on symbiotic associations between animals and their bacterial partners has demon- strated that symbiont population density is regulated in a variety of ways. These control mechanisms can be grouped into two broad categories that work either alone or in concert: control of bacterial growth rate and elimina- tion of excess bacterial cells. In the former mode, the host environment, while sustaining the viability of the symbionts, presents a biochemical milieu that attenuates bacterial cell division. In the latter, elimination of super- numerary symbionts is typically accomplished by the reg- ular venting of the bacterial cells from the host tissues, the digestion by the host of the bacterial cells, or both. Digestion can be either extracellular or intracellular, the latter typically following the engulfment of the bacterial cell by a phagocytic host cell. Previous research and the results presented here provide evidence for a multifaceted control of the symbiont population in the squid-vibrio symbiosis. Lee and Ruby (1994) demonstrated that the bacterial growth rate in the adult light organ (average doubling time of approximately 4.8 h) is suppressed in comparison with the physiological potential for growth seen under culture conditions (average doubling time of 0.5 h; Ruby and Asato, 1993). However, the former growth rate is still sufficiently high that, although the numbers of bacteria are set back at dawn to a lower popu- lation density by the venting behavior, the light organ will be repopulated with symbionts over the course of the day. Slow growth rates, coupled with shedding of symbionts into the environment, have been documented previously in symbioses between animal hosts and their 96 S. V. NYHOLM AND M. J. McFALL-NGAI luminous bacterial partners, although no rhythm or depen- dency on cues was noted (Haygood et al.. 1984). In addition to the suppression of growth rate and the diel venting of symbionts, the phagocytosis of bacterial cells by host macrophage-like cells that we observed in this study (Fig. 6) may represent yet another level of control of the symbionts in these associations. Tebo et nl. ( 1979) showed bacteria in host cells in the light organ of the monocentrid fish Monocentris japonicux, whose light organ contains the symbiont V. fischeri. However, those bacterial cells were in the epithelium rather than in the macrophage-like cells seen in the light organ of E. scolopes. At this point, neither they nor we have deter- mined whether the bacteria within host cells are V. fischeri or other bacterial species that are being eliminated to maintain V. fischeri as the sole symbiont; i.e.. we do not know whether the macrophage-like host cells in the squid light organ function principally in control of V. fischeri symbiont number or in maintenance of light organ speci- ficity, or both. Thus, the determination of the precise function of these cells in the dynamics of this symbiosis awaits their further characterization. However, to our knowledge, this report represents the first documentation of macrophage-like cells in direct association with the symbionts in a light organ. Whatever the function of these host macrophage-like cells in the crypt spaces, their association with the popula- tion of bacterial symbionts is not unexpected. In molluscs, macrophage-like hemocytes are reported to be involved in phagocytosis and digestion of pathogens in many tis- sues and their associated lumina (Cheng, 1981; Sminia, 1981; Cowden and Curtis. 1981). Vertebrate macro- phages, which are thought to be derived evolutionarily from this invertebrate cell type (Ottaviani and Franceschi, 1997). are also found in similar circumstances. For exam- ple, mononuclear phagocytes are released into the blood stream and spread to all tissues including epithelia (Stew- art et til., 1994), and lymphocytes are a common constit- uent of the cellular community that lines the mammalian intestine (James and Zeitz, 19941. The other animal cells in the crypt environment that do not resemble healthy, functioning macrophage-like molluscan hemocytes may be dead or dying hemocytes, or they may be epithelial cells shed from the lining of the crypt spaces. The shedding of epithelial cells in re- sponse to interaction with bacteria or bacterial by-prod- ucts is known to occur in other instances. For example, bacterial lipopolysaccharide will induce shedding of mu- rine uroepithelial cells, which is believed to be a mecha- nism by which the host evades pathogenic bacteria ( Aron- son et id., 1988). The exact definition of the host cell types in the crypt space and the origin of these cells, thus, also awaits future studies. All of the bacterial and animal cells contained in the exudate are suspended in a thick, heterogeneous matrix. SEM showed that this matrix resembles other bacteria- associated biofilms, including those associated either with inorganic substrates or animal tissues (Pearl, 1985). TEM revealed a complex mixture of membranes and particulate matter. Matrix ultrastructure of the squid light organ appears similar to that described in the light organs of the macrourid fish Coelorhincns (W. Loh, University of Sydney, pers. comm.). The exact nature of the constituents, as well as the relative contributions of the animal host and the bacterial symbionts to the matrix material, remain to be determined. To date, only one constituent has been defined, i.e., the host-derived amino acids that supply the growing symbiont popula- tion with these metabolic building blocks (Graf and Ruby, 1998). The finding that venting behavior can be induced exper- imentally during the 12 h preceding the natural expulsion event at dawn paves the way for examination of the micro- environment of the crypts during this portion of the diel cycle of the symbiosis. Boettcher et al. ( 1996), in studies of the diel rhythm of the light organ, showed that this 12-h period contains the highest and lowest per cell lumi- nescence of V. fischeri. They interpreted this finding to suggest that significant fluctuations in the biochemistry and physiology of the crypt environment occur between the hours around dusk and the hours around dawn. After venting, the remaining 10% of the symbiont population divides and repopulates the light organ, so that by dusk, there is once again a full complement of bacteria in the crypts. Whether the animal delivers nutrients steadily over the period of about 1 2 h following venting, sustaining a slow bacterial growth rate over this period, or whether it maintains a low bacterial population until the late after- noon, when it delivers a large pulse of nutrients that stim- ulates rapid bacterial growth, remains to be determined. Defining changes in the biochemistry of the microenvi- ronment during the day may provide insight into the mechanisms by which luminescence is controlled. In ad- dition, such analyses will define other types of metabolic and regulatory changes that accompany the diel rhythm of this symbiosis. At present, we cannot experimentally induce venting of the crypt contents during the light por- tion of the animal's cycle. Thus, a full understanding of the dynamics of symbiont population growth in the light organ over the entire day awaits refinement of our current method or reliance on alternative methods of sampling this environment. The accessibility of the bacterial microenvironment in this symbiosis makes a rich frontier available for the study of the dynamics of an animal-bacterial association. Future studies will focus on defining both the biochemical con- stituents of the matrix material and the nature of the ani- mal cells that occur among the bacterial symbionts. SAMPLING OF A SYMBIOTIC MICROENVIRONMENT 97 Acknowledgments We thank B. DeCaires, J. Doino, J. Foster. J. Kimbell, L. Lamarcq, C. Phillipson, E. Ruby. E. Stabb, and K. Visick for helpful comments on the manuscript. We are also grateful for technical assistance from A. Thompson and W. Ormerod of the Center for Electron Microscopy and Materials Analysis at the University of Southern Cali- fornia. This work was supported by NSF grant IBN 96- 01155 to MMN and E.G. Ruby and NIH grant RO1 RR10926-O1A1 to E. G. Ruby and MMN. Literature Cited Aronson, ML, O. Medalia, D. Amichay, and O. Nativ. 1988. Endo- toxin-induced shedding of viable uroepithelial cells is an antimicro- bial defense mechanism. Infect, lininim. 56: 1615-1617. Boettcher, K. J., E. G. Ruby, and M. J. McFall-Ngai. 1996. Biolumi- nescence in the symbiotic squid Euprymna scolopes is controlled by a daily biological rhythm. J. Comp. Physiol. A. 179: 65-73. Breznak, J. A. 1982. Intestinal microbiota of termites and other xy- lophagous insects. Anint. Rev. MicrobioL 36: 323 343. Breznak, J. A., and A. Brune. 1994. Role of microorganisms in the digestion of lignocellulose by termites. Aniui. Rev. Entonwl. 39: 453_487. Cheng, T. C. 1981. Bivalves. Pp. 233-300 in Invertebrate Blood Cells. N. A. Ratcliffe and A. F. Rowley, eds.. Academic Press, New York. Cowden, R. R., and S. K. Curtis. 1981. Cephalopods. Pp. 301 -323 in Invertebrate Blood Cells. N. A. Ratcliffe and A. F. Rowley, eds.. Academic Press, New York. Diwu, Z., Y. Z. Zhang, and R. P. Haugland. 1994. Novel site-selec- tive fluorescent probes for lysosome and acidic organelle staining and long-term tracking. Cytometry 77: 426B. Flint, H. J. 1997. The rumen microbial ecosystem some recent de- velopments. Trends Microhinl. 5: 483 188. Graf, J., and E. G. Ruby. 1998. Host-derived amino acids support the proliferation of symbiotic bacteria. Proc. Nut!. Acad. Sci. USA 95: 1818-1822. Haygood, M. G., B. M. Tebo, and K. H. Nealson. 1984. Luminous bacteria of a monocentnd fish (Moiiocentris japonicus} and two anomalopoid fishes (Photoblepharon palpebratus and Kryptopha- naron alfrediY. population sizes and growth within the light organs, and rates of release into the seawater. Mar. Biol. 78: 249-2S4. James, S. P., and M. Zeitz. 1994. Human gastrointestinal mucosal T cells. Pp. 275-285 in Handbook of Mucosal Immunology, P. L. Ogra, M. E. Lamm. J. R. McGhee, J. Mestecky, W. Strober. and J. Bienenstock. eds.. Academic Press, San Diego. Lee, K., and E. G. Ruby. 1994. Effect of the squid host on the abun- dance and distribution of symbiotic Vibrio fischeri in nature. Appl. Em-iron. Microbiol. 60: 1565-1571. Lloyd, D., and A. J. Hayes. 1995. Vigour, vitality, and viability of microorganisms. FEMS MicrobioL Let!. 133: 1-7. McKall-Ngai, M., and M. K. Montgomery. 1990. The anatomy and morphology of the adult bacterial light organ of Euprymna scolopes Berry (Cephalopoda: Sepiolidae). Biol. Bull. 179: 332-339. McFall-Ngai, M. J., and E. G. Ruby. 1998. Sepiolids and vibrios: when first they meet. BioScience 48: 257-265. Ottaviani, E., and C. Franceschi. 1997. The invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroen- docrine systems, linmunol. Todav 18: 169-174. Pearl, H. W. 1985. Influence of attachment on microbial metabolism and growth in aquatic ecosystems. Pp. 363-400 in Bacterial Adhe- sion: Mechanisms and Physiological Significance, D. C. Savage and M. Fletcher, eds.. Plenum Press, New York. Ruby, E. G. 1996. Lessons from a cooperative bacterial-animal asso- ciation: the Vibrio fischeri-Euprymna scolopes light organ symbiosis. Annu. Rev. Microbiol. 50: 591-624. Ruby, E. G., and L. M. Asato. 1993. Growth and flagellation of Vih- rio fischeri during initiation of the sepiolid squid light organ symbio- sis. Arch. Microbiol. 159: 160-167. Sniinia, T. 1981. Gastropods. Pp. 191-232 in Invertebrate Blood Cells. N. A. Ratcliffe and A. F. Rowley, eds.. Academic Press, New York. Spurr, A. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26: 31-43. Stewart, C. C., M. C. Riedy, and S. J. Stewart. 1994. Induction: the proliferation and differentiation of macrophages. Pp. 3-27 in Macrophage-Pathogen Interactions. B. S. Zwilling and T. K. Eisenstein. eds.. Marcel Dekker, New York. Tebo, B. M., D. S. Linthicum, and K. H. Nealson. 1979. Luminous bacteria and light emitting fish: ultrastructure of the symbiosis. Bio- Svstems 11: 269-280. Reference: Bid/. Bull. 195: 98-106. (October, 1998) Transplantation of Fu/HC-Incompatible Zooids in Botryllus schlosseri Results in Chimerism BARUCH RINKEVICH', IRVING L. WEISSMAN : . AND ANTHONY W. DE TOMASO : * * National Infinite of Oceanography, Tel Shikmona, P.O. Box 8030, Haifa 31080. Israel: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305: and Hopkins Marine Station, Pacific Grove, California 93950 Abstract. The colonial urochordate Botryllus schlosseri undergoes a genetically defined, natural transplantation reaction that is controlled by a single Mendelian locus (called the Fu/HC). This Fu/HC-based allorecognition system is initiated when peripheral elements of the vascu- lature interact on the edges of two asexually expanding colonies. To better understand the spatial organization of the cellular elements responsible for Fu/HC-based allo- recognition, we bypassed the normal site of interaction (the ampullae) and experimentally transplanted zooids be- tween Fu/HC-noncompatible Botryllus schlosseri pairs. The results show that ( 1 ) instead of the expected rejec- tions (tissue necroses) that develop after natural contacts between peripheral blood vessels, the transplanted organs are morphologically eliminated within a few days in con- junction with the normal blastogenic cycle; and (2) donor- recipient chimerism is established after complete morpho- logical elimination of transplanted tissues. These results suggest that Fu/HC-based allorecognition responses in Botryllus schlosseri occur exclusively at the ampullae and that once cells have crossed this barrier, they are able to survive and proliferate in the new host colony. Introduction Colonies of the urochordate Botryllus schlosseri un- dergo a genetically defined, natural transplantation reac- lion following allogeneic contacts between peripheral blood vessels. Upon contact, the allogeneic vessels either fuse to form a single colony with a common blood supply or reject each other in a blood-based, inflammatory reac- tion, after which the two colonies no lonuer interact. His- Received 5 December 1997; accepted 8 June 1998. * To whom correspondence should be addressed. E-mail: leland.stantord.edu tdet(s' tocompatibility discrimination resides in a single, highly polymorphic fusion/histocompatibility locus (called the Fu/HC; Scofield el al., 1984) with a large number of codominantly expressed alleles (Rinkevich et al., 1995). Allogeneic fusion occurs between colonies that share at least one Fu/HC allele; in contrast, partners will reject each other if they share no Fu/HC alleles (Bancroft, 1903; Oka and Watanabe. 1957, 1960; Sabbadin, 1962; Scofield et al.. 1982). Several interesting phenomena may occur after two colonies have undergone a Fu/HC-mediated fusion event. In the laboratory, we have observed that, after fusion, the genetic colonial descendants (zooids) from one partner in the chimera cease normal development, and these dying zooids are resorbed by massive phagocytosis, leaving the zooids of the other colony intact (Rinkevich and Weiss- man, 1987). This phenomenon, called colony resorption, occurs at the end of the synchronized weekly blastogenic cycle in which the old generation of zooids dies through a programmed apoptotic event (Lauzon et al.. 1993). We have used genetically defined laboratory colonies to show that the ability of one colony to resorb another is consis- tent, and that different genotypes can be grouped into a reliable hierarchy. However, analysis of the segregation of these traits in defined crosses suggests that it is a complex phenomenon involving at least several loci, possibly in- cluding the Fu/HC locus (Rinkevich et al.. 1993). Thus, resorption in Botryllus has been compared to the minor histocompatibility loci seen in the vertebrates (Rinkevich, 1993). Several laboratories have also shown that, after fusion, each colony can exchange germ and somatic stem cells which are then able to survive and replicate in the alloge- neic colony. In fact, as first described by Sabbadin and Zaniolo ( 1979), the germ cells from one colony can actu- ally completely parasitize the other colony in a fused 98 A I I OKI ((HiNIIION IN A I'I )T( )C'H( )KI > \ I I 99 chimera, such that only one of the genotypes is repre- sented in the mature germ cells. This can occur up to a month after two fused colonies have been experimentally separated, demonstrating that cells from one colony sur- vive and proliferate in the other (Sabbadin and Zaniolo, 1979; Pancer et ai, 1995; Stoner and Weissman, 1996). These two postfusion events appear to be opposite in nature and are difficult to reconcile. On one hand, the resorption phenomenon suggests that in an allogeneic chi- mera there is still a form of directed allorecognition oc- curring, and the ability to demonstrate a hierarchy among laboratory-bred colonies suggests a genetic component to this process. Conversely, the precursors of germ and somatic cells clearly can proliferate inside an Fu/HC- matched, allogeneic colony, suggesting that there is not a secondary form of allorecognition occurring, particu- larly on a global scale. This is further complicated by recent observations that, in a fused colony in which one of the partners had been resorbed. only the genotype of the resorbed partner was present in the germ line. Thus a somatic loser (by resorption) can be a gametic winner (by successful germ cell competition) (Pancer el /., 1995: Stoner and Weissman, 1996). The above observations led us to ask whether any allo- geneic effector systems are present throughout the entire colony. To investigate this question, we decided to cir- cumvent the natural manifestation of incompatibility, which occurs when peripheral blood vessels interact, by grafting whole zooids between colonies with rejecting Fu/ HC genotypes. Results from these experiments provide further evidence that Fu/HC-mediated allorecognition. the rejection effector system, or both, may be restricted to the ampullar tips, suggesting that allorecognition re- sponses are not a colony-wide phenomenon in Botrylhis. Materials and Methods Animals We used colonies of B. schlosseri that were originally collected from the Monterey Marina (California) but have been growing in the laboratory at Haifa. Israel, and at Hopkins Marine Station, Pacific Grove, California. Colo- nies were born and reared separately on glass slides (5 x 7 cm) in glass staining racks within 1 7-1 tanks as described (Boyd et al., 1986). Subclones of individual colonies were Fu/HC phenotyped using a cut colony assay (Rinkevich et til.. 1993), and rejecting pairs of colonies were identified. These subclones were not used in the transplantation assays. Transplantation Reciprocal transplantation of whole zooids between re- jecting B. schlosseri pair genets was performed during developmental stages A-C of the blastogenic cycle (de- scribed in Milkman, 1967). A longitudinal incision was made (with a thin needle) between the atrial and branchial siphons of a single zooid (highlighted in Fig. Ib). Both edges of the incision were then retracted with fine forceps. The incision went through the tunic and upper body wall of the individual zooid, and retraction of the edges ex- posed the body cavity, which contained the body of the zooid, the bud, and the gonads (see Berrill, 1941, for an in-depth description of B. schlosseri anatomy). The zooid was lifted and removed with a tine needle and forceps, leaving behind a "cup" consisting of the outer and lower tunics lined with epithelium, connective tissues, cut blood vessels, and. in many cases, all or part of the buds and the gonads. Bleeding from the cut blood vessels (either from the removed zooids or from the leftover vessels) was always seen. The removed zooid was then replaced by another zooid taken from a naive subclone of a re- jecting genet, or in the case of controls, from another subclone of the same genet. A control zooid and the ex- perimental zooid were transplanted within the same sys- tem, separated by 1-3 zooids (Exps. I-III, Table IA) or in adjacent systems (Exps. IV-VI, Table IB). The transplanted zooids were carefully inserted within the empty cups, one zooid per cup, pushed slightly inside with the blunt end of a forceps, and covered with the almost enclosing, cut surface of the outer tunic layer. In all cases of transplantation a mixture of blood cells com- ing from the introduced zooid and from the local bleeding was documented. Bleeding stopped within a few minutes. Tissue sampling Amplified fragment length polymorphism (AFLP) analysis was performed on tissue samples from Exps. I- III. Exps. lid and Hid died prematurely. Sampling was usually done 3-4 weeks after the final transplantation. Before tissue was sampled, each subclone was carefully checked under a dissecting microscope to determine whether the transplanted zooids had been completely re- sorbed. In one case (Exp. He. Table I) the whole subclone, which started to degenerate, was isolated as is. All other seven subclones were photographed, their general struc- ture was outlined, and three to seven tissue samples per subclone were separated with a razor blade and individu- ally snap-frozen in liquid nitrogen. Amplified fragment length polymorphism (AFLP) analysis All enzymes were purchased from New England Bio- labs (Beverly. Massachusetts), and the chemical reagents were purchased from Sigma (St. Louis, Missouri). Oligo- nucleotides were synthesized at the PAN facility at the Stanford University Medical School. Frozen tissue sam- ples were ground to a fine powder with a mortar and pestle. DNA was extracted on silica columns (Nucleo- 100 B. RINKEVICH ET AL. Table I Experimental procedures and major results fur allogt'nt'ic and isogeneic zooid transplantation Experiment # Allogeneic* combination Pair # Donor genotype Initial si/,e (# zooids)t Recipient Exp. system Major outcomes! A = One-month experiments I vs. 2 a I 3 vs. 4 III B = Two-month experiments IV VI 5 vs. 6 7 vs. 8 9 vs. 10 11 vs. 12 1 9 10 23 20 20 II 30 32 16 15 31 13 21 26 27 19 22 IS 30 31 1(1 4 repeated zooid transplantations (within 21 days from first event), all resorbed together with isogeneic controls and before the takeover phase of blastogenesis. No single case of FOR. The ramet died during the 5th transplantation, before completion. 9 Same as la. 6 sets done within 1 month. Resorption of transplanted allogeneic and isogeneic zooids in all cases stalled after transplantation and terminated during takeover. No FOR. Sampled for AFLP analysis. 7 Same as Ic: sampled for AFLP analysis. 13 Same as Ic; sampled for AFLP analysis. 10 Same as Ic; sampled for AFLP analysis. The first 4 sets of transplantations as in Exp. la. During the 5th set (day 25). the ramet degenerated and was sacrificed for AFLP analysis as one sample. No POR. 10 Morphology same as Ic. died on day 25, no further sampling. 7 Same as Ic; sampled for AFLP analysis. 6 Same as Ic; sampled for AFLP analysis. 10 Same as Ic; sampled for AFLP analysis. 12 Same as Ic; died prior to sampling. I 1 Same as Ic; but zooids transplanted within two months. No AFLP sampling was done on these experiments. 9 Same as IVa. 8 Same as IVa. 7 Same as IVa. 9 Same as IVa. 8 Same as IVa. * Each of the 12 genets was used in only one set of experiments. t The size of the recipient subclone at the day of the first transplantation, and the size of the experimental system within the recipient subclone on which both transplanted zooids (experimental and control) were introduced. Follow-up transplantations were performed on daughter zooids. produced through blastogenesis from intact buds, of these same systems. $ POR. point of rejection; AFLP, amplified fragment length polymorphism. bond C+T Kit. Macherey Nagel. Duren, Germany) using proprietary buffers according to the manufacturer's in- structions. AFLPs were performed as described pre- viously (Vos et al., 1995). Briefly, 200 ng of DNA was cut to completion with restriction enzymes Eco RI and Mse I for 2 h at 37C in a 30-^/1 reaction volume. Oligonu- cleotide adaptors, 1 mM ATP, and T4 DNA ligase were then added (total volume. 40 p\), and the incubation was continued for 3 h. The DNA was preamplified with one selective nucleotide on each primer (Eco RI = A; Mse I = T). The preamplirication mix was diluted 1:20 and 3 /jl was used for AFLP fingerprinting with each primer containing three selective nucleotides (Eco RI = ATg; Msel = TCg). The Eco RI primer was end-labeled with "P-ATP (New England Nuclear) using polynucleotide ki- nase. PCR reactions were diluted 1:1 in stop solution (98% formal-Hide, 10 mM EDTA pH 8, 0.1 <7r bromophe- nol blue, 0.1% xylene cyanol). denatured for 5 min at ALLORECOGNITION IN A PROTOCHORDATE 101 95C, and resolved on a standard sequencing gel at 70 W for 2.5 h. Gels were dried, and the autoradiograms were exposed for 36 h. Each experiment was repeated two times to ensure that the AFLP fingerprints were consis- tent. In some sets, different primer sets were used, and the results were equivalent (not shown). Results Twelve B. schlosseh colonies were organized into six rejecting pairs, providing six independent experiments (see Table I). Within each pair, two types of trans- plantations were done in parallel (described in the Meth- ods). Zooids were reciprocally transplanted between the two rejecting colonies to make allografts; and zooids were transplanted from another ramet of each colony to make isografts. Allograft and isograft transplantations were car- ried out within a single system of the recipient, usually a few zooids apart (see Fig. 1). There were no observed differences in response if allografts or isografts were done independently (not shown). The initial transplantation procedure was followed by four to six sequential transplantations over the course of 1 (Exps. I-III) or 2 (Exps. IV-VI) months (Table I). Multiple transplantations into the same ramet were done to test for the induction of a rejection response after re- peated exposures to the same allogeneic tissue. Trans- plantations were also done at different points of the blas- togenic cycle (Milkman. 1967) to test for any variability in the alloresponse. In Exps. I-III (Table IA) the subse- quent transplantation was performed immediately after the takeover phase of blastogenesis (Milkman, 1967), while in Exps. IV-VI (Table IB), one full blastogenic cycle separated the two sequential transplantations. Apart from the zooids that were completely excised during the transplantation procedure, other zooids in the experimental system and in all the other systems within the same colony were usually not affected. A few hours after transplantation, the implanted tissues were covered by the matrix of the cut tunic, sealing them within the recipient colony. Under normal conditions in a Botiyllns colony all zo- oids and buds are connected by vascular outgrowths to the colonial circulatory system (Milkman, 1967). Vascular anastomoses were not observed in any of the 1 14 alloge- neic and isogeneic zooids transplanted in these experi- ments. Although there was no long-term vascularization. hemocytes from the donor and recipient were in contact for several hours following the transplantation procedure: there was bleeding from the cut vasculature of the recipi- ent colony, as well as from the open circulatory system of the donor zooid. This allowed mixing of host hemo- cytes with donor hemocytes and tissue for several hours. After 12-24 h, the disconnected blood vessels of the colony regenerated, sometimes forming a circular pattern of blood vessels around the transplanted zooid ( not shown). Thus the allogeneic interactions in these experi- ments can be summarized as follows: the donor zooid was in contact with the recipient hemocytes, while the donor hemocytes were allowed to mix with the colonial circulation of the recipient, analogous to injection of he- mocytes across a Fu/HC incompatible barrier. Within 24 h, many of the implants were completely covered by the upper tunic wall (Fig. la). In some cases, especially when a large zooid was transplanted, part of the zooid extended out of the "cup." closed around by a "collar" made of the colony epidermal wall (not shown). Within the next few days, all of the implants degenerated, a phenomenon coinciding with the regular colony blasto- genesis (Fig. Ib) in which all zooids go through a sys- temic programmed apoptotic cycle, followed by massive phagocytosis (Lauzon et ai, 1992). There was no observ- able difference between resorption of isografts or allo- grafts (Fig. la, c). For instance, in Figure Ic, the isograft appears to be resorbing faster than the allograft. Any variations in the time scale of resorption (2-5 days) were related to the blastogenic cycle. More importantly, no visible points of rejection (FOR; Table I) were ever ob- served in these transplantations (Fig. la, b), although nat- ural contact assays done on other ramets of the same six pairs of genets (Table I) always resulted in typical, distinct FOR within 24 18 h after first ampullae contacts (not shown). After the takeover phase of blastogenesis, all or most parts of the allogeneic and isogeneic tissues were resorbed and had disappeared, leaving behind a clear tunic matrix or a space occupied by the new generation of developing zooids (Fig. Ib). In some cases, remnants were found trapped in the bare tunic (Fig. Id) in a manner similar to that recorded in regular colonies (Rinkevich and Weissman, 1987). Two major morphological variations in the outcomes of transplantation were documented, although neither was related to the type of transplant (autograft or allograft). The first was a partial resorption of an intact zooid border- ing the area of transplantation (Fig. Ic), probably resulting from the experimental manipulation of zooid excision and transplantation. Morphological resorption as a result of stress conditions has already been documented in Botiyl- Ins (Rinkevich etui., 1993). This type of partial resorption (Fig. Ic) occurred within 24 h of zooid transplantation, but was never completed before the takeover phase (Lau- zon et nl.. 1992) of blastogenesis. The second type of variation was the resorption rate of the implant. In some cases, the implants were resorbed completely or mainly within 24 h of transplantation (Fig. le, f). Where partial resorption was recorded (Fig. If), the leftover parts re- mained within the tunic for the whole blastogenic cycle and were completely resorbed only during the takeover stage. To analyze the possibility for donor cell proliferation 102 B. RINKEVICH ET AL Figure 1. Morphological outcomes for zooid transplantation in Fu/HC-noncompatible colonies of Bo- riyllux xcliloxxi'ri. al = allogeneic transplant, am = ampulla, b = bud, is = isograft, v = blood vessel, z = zooid. Length of each zooid = 2 mm. (a) Results from Exp. Ic (Table I): The second set of allogeneic and isogeneic grafts, 24 h after implantation, when the colony is at blastogenic stage C. (b) The same area, photographed 72 h later, following a takeover event. Both implanted zooids are completely resorbed, and there is no difference between control and allograft; the zooid between them as well as adjacent buds are unaffected. After the takeover process, all transplanted materials were morphologically eliminated, no point of rejection (FOR) is observed. Note the top right zooid: the incision for transplanting the zooids is highlighted; white arrows point to the atrial and branchial siphons, and the white line highlights the incision made for the transplantation procedure (see Methods), (c) Results from Exp. Id (Table I| 24 h after first set of implants was established. The control isograft appears to be resorbing faster than the allograft. The zooid to the right of the isograft is also partly resorbed. The colony is at blastogenic stage C. and the developed buds in the area of implantation are not affected, (d) Exp. Ib, after resorption of most of the allogeneic implant. Resorption is not complete, and a remnant of the graft (arrow) is trapped in the tunic matrix, (e, f) Results from Exp. 5a (Table I): The third set of allografts, immediately after implantation (a), and 24 h later (f). By 24 h. most of the graft has been resorbed and only a small part of it (arrow) is left. This remnant remained for an additional 3 days, and was resorbed during the takeover stage of blastogenesis. in the recipient (Sabbadin and Zaniolo, 1979; Pancer et a/.. 1995: Stoner and Weissman, 1996). all the available subclones from the 1 -month experiment (rive or six con- secutive transplantation events; Table IA) were sampled (1-7 fragments/subclone) 3 to 4 weeks after complete resorption of the donor's zooids (except for Exp. Ic. see Table I). The genotype of the recipient was then analyzed using amplified restriction fragment polymorphisms (AFLPs; Vos et at.. 1995). AFLPs identify DNA poly- morphisms between individuals; these polymorphisms can then be used as molecular genetic markers. AFLP polymorphisms are often single base-pair substitutions, and since these are the most abundant polymorphisms available, a large number of AFLP genetic markers can be identified, even between closely related colonies (Fig. 2). In these experiments. 6 to 12 unique AFLP markers were first identified in the naive donor subclones. Follow- ing a transplantation, we then looked for the unique donor AFLPs in samples of recipient DNA. Tissue samples were taken such that the transplanted area (including the relevant system of zooids and the surrounding tissue matrix) was isolated from the rest of ALLORECOGNITION IN A PROTOCHORDATH 103 A b B I , * **fr|- b 234 12 34 56 c Control Genotype Mix (donorredpient) 100:1 50:1 25:1 10:1 5:1 1:1 1:5 1:25 1:50 1:100 <** Figure 2. Amplified fragment length polymorphism (AFLP) fin- gerprints reveal the presence of donor alleles in the recipient after zooid transplantation. Panels a and b correspond to Experiment II, pair number a and b, in Table I. (A) Photographs of the recipient ramets after six sets of zooid transplantation and before subcloning. The areas sampled for DNA extraction and AFLP analysis are delineated and numbered. An asterisk indicates the site to which the donor zooids were originally transplanted; boxed numbers indicate a sample that contained peripheral vascular tissue only. (B) AFLP fingerprints of the donor, the naive recipient, and samples taken from the recipient after transplantation, as shown in A. Arrows indicate polymorphic AFLP alleles present in the donor and absent in the naive recipient. The appearance of these bands in the recipient ramets after transplantation indicates the presence of donor DNA. The recipient bands are invariable, indicating that the recip- ient genotype is stable and the resulting animal is a chimera. A section of the complete gel is shown for clarity. (C) A control experiment showing the independent sensitivity of each polymorphic AFLP donor locus in known mixtures of genotypes. DNA from the donor and naive recipient were mixed in the indicated ratios and the corresponding AFLP fingerprints were produced. The closed arrows indicate the same poly- morphic donor loci as shown in B. The asterisk denotes a DNA mixture in which the top allele is present and the bottom allele is very faint. In this section of the gel a recipient locus can also be seen (arrow plus asterisk) and demonstrates that polymorphic loci can differ as much as 10-fold in their ability to be amplified from low concentrations. the subclone. Then each subclone was further divided into several parts, including bare-tunic ampullae zones. Figures 2 and 3 depict the sampling details for seven subclones. One subclone (Exp. He) was not separated, but was used whole for a single AFLP analysis. In seven of the eight subclones (Fig. 2, Fig. 3 except Exp. IIIc) donor AFLPs were clearly documented in all samples, including zooid- free areas and zones away from the trans- plantation areas. However, the degree of chimerism was not consistent among all parts of the recipients; different regions showed a higher or lower percentage of the donor genotype. In other words, not all of the donor AFLP markers were amplified from each recipient DNA sample (Fig. 2b). This is because the ability to amplify a particular AFLP marker from a mixed sample of DNA is unique for that marker; some AFLP markers can be identified when they represent less than \ c /c of the total DNA, while others are less sensitive (Fig. 2c). Thus, if the amount of chimerism is very low (e.g., donor DNA < 1% of the total DNA in the sample), only the most sensitive donor AFLP marker will be identified (see Fig. 2c). In contrast, in a sample that contains a higher amount of donor cells, the other, less sensitive AFLP markers will also be identi- fied (Figs. 2 and 3). This provides a nice tool for estimat- ing the amount of chimerism, but because the AFLP tech- nique includes an initial PCR amplification of the total genomic DNA sample (Vos et ai. 1995), these compari- sons are relative and can only be made in a side-by-side comparison to the controls (Fig. 2b. c). Chimerism was detected globally in the recipient colo- nies more than 3 to 4 weeks after the final transplantation, and the degree of chimerism was variable in different samples. This suggests that donor cells proliferated in the host colonies; the small number of hemocytes from sev- eral transplanted zooids are unlikely to have been pas- sively circulated and then detected several weeks later, after several rounds of blastogenesis. Exp. IIIc (Table I; Fig. 3) was the only case in which we did not observe the donor genotype in any sample, although in the reciprocal tests (Exp. Ilia, b; Fig. 3) the other partner's DNA was clearly evident in all tissue sam- ples. This is reminiscent of the directionality observed in resorption, where hierarchies were demonstrated in labo- ratory-reared colonies (Rinkevich et ai, 1993). In summary, chimerism between the donor and recipi- ent appeared to be the rule rather than the exception in these experiments. In 1 1 of 1 2 cases, donor AFLP markers could be identified in the recipient more than 3 to 4 weeks after the donor transplant had been eliminated. Further- more, high amounts of chimerism were detected through- out the recipient colony. This suggests that once donor cells have crossed the ampullar barriers, they are able to survive and proliferate in the recipient colony, and are not being eliminated by an Fu/HC-based, or any other. allorecognition system. 104 B. RINKEVICH ET AL. Expt, I: Poire Expt, I: Poire! Expt. Ill: Pair a Expt. Ill: Pair b Expt. Ill: Pairc Figure 3. A summary of amplified fragment length polymorphism (AFLP) data from experiments described in Table I. Photographs show rive recipient ramets after transplantation. The areas sampled for DNA extraction and AFLP analysis are delineated and numbered. Asterisks indicate the sites to which the donor zooids were originally transplanted: boxed numbers indicate a sample that contained vascular tissue only. Because of the independent sensitivity of each polymorphic donor locus (illustrated in Fig. 2C). not all of the polymorphic donor loci were seen in each of the transplanted recipient samples. Samples were scored as ( + + ) if they contained over 50% of the polymorphic donor loci, ( + ) it under 50%, and (-) if none were seen, "n.d." indicates that the sample was not determined. Discussion This study produces two interesting results. First, zooid transplantation between noncompatihle Fu/HC B. schlos- seri genotypes does not result in the typical formation of visible PORs (reviewed in Weissman et ai, 1990). Rather, transplantation is followed by a morphological resorption similar to the allogeneic resorption that takes place after fusion between Fu/HC-compatible colonies (Rinkevich and Weissman, 1987). Second, the Fu/HC-noncompatible genotypes continue to thrive within the host, even away from the transplantation zone; this phenomenon had pre- viously been recorded only from Fu/HC-compatible en- counters (Sabbadin and Zaniolo, 1979; Pancer ft - senbergii larvae were within the physiological range (Ta- kac, pers. comm. and Abdu et cii, 1998). the possibility of nonspecific toxicity could not be entirely ruled out. In crustaceans, intermediate larval forms may be produced by eyestalk removal during a "critical period," as has been shown for Rhithropanopeus harrisii (Costlow. 1966a), Sesartnci reticiiliittini (Costlow, 1966b). H. anieri- canus (Charmantier and Aiken, 1987), Pahu'immetes vnr- ians (Le Roux, 1984). andAlphens heterochaelis (Knowl- ton, 1994). Snyder and Chang ( 1986) suggested that inter- mediate larval forms in H. umericuinis were probably a manifestation of inadequate nutrition or other stresses. However, Knowlton ( 1994) showed that the appearance of intermediate forms in A. heterochaelis larvae, which have a Iccithotropic mode of nutrition, was due to direct or indirect hormonal control from the eyestalk. The re- ports of the isolation and characterization of mandibular organ-inhibiting hormone (MOIH) from the sinus gland in the eyestalk (Liu and Laufer. 1996; Wainwright et al, 1996), together with the present work, suggest that MF could be the direct hormonal agent controlling metamor- phosis in crustaceans through an indirect effect of eyestalk neuropeptides. MF has recently been described as a "crustacean juve- nile hormone in search of functions" (Homola and Chang, 1997b). The findings of the present study the delay in larval development and metamorphosis in M. rosenbergii, together with the increase in the abundance of intermediate specimens with the increase in exogenous MF contribute to the debate over the physiological function of MF, supporting its possible role as a juvenile hormone in the development and metamorphosis of crus- taceans. Acknowledgments We thank Ms. Hanna Ben-Galim and Ms. Galit Yehez- kel for their technical assistance, and Ms. Inez Mureinik for editorial review of the manuscript. A. S. is the incum- bent of the Judith and Murray Shusterman Chair for Ca- reer Development in Physiology. Literature Cited Ahdu, U. 1996. The involvement of methyl tarnesoate. administered through Anemia vector, in the endocrine regulation of larval devel- opment and metamorphosis in the freshwater prawn Macrobrachium rosenbergii. M.Sc. Thesis. Ben-Gurion University of the Negev, Israel. 55 pp. Ahdu. U., P. Takac, G. Yehezkel. R. Chayoth, and A. Sagi. 1998. Administration ot methyl tarnesoate through theArtemia vector, and its effect on Macrobrachium rosenbergii larvae. /.ST. J. Ai/iuiciilt. 50(2): 73-81. Borst, D. W., H. Laufer, M. Landau, E. S. Chang, W. A. Hertz, F. C. Baker, and D. A. Schooley. 1987. Methyl famesoate and its role in crustacean reproduction and development. Insect Biochem. 17: 1123-1127. Kruning. K., R. R. Shivers, and B. Lanzrrin. 1985. Methyl tarne- soate and juvenile hormone III in normal and precocene-treated em- bryos of the ovoviviparous cockroach Nauphaeta cinereu. Int. ./. Im-cru-hr. RcproJ. Ocv. 8: 269-278. C'hang, E. S., M. J. Bruce, and S. L. Tamone. 1993. Regulation of crustacean molting: a multi-hormonal system. Am. Zoo/. 33: 324- 329. Charmantier, G., and D. E. Aiken. 1987. Intermediate larval and postlarval stages of Homarus americanus H. Milne Edwards. 1837 (Crustacea: Decapoda). J. Crust. Biol. 7: 525-535. Charmantier, G., M. Charmantier-Daures, and D. E. Aiken. 1988. Larval development and metamorphosis of the American lobster Homarus nmericamis (Crustacea: Decapoda): effect of eyestalk abla- tion and juvenile hormone injection. Gen. Comp. Endocrinol. 70: 319-333. Christiansen, M. E., J. D. Costlow, Jr., and R. J. Monroe. 1977a. Effects of the juvenile hormone mimic ZR-515 (Altosid) on larval development of the mud-crab Rhithropanopeus liarri.sii in various salinities and cyclic temperatures. Mar. Biol. 39: 269-27. Christiansen, M. E., J. D. Costlow, Jr., and R. J. Monroe. 1977h. MF AND METAMORPHOSIS IN MACROBRACHIUM 19 Effects of the juvenile hormone mimic ZR-512 (Altozar) on larval development of the mud-crab Rhithropanopeus harrisii in various salinities and cyclic temperatures. Mar. Biol. 39: 281-288. Costlow, J. D., Jr. 1966a. The effect of eyestalk extirpation on larval development of the mud crab. Rhithropanopeus harrisii (Gould). Gen. Comp. Endocrinol. 3: 120130. Costlow, J. D., Jr. 1966b. The effect of eyestalk extirpation on larval development of the crab Sesarma reticiilanim Say. Pp. 209-224 in Sonit' Contemporary Sunlit'* in Marine Science. H. Barnes, ed. Appleton-Century-Crofts. New York. Daniels, W. H., L. R. D'Abramo, and L. D. Parseval. 1992. Design and management of a closed, recirculating "clearwater" hatchery system for freshwater prawns, Macrobrachium rosenbergii De Man, 1879. J. Shellfish Res. 11: 65-73. Gomez, E. D., D. J. Faulkner, VV. A. Newman, and C. Ireland. 1973. Juvenile hormone mimics: effects on cirriped crustacean metamor- phosis. Science 179: 813-814. Hertz, \V. A., and E. S. Chang. 1986. Juvenile hormone effects on metamorphosis of lobster larva. Int. J. Invertebr. Reprod. Devel. 10: 71-77. Homola. E., and E. S. Chang. 1997a. Distribution and regulation of esterases that hydrolyze methyl farnesoate in Homarus americam/s and other crustaceans. Gen. Comp. Endocrinol. 106: 62-72. Homola, E., and E. S. Chang. 1997b. Methyl farnesoate: crustacean juvenile hormone in search of functions. Comp. Biochem. Physiol. 117B: 347-356. King, L. E.. Q. Ping, G. D. Prestwich, and S. S. Tobe. 1993. The characterization of haemolymph methyl farnesoate binding protein and the assessment of methyl farnesoate metabolism by the haemo- lymph and other tissues from Procambarus clarkii. Insect Binchem. Mul. Biol. 25: 405-501. Knowlton, R. E. 1994. Effects of larval eyestalk extirpation on mor- phogenesis and molting in the snapping shrimp Alplteus hetero- cliaelis Say. J. E.\p. Zool. 270: 162-174. Landau, M., H. Laufer, and E. Homola. 1989. Control of methyl farnesoate synthesis in the mandibular organ of the crayfish Pro- cambarus clarkii: evidence for peptide neurohormones with dual functions. Invertebr. Reprotl. Devel. 16: 165-168. Lanzrein, B., H. Imboden, C. Burgin, E. Bruning, and H. Gf'eller. 1984. On tilers, origin, and functions of juvenile hormone III, methyl farnesoate, and ecdysteroids in embryonic development of the ovoviviparus cockroach Nauphoeta cinerea. Pp. 454-465 in Biosynthesis, Metabolism and Mode of Action of Invertebrate Hor- mones. J. Hoffman and M. Porchet, eds. Springer-Verlag. New York. Laufer, H., and K. H. Albrecht. 1990. Metabolism of methyl farne- soate in vitro by peripheral tissues of the spider crab Libiniii I'liiargi- nata (Decapoda). Pp. 217-222 in Advances in Invertebrate Repro- duction 5, M. Hoshi and O. Yamashita. eds. Elsevier, Amsterdam. Laufer, H., and D. W. Borst. 1983. Juvenile hormone and its mecha- nism of action. Pp. 203-216 in Endocrinology of Insects. R. G. H. Downer and H. Laufer. eds. Alan R. Liss, New York. Laufer, H., and D. W. Borst. 1988. Juvenile hormone in Crustacea. Pp. 305-313 in Endocrinology of Selected Invertebrate Types. R. G. H. Downer and H. Laufer, eds. Alan R. Liss, New York. Laufer, H.. D. W. Borst, F. C. Baker, C. Carrasco, M. Sinkus, C. C. Reuter, L. Tsai, and D. A. Schooley. 1987. The identification of a crustacean juvenile hormone. Science 235: 202-205. Laufer, H., P. Takac, J. S. B. Ahl, and M. R. Laufer. 1997. Methyl farnesoate and the effect of eyestalk ablation on the morphogenesis of the juvenile female spider crab Libinia emarginata. Invertebr. Reprod. Dcvel. 31: 63-68. Le Roux, A. 1984. Quelques effets de 1'ablation des pedoncules ocu- lairs sur les larves et les premiers stades juveniles de Palaemonetes various (Leach) (Decapoda. Palaemonidae). Bull. Soc. Zool. Fr. 109: 43-60. Ling, S. W. 1969. The general biology and development of Macmbra- chium rosenbergii. De Man. FAO Fish. Rep. 57: 589-606. Liu, L.. and H. Laufer. 1996. Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hy- perglycemic effects from the spider crab. Libinia emarginata. Arch. Insect Biochem. Physiol. 32: 375-385. McKenney, C. L., Jr., and D. M. Celestial. 1993. Variations in larval growth and metabolism of an estuarine shrimp Palaemonetes ptigio during toxicosis by an insect growth regulator. Comp. Biochem. Physiol. 105C: 239-245. Prestwich, G. D., H. VVojtasek, A. J. Lentz, and J. M. Rabinovich. 1996. Biochemistry of proteins that bind and metabolize juvenile hormone. Arch. Insect Biochem. Physiol. 32: 407-419. Ramenofsky, M., D. J. Faulkner, and C. Ireland. 1974. Effects of juvenile hormone on cirriped metamorphosis. Biochem. Biophys. Res. Com/mm. 60: 172-178. Riddiford. L. M. 1994. Cellular and molecular actions of juvenile hormone I. General considerations and premetamorphic actions. Pp. 213-274 in Advances in Insect Physiology vol 24. P. D. Evans, ed. Academic Press, London. Sagi, A., E. Homola, and H. Laufer. 1991. Methyl farnesoate in the prawn Macrobrachium rosenbergii: synthesis by the mandibular organ in vitro, and liters in the hemolymph. Comp. Biochem. Physio/. 99*8: 879-882. Sehnal, F. 1983. Juvenile hormone analogues. Pp. 657-672 in Endo- crinology of Insects, R. G. H. Downer and H. Laufer. eds. Alan R. Liss. New York. si. mi, i. K. 1971. Insect juvenile hormone and analogues. Annu. Rev. Biochem. 40: 1079-1102. Slama, K. 1995. The present status of the mode of action of insect juvenile hormone. Netli. J. Zool. 45: 71-78. Snyder, M. J., and E. S. Chang. 1986. Effects of eyestalk ablation on larval molting rates and morphological development of the American lobster, Homarus americanus. Biol. Bull. 170: 232-243. Sollaud, M. E. 1923. Le developpement larvaire des "Palaemoni- nae". I. Partie descriptive. La condensation progressive de 1'onto- genese. Bull. Biol. 57: 509-603. Takac, P., J. S. B. Ahl, and H. Laufer. 1997. Seasonal differences in methyl farnesoate esterase activity in tissues of the spider crab Libinia emarginata. Invertebr. Reprod. Devel. 31: 211-216. Tamone, S. L., G. D. Prestwich, and E. S. Chang. 1997. Identifica- tion and characterization of methyl farnesoate binding proteins from the crab. Cancer magister. Gen. Comp. Endocrinol. 105: 168-175. Tighe-Ford, J. D. 1977. Effects of juvenile hormone analogues on larval metamorphosis in the barnacle Eliminus modestus Darwin. (Crustacea: Cirripedia). J. E.\p. Mar. Biol. Ecol. 25: 163-176. Uno, Y., and K. C. Soo. 1969. Larval development of Macrobrachium rosenbergii De Man, reared in the laboratory. J. Tokyo Univ. Fish. 55: 179-190. Wainwright, G., S. G. Webster, M. C. Wilkinson, J. S. Chung, and H. H. Rees. 1996. Structure and significance of mandibular organ- inhibiting hormone in the crab. Cancer pagurus. J. Biol. Client. 271: 12749-12754. Wigglesworth, V. B. 1970. Insect Hormones. University Reviews in Biology, p. 159. Oliver and Boyd, Edinburgh. Reference: Biol. Bull 195: 120-125. (October, 1998) Body Polarity and Mineral Selectivity in the Demosponge Chondrosia reniformis GIORGIO BAVESTRELLO', UMBERTO BENATTI : . BARBARA CALCINAI', RICCARDO CATTANEO-VIETTI 1 , CARLO CERRANO 1 . ANNA FAVRE 3 , MARCO GIOVINE 2 , SERENA LANZA 1 , ROBERTO PRONZATO 1 , AND MICHELE SARA 1 Istititto di Zoologia dell'Universita di Genova, Via Balbi, 5 1-16126 Genova; ~ Istititto di Chimica Biologicu dell'Universita di Genova, Viale Benedetto XV. 8 1-16132 Genova; and 3 Istititto di Anatomia Patologica deU'Ospedale G. Gaslini, Lg. G. Gaxlini 5, 1-16100 Genova Abstract. The skeleton of the common Mediterranean demosponge Chondrosia reniformis lacks endogenous spicules; but exogenous siliceous material is selectively incorporated into its collagenous ectosome, strengthening this layer. Nevertheless, the settling of sponge buds during asexual reproduction necessitates an active incorporation of the calcareous substratum through the sponge lower ectosome. This fact suggests the presence of a polarity in the sponge, with the lower surface selecting primarily carbonates, and the upper surface selecting exclusively silicates and quartz. Our observations under experimental conditions showed that the strong selectivity of the upper ectosome is realized only when the sponge is fixed to the substratum; if detached, the sponge incorporates both quartz and carbonates. In laboratory experiments, the in- capacity of both kinds of ectosome to regenerate into a new complete sponge suggests that this polarity arises early in ontogeny. Introduction Chondrosia reniformis is a cushion-shaped, Atlanto- Mediterranean demosponge that usually lives on shallow rocky bottoms. A section through the sponge reveals two distinct regions: a cortical zone called ectosome, and an internal zone, the choanosome, which contains the cho- anocyte chambers. The ectosome is composed of a layer of flattened cells, exopinacocytes, that surround dense interwoven bundles of fibrils of collagen. In many circum- stances the pinacocyte layer is loose, and the collagen Received 7 August 1997; accepted 10 July 1998. fibrils can be in direct contact with water (Garrone et ai, 1975). C. reniformis lacks the opaline spicules that are the main constituents of the skeleton of other demosponges; rather, the collagenous ectosome is strengthened by sand grains and exogenous spicules, which are actively incor- porated by the sponge (Bavestrello et ai. 1995). Studies of foreign matter incorporation have been carried out on the sponge Dysidea etheria. In this species, the particles are incorporated by contraction of the dermal membrane, which probably separates or disrupts the thin exopinaco- cyte layer on the dermal membrane surface (Teragawa, 1986a, b). The ectosome of C. reniformis behaves simi- larly (Bavestrello et al., in press). In C. reniformis, the upper ectosome is able to select the minerals that settle on the sponge: thus, siliceous ma- terial is engulfed while the calcareous fragments that are the main sediments available in the surrounding environ- ment are eliminated (Bavestrello et al., 1996). In contrast, however, C. reniformis settles on calcareous rocks through the partial incorporation of outgrowths of this substratum by the lower ectosome. This process suggests a polarization in the sponge body, with a specificity for the incorporation of minerals that varies from the lower ectosome to the upper one. Indeed, the polarization of the adult sponge body was already demonstrated 45 years ago with Sycon raphanus. When specimens of these sponges were bisected trans- versely, both halves could develop a complete new animal with the same polarity, from osculum to base, as the original (Tuzet and Paris, 1963). The polarization of sponges relative to their position on the substratum probably arises in the larval stage. In 120 POLARITY AND PARTICLE SELECTION IN A SPONGE 121 the amphiblastula larvae of Calcarea, the flagellate cells of the anterior pole make the initial contact with the sub- stratum (Bergquist and Green, 1977). In Demospongiae, several authors have suggested that the coeloblastula or parenchymella larvae express an existing polarity in their attachment to the substrate. But such views are highly speculative, because distinguishing between an anterior and a posterior hemisphere in these animals is difficult (see Simpson, 1984, for a review). The aims of this work are to verify through labora- tory experiments and scanning electron microscopy (SEM) the capacity of both kinds of ectosome of Chon- drosia reniformis to develop specificity towards siliceous and calcareous materials, and to demonstrate the cellular basis of that specificity. Materials and Methods Along the rocky cliff of the Portofino Promontory (Li- gurian Sea, Italy) Chondrosia reniformis lives from the surface to the base of the cliff (about 50 m depth). The specimens used in this study were collected during Janu- ary 1997, at 10 m depth, on calcareous substrates. We performed our experiments with specimens having a surface area of 11 2- 156 cm 2 . These specimens were reared at 15C in 200-1 aquaria containing natural seawa- ter with a salinity of 37%o. The medium was aerated by bubbling, and it was replaced twice a week. The collected sponges attached to the aquarium bottom in about 10 days. The sandy materials used in testing sponge selectivity were white polycrystalline quartz with a particle size of 0.25-0.5 mm (BDH laboratory sand); red calcareous sand of the same particle size obtained from the organ-pipe coral Tubipora niusica; and fragments of a coralline alga, Lithothamnium sp., 3-5 mm in size. To test the differences in behavior between the upper and lower surfaces of the sponge ectosome, a thin layer of a mixture (1:1) of the BDH siliceous and Tubipora calcareous sands was laid down on the upper ectosome of five specimens that had attached to the bottom of an aquarium covered by the same mixture. Two experiments were carried out with these specimens. First, cores through the sponge, from upper to lower surface, were inverted and then transplanted, upside-down, inside the same specimen (five replicas). Second, 40 half-cores (20 mm in diameter) were taken from the upper and lower ectosome and reared for 3 months (see Fig. 1 ). Observations made with SEM allowed us to distinguish differences in the organization of the two ectosomal sur- faces. The samples used for these observations were col- lected by scuba divers and fixed in situ in 10% formalin. Upside-down reinsertion Free semi-cores regeneration Upper ectosome Choanosome Lower ectosome B regeneration c Figure 1. Scheme of coring experiments in Chondrosia reniformis. (A) Showing that differences in the mineral selectivity of the upper and lower sides of the ectosome are related to their histology. Cores (about 5-6 cnr ) were cut from the upper to the lower surface and reinserted upside-down in the hole. The central portion of the resulting reconstitu- ted sponges thus had an upper-lower orientation that was reversed. (B, C) Determining whether isolated upper and lower ectosomes can reconstitute an entire animal. Cores were produced and cut transversely to make half-cores. After 2 weeks' regeneration, the upper and lower ectosomes proliferated, enveloping the half-cores. Results When a mixture of siliceous and calcareous sand was allowed to settle on the upper ectosome of specimens that had attached to the bottom of the aquarium, only the quartz fragments (white) were incorporated. The calcare- ous grains (red) were never engulfed; rather they were quickly removed from the sponge surface (Fig. 2a). In contrast, the lower ectosome showed a remarkable prefer- ence for incorporating the calcareous grains of the mixture lying on the bottom (Fig. 2b). The upper ectosome of newly collected specimens that had not yet attached to the aquarium bottom actively in- corporated both kinds of minerals (Fig. 2c). Ten days later, after attachment, the upper surface of all the tested specimens began to select siliceous material exclusively. To verify the difference in mineral selectivity between the two sides of the ectosome, cores (about 5-6 cm 2 ) extending from the upper to the lower surface were cut from five large specimens and reinserted upside-down, to produce sponges with a portion of their lower surface having an upward orientation (Figs. 1, 2e). A week later, when the explants were perfectly fused in their anomalous positions and the sponges were attached to the aquarium floor, 5 g of Lithothamnium calcareous sand was laid on their surface (Fig. 2f ). The subsequent behavior of the two types of surface was very different. The upper ectosome (brown) behaved normally, quickly removing calcareous particles (Fig. 2g-h); but the inverted, originally lower 122 BAVESTRELLO ET AL Figure 2. Mineral selectivity experiments. A 1:1 mixture of calcareous (red) and quart/ (white) grains were allowed to settle on the upper and lower ectosome of Chondrosia reniformis specimens, (a) The upper ectosome (brown) has incorporated several quartz grains (arrows), while almost all the calcareous particles have been eliminated, (b) The opposite happens in the lower ectosome (white), which incorporates the calcareous fraction. Arrows indicate the thin collagenous sheet growing on the calcareous fragments, (c) The upper ectosome of a free, unattached specimen incorporates both quartz (arrows) and carbonatic particles (arrow heads), (d) Asexual reproduction in C. remformis. The underwater photograph shows a long ( 1 m), stretched filamentous outgrowth hanging from a mother sponge (ms) that lives attached to the vault of a cave. A new sponge has formed at the free tip of the filament, (e) A core (about 6 cnr) cut and newly inserted upside-down in a large specimen of C. rcnijunms. When the explant was perfectly fused in Us anomalous position, and the sponge had attached to the aquarium floor, calcareous grains were laid on its surface tf ). Afterwards, the behavior of the two portions was very different. The upper surface (brown) behaves normally, quickly removing calcareous particles; shown at 24 h (g) and 48 h (h). The section that was originally the lower surface (whitish) did not move the particles. Scale bars: a-c = I mm: d = 10cm; e-h = 1 cm POLARITY AND PARTICLE SELECTION IN A SPONGE 123 Figure 3. (a) The upper ectosome of Clumdrosia reniformis. brown in color due to the presence of numerous melanocytes. and (b) the lower whitish one. SEM observations reveal differences between the two zones, (c) The upper surface is covered by polygonal flattened exopinacocytes (ep) and pierced by the incurrent openings, or ostia (o). (dl The lower surface shows no pores, and the basopinacocytes (bp) are covered by a collagenous sheet. Twelve weeks after preparation, the half-cores contain only an upper (e) or lower (f ) ectosome. Two half-cores (g) derived from the upper (left) and lower (right) ectosomes. brought together and fused maintaining the characteristics of their original position. Scale bars: a-b. e-g = 1 cm; c-d = 50 ^m. surfaces (whitish) did not move the particles, and 3 months later, they were all incorporated. The gross morphological differences between the upper (Fig. 3a) and lower surfaces of the sponge (Fig. 3b) were supported by SEM observations: the upper ectosome is entirely covered by polygonal flattened pinacocytes and perforated by the incurrent openings (ostia) (Fig. 3c). whereas the pinacoderm of the lower surface is covered by a collagenous sheet that lacks ostia (Fig. 3d). To verify that the polarization between the upper and lower ectosome arises very early in development, experi- ments were performed with half-cores; these are fractions of sponge tissue that contain a portion of choanosome covered on one side by either upper or lower ectosome 124 BAVESTRELLO ET AL. (see Fig. 1 ). In the first 2 weeks of culture, the ectosome of both kinds of free half-cores actively proliferated, and the pieces assumed a spherical shape (Fig. 3e-f). The half-cores covered with lower ectosome (Fig. 3f ) settled on the smooth bottom of the aquarium in 3-4 weeks, whereas those with the upper ectosome (Fig. 3e) settled after about 10-12 weeks. Furthermore, 3 months after the beginning of the experiment, the half-cores with lower ectosome were covered by a collagenous, non-cellularized layer that remained white and formed no osculum; the half-cores with the upper ectosome produced a new oscule in 12-15 weeks and showed a normal pinacoderm perfo- rated by pores. Sometimes, when half-cores deriving from the upper and lower ectosome came in contact, they fused together; but a normal sponge was never reconstituted, although the two kinds of ectosome remained distinct on the opposite sides of the sponge (Fig. 3g). Discussion The structural and functional differences between the two sides of the ectosome of the sponge Clwndrosia reni- formis suggest a strong polarization upper pole versus lower pole along the axis of the sponge. The activity of the upper ectosome is likely due to the pinacocyte- mineral interaction. More problematic is the basis for the preferences of the lower ectosome for calcareous sub- strata. In other sponges, a nonspecific attachment to the substratum is probably due to the secretion by the basopi- nacocytes of a complex basal lamella (Pavans de Cec- catty, 1981) that anchors the sponge but prevents any contact between the cells and the substratum. The ability of the upper ectosome to discriminate be- tween silica and carbonates is present only in attached specimens and vanishes in free, nonattached ones, which incorporate both materials indiscriminately. This distinc- tion is interpretable if we consider the asexual reproduc- tive strategy of the species: Sponges living on overhang- ing ledges or on the vaults of submarine grottos give rise to long, thin pendant filaments (Fig. 2d). Cell reorganiza- tion within the apical region of these filaments produces a new, functional, but suspended animal. When the filament breaks, the bud is separated from the maternal sponge (Gaino et ai, 1995); it falls and must attach quickly irre- spective of the side of the ectosome that comes in contact with the substratum. This behavior indicates, not only that mineral receptors are distributed evenly on the sponge surface, but also that these receptors may be activated or deactivated under particular conditions by an environ- mental switch. We suppose that the mineral discrimina- tion of the upper ectosome is switched on by the adhesion of the sponge to the bottom. Our studies indicate two modalities of mineral incorpo- ration that are associated with the two sides of the ecto- some. The upper side collects quartz and silicates, which strengthens the collagenous structure; this is a dynamic process comprising incorporation of the particles and re- sizing of the quartz grains, with their elimination via the aquiferous system (Bavestrello et ai, 1995). The lower side specifically engulfs the calcareous substrata, thus fixing the sponge to the bottom. There is a rich literature about the very wide potential for cytodifferentiation in sponges (see Simpson, 1984). Our data indicate that, at least in C. reniformis, the mor- phological differences between the upper and lower re- gions of the ectosome are sharp, probably arising in a very early stage of sponge ontogeny; that is, a functionally complete specimen cannot be reconstituted from a portion of only upper or lower ectosome with its adjacent choano- some. Connes (1966, 1968) has demonstrated that the ectosome and choanosome of Tethya aurantium have dif- ferent potentials for reconstructing an entire sponge, but our data provide the first indication of differences in this process associated with distinct zones of the same ecto- some. In higher metazoans, the spatiotemporal development of morphological structures is regulated by homeobox genes (Lawrence, 1992). These genes have also been ob- served in lower metazoans such as sponges (Kruse et ai, 1 994; Coutinho et ai, 1994; Degnan et ai, 1995; Seimiya et ai, 1997), but their meaning has been obscure until now. We hypothesize that the acquisition of an axial po- larity in the sponge may be controlled by these genetic structures. Acknowledgements This research was supported by Italian MURST funds. Literature Cited Bavestrello, G., A. Arillo, U. Benatti, C. Cerrano, R. Cattaneo-Vietti, L. Cortesogno, A. Gaggero, M. Giovine, M. Tonetti, and M. Sara. 1995. Quartz dissolution by the sponge Chondrosia reni- formis (Porifera, Demospongiae). Nature 378: 374-376. Bavestrello, G., C. Cerrano, R. Cattaneo-Vietti, M. Sara, F. Cala- bria, and L. Cortesogno. 1996. Selective incorporation of foreign material in Clionilrosia reniformis (Porifera, Demospongiae). Ittil. J. Zixil. 63: 215-220. Bavestrello, G., A. Arillo, B. Calcinai, C. Cerrano, R. Cattaneo- Vietti, S. Lanza, and M. Sara. Interaction between different kinds of silica and the exo-pinacocytes of the demosponge Chondrosia renifiirmi.i. Ital. J. Znnl. (in press). Bergquist, P. R., and C. R. Green. 1977. An ultrastructural study of settlement and metamorphosis in sponge larvae. Cah. Biol. Mar- 18: 289-302. Connes, R. 1966. Aspects morphologtques de la regeneration de Telhva iMiciiriiun Lamarck. Bull. Sue. Ziiol. Fr. 91: 43-53. Connes, R. 1968. Etude histologique. cylologique et expenmentale de la regeneration et de la reproduction asexuee chez Tethya lyncurim Lamarck (= Tethyu aurantium Pallas) (Demosponges). Thesis. Univ. Montpellier. France. 193 pp. POLARITY AND PARTICLE SELECTION IN A SPONGE 125 Coutinho, C., S. Vissers, and G. Van de Vyver. 1994. Evidence of homeobox genes in the freshwater sponge Ephydatia fluviatilis. Pp. 385-388 in Sponges in Time and Space, R. W. M. van Soest, Th. M. G. van Kempen. and J. C. Braekman, eds. Balkema, Rotterdam, Degnan, B. M., S. M. Degnan, A. Giusti, and D. E. Morse. 1995. A hox/hom homeobox gene in sponges. Gene 155: 175-177. Gaino, E., R. Manconi, and R. Pronzato. 1995. Organizational plas- ticity as a successful conservative tactics in sponges. Aniin. Bioi 4: 31-43. Garrone, R., A. Hue, and S. Junqua. 1975. Fine structure and physi- cochemical studies on the collagen of the marine sponge Chondrosia reniformis Nardo. J. Ultrastruct. Res. 52: 261-275. Lawrence, P. A. 1992. The Making of a Fly: The Genetics of Animal Design. Blackwell Scientific, Oxford. Kruse, M., A. Mikoc, H. Cetkovic, V. Gamulin, B. Rinkevich, I. M. Muller, and W. E. Muller. 1994. Molecular evidence for the pres- ence of a developmental gene in the lowest animals: identification of a homeobox-like gene in the marine sponge Geodia cydonium. Mech. Ageing Dev. 77: 43-54. Pavans de Ceccatty, M. 1981. Demonstration of actine filaments in sponge cells. Cell Bin/. Int. Rep. 5: 945-952. Seimiya, M., H. Ishiguro, K. Miura, Y. Watanabe, and Y. Kurosawa. 1997. Homeobox-containing genes in the most primitive metazoa, the sponges. Eur. J. Biochem. 221: 219-225. Simpson, T. L. 1984. Cell Biology of Sponges. Springer, New York. Teragawa, C. K. 1986a. Particle transport and incorporation during skeleton formation in a keratose sponge: Dysidea etheria. Biol. Bull. 170: 321-334. Teragawa, C. K. 1986b. Sponge dermal membrane morphology: his- tology of cell-mediated particle transport during skeletal growth. J. Morphol. 190: 335-347. Tuzet, O., and J. Paris. 1963. Recherches sur la regeneration de Sycon raphanus. Vie Milieu 14: 285-298. Reference: Biitl. Bull. 195: 126-135. (October. 1998) Effect of Larval Swimming Duration on Growth and Reproduction of Bugiila neritina (Bryozoa) Under Field Conditions DEAN E. WENDT Department of Organismic and Evolutionary Biology. Harvard University. Cambridge, Massachusetts 02138 Abstract. A growing body of evidence indicates that even subtle events occurring during one portion of an animal's life cycle can have detrimental, and in some cases, lasting effects on later stages. Using a laboratory- Held transplant design, postmetamorphic costs associated with the duration of larval swimming were investigated in the bryozoan Biigitla neritina. Larvae were induced to metamorphose in the laboratory after swimming for either less than 1 h or between 23 and 24 h; colonies that devel- oped from these two groups of larvae are referred to hereafter as " 1-h colonies" and "24-h colonies." respec- tively. After completing metamorphosis, individuals were transplanted to the field, where rates of growth and repro- duction were monitored. In a study of the interaction between colony orientation (up or down) and larval swim- ming duration, both factors significantly affected the num- ber of autozooids produced. For example. 14 days after metamorphosis, 1-h colonies facing up were approxi- mately 40% smaller than 1-h colonies facing down. In another study, the effects of larval swimming duration, orientation, and a neighboring conspecitic colony on growth and reproduction were examined. In this experi- ment, proximity to a conspecific colony and orientation did not significantly affect growth or fecundity, whereas increased larval swimming duration significantly reduced both. For example, 14 days after metamorphosis, the 24- h colonies were 35% smaller than 1-h colonies. Further- more, from the time metamorphosis was initiated, the onset of reproduction was delayed by about 1 .5 days in 24-h colonies when compared to 1-h colonies; and a slight delay (cci. I day) was associated with proximity of a Received 9 October 1997; accepted 26 June 1998. E-mail: dwendt@oeb.harvard.edu developing conspecific in 1-h and 24-h colonies. In addi- tion. 17 days after metamorphosis, 24-h colonies had about half as many brood chambers (an index of fecun- dity) as 1-h colonies. Costs associated with increasing the larval swimming phase by only 24 h are significant in postmetamorphic individuals, and they clearly compro- mise colony fitness. Introduction Possession of a larval stage is common in a wide range of animals, including many fish, amphibians, and both terrestrial and aquatic invertebrates. Occurrence across such an array of taxa suggests that some benefits are associated with a motile larval stage (Strathmann. 1993: Havenhand, 1995; Wray, 1995). For marine invertebrates, a major benefit is dispersal ability, which, for example, reduces parent-offspring competition and facilitates the recolonization of disturbed habitats. In species with sed- entary or sessile adults, larvae help to increase gene flow between geographically separated populations and extend species' ranges. However, there are also costs associated with a free-living larval stage (Pechenik. 1990). These costs can be lethal (advection from suitable habitats, pre- dation, and loss of metamorphic competence), or sublethal (slower growth after metamorphosis and delayed onset of reproduction). Thus, by severely limiting dispersal, these costs probably contribute to the speciation of marine in- vertebrates with aplanktotrophic larvae (see Wendt, 1996, for a discussion of this term). Models used to examine the life-history strategies and population dynamics of marine invertebrates have fo- cused on the lethal costs of dispersal (e.g., Vance. 1973; Strathmann. 1985; Roughgarden et ai. 1988): the suble- 126 LARVAL SWIMMING DURATION 127 thai costs have been largely overlooked. Recent work demonstrates that sublethal effects can dramatically in- fluence juvenile growth and survival under laboratory conditions (Woollacott et al.. 1989; Pechenik and Cerulli. 1991; Pechenik et al., 1993; Wendt, 1996). This study assesses the costs of larval swimming duration on growth and reproduction under field conditions in the cheilostome bryozoan Bugitla neritina. Larvae of marine invertebrates commonly metamor- phose in response to cues indicative of a favorable habitat for the adult (Scheltema, 1974; Hadfield, 1978; Crisp. 1984; Chia. 1989; Pawlik, 1992). Once physiologically competent to metamorphose, a larva can remain in the swimming phase because it has not encountered a suitable cue to trigger metamorphosis or because it does not re- spond to the normal metamorphic cue as a consequence of additional factors. For example, Young and Chia ( 1981 ) demonstrated that competent larvae of Bugiila pacifica delay metamorphosis in the presence of extracts of the compound ascidian Diplosoma macdonaldi, a dominant competitor. An extended larval swimming period occurs when an individual becomes physiologically capable of responding to cues that elicit metamorphosis, but instead continues swimming. The benefit of remaining in the swimming phase is the increased likelihood of synchro- nizing the onset of metamorphosis with encountering a favorable adult site. On the other hand, the longer a larva swims the greater its exposure to the potentially lethal and sublethal effects of a planktonic existence (Rumrill, 1990; Morgan, 1995). The adverse effects of an extended larval swimming phase are well documented in laboratory studies of marine invertebrates. Increasing larval swimming time in the polychaete Capitella sp. I significantly decreased postset- tlement survivorship from 100% to 12.5% over 216 h of larval swimming (Pechenik and Cerulli, 1991 ). In bryozo- ans identified as Bugula spp., the ability to initiate and complete metamorphosis was inversely proportional to larval swimming duration (Woollacott et al., 1989; Hunter and Fusetani, 1996; Wendt, 1996). Furthermore, a loss of metamorphic competence was observed after 24 h of larval swimming in Celleporella hyalina, another cheilostome bryozoan (Orellana and Cancino. 1991 ). The size of postmetamorphic individuals is affected by the duration of larval swimming. Unusually small ancestru- lae developed from larvae of the bryozoan Hippodiplosia insculpta that swam for longer than 6 h (Nielson, 1981). Wendt (1996) quantitatively extended Nielson's qualitative observations on H. insculpta to B. neritina, showing that ancestrulae developed from larvae that swam for 28 h had lophophores (the feeding apparatus) 25% smaller in height, 40% smaller in surface area, and 50% smaller in volume, compared to ancestrulae that developed from larvae induced to metamorphose within 1 h of release. Only short-term effects of increased larval swimming duration on growth have been assessed. For example, in 12 out of 14 cases, Woollacott et al. (1989) found that after 1 1 h of swimming, larvae of B. stolonifera developed into juveniles that grew significantly slower than juveniles from larvae that swam for only 6 h. Likewise, for the barnacle Balanus amphitrite, increasing the swimming period of cyprids for 3-5 days depressed juvenile growth rate compared to controls (Pechenik et al., 1993). Long- term effects on growth and reproduction have not been explored. Effects associated with increased swimming duration are common, but not universal. For example, Highsmith and Emlet ( 1986) found no significant correlation between "delay time" and juvenile growth rate in the sand dollar Echinarachnius panmi, which has planktotrophic larvae. In the gastropod Crepidula fornicata, which also has a planktotrophic larva, no significant differences were ob- served in average rates of survival, feeding, respiration, or growth between juveniles that were induced to meta- morphose shortly after attaining competence and those that kept swimming until metamorphosis occurred sponta- neously (Pechenik and Eyster, 1989). In general, the adverse effects associated with larval swimming duration are common in species with aplankto- trophic larvae, whereas species with planktotrophic larvae typically are buffered from these costs. Information on the long-term effects of larval swimming duration on adults are confined to a single laboratory study (Pechenik and Cerulli, 1991) of a polychaete. No study has yet evaluated the performance of individuals in the field. I assess, under field conditions, the long-term costs of in- creasing larval swimming duration on colony growth and reproduction of Bugiila neritina. In addition, I investigate the effects of colony orientation and intraspecific competi- tion in relation to larval swimming duration. Materials and Methods Collection of specimens Gravid colonies of Bugula neritina were collected from the undersides of floating docks near the Smithsonian Marine Station at Link Port in Fort Pierce. Florida, during February and March 1997. Colonies were maintained in light-tight, flow-through plastic containers. Natural sea- water from the Indian River (salinity ca. 32 ppt) was con- tinuously pumped through the containers, providing the colonies with ambient levels of food and oxygen. Laivul release Larvae were obtained from several colonies to foster genetically heterogeneous populations for experiments, and larvae used in experiments were obtained only from 128 D. E. WENDT parent colonies kept in the laboratory less than 5 days. There were no qualitative differences between colonies kept in the light-tight boxes for 1 day and those kept for 5 days; in fact, colonies stayed healthy under these conditions for several weeks after the experiments. Colo- nies were removed from the light-tight containers, placed in glass bowls with 1 .0 1 of seawater, and exposed to fluorescent light. Larvae appeared within 10 min of illu- mination, and release was complete by 1 h. As B. neritina larvae are positively phototactic on release, they aggre- gated at the illuminated side of the dishes, a behavior that facilitated their collection. Lan'al swimming Following release, larvae were transferred to an auto- claved, 1.5-1 glass finger bowl containing about 1.01 of 0.2-^m filtered seawater. Larvae were prevented from initiating metamorphosis by continuous exposure to bright, fluorescent illumination accompanied by stirring (Wendt, 1996). The bowl was placed on an acrylic plastic table to reduce UV exposure and illuminated from below with four 20-W, 24-in. full-spectrum DayCycle lamps. An additional two 20-W, 24-in. fluorescent lamps were used to increase the overall lighted area. Pieces of alumi- num foil were placed around the finger bowl to create a constant reflection and constant levels of illumination from all directions. Illumination levels ranged from 130 to 170 |U.E irT 2 s '. Fans were installed under the acrylic- table to maintain ambient room temperatures (ca. 22C) during larval swimming. Metamorphosis Groups of larvae were induced to metamorphose in small polystyrene dishes by adding 10 mM excess KC1 to the seawater (Wendt and Woollacott, 1995). Metamor- phosis is the time from eversion of the larval metasomal sac to eversion of the lophophore of the ancestrular polyp- ide. To synchronize completion of metamorphosis for 1-h and 24-h individuals, larvae released on two consecu- tive days were used for each experiment. Those released on the first day were kept swimming for 24 h before metamorphosis was initiated. On the second day, the same adult colonies were used for another release of larvae. This release was 6 h later than on the previous day to allow for the increased time individuals take to metamor- phose after swimming for 24 h (Wendt, 1996). This re- lease schedule ensured that 1-h and 24-h individuals fin- ished metamorphosis at about the same time (ca. 48 h). Spontaneous metamorphosis was generally rare and oc- curred at very low levels. If more than 5% of the larvae metamorphosed during larval swimming, the experiment was aborted. Growth in these experiments was estimated by counting the number of autozooids and bifurcations in a colony. Bryozoans grow by asexual reproduction of modular units, zooids. from a sexually produced individual, the ancestrula. Zooids are connected to one another by a strand of tissue, the funiculus. Most generally there are two types of zooids: autozooids, which are present in all species, are specialized for feeding and digestion; hetero- zooids, which are not found in all species, function in defense, attachment, and reproduction. In B. neritina, brood chambers are the sole type of heterozooid. Thus, the number of autozooids is a good estimator of colony growth and has an advantage over dry weight or colony length in that it can be determined nondestructively over many days in the same individuals. Effect of lan'al xwimming duration and colon\ orientation on adult growth Larvae were released and metamorphosed as described above. Two polystyrene dishes were attached with low- temperature hot glue to a clear acrylic plastic plate. Three to five larvae were pipetted into dishes to ensure success- ful metamorphosis of at least one. On completion of meta- morphosis, all but a single individual were removed and the sides of the dish were trimmed away so that only the flat bottom portion remained. Each replicate consisted of two plates, each with two dishes and a total of four individuals: colony orientation was up (high siltation) or down (low siltation), and each plate had a 1-h and a 24-h individual (Fig. 1). The relative positions of the individuals were changed between replicates so as to nul- lify any micro-environmental effects associated with the plates. The replicate plates were then attached to nylon 10 cm ' A 1 h A 24 h Figure 1. Experimental apparatus for assessing the effect of colony orientation and swimming duration on growth. See text for details on the placement of ancestrulae ( Y ). The colonies were placed far enough apart so that no competition for food and space occurred. Colonies grew lor 14 days and then were returned to the laboratory for scoring. LARVAL SWIMMING DURATION 129 line at intervals of about 35 cm and suspended from float- ing docks. Lead ballasts weighing 0.90 kg were hung 0.5 m below the plates to keep them level, and the plates were submerged about 1 m below the surface. A rain gauge modified to serve as a sediment trap was submerged at the same level as the plates to provide a rough estimate of the amount of sediment accumulation over the course of the experiment. After 14 days, the plates were returned to the laboratory and the number of autozooids and bifur- cations counted for each colony. Each condition started with 18 replicates, totaling 72 individuals. Effect of larval swimming duration, orientation, and the presence of a conspecific colony on growth and reproduction Larvae were released and metamorphosed as described above. About 50 individuals were pipetted into polysty- rene dishes and allowed to metamorphose. The dishes were carved into thin strips such that each strip had a newly metamorphosed individual on its end (Fig. 2). The experimental apparatus was a plastic box that contained "lanes" with walls made of dense, chemically inert foam. Tiny slits were made in the foam, which allowed one end of the strip to be inserted in the wall of the lane. Another individual was placed in a slit directly opposite the first, so that the individuals shared space in the center of a lane. For each apparatus there were eight individuals in a total of four lanes. The individuals were either in the presence or absence of competition with a neighboring conspecific (i.e., with a conspecific in a slit directly across the lane facing the same direction) and were either 1-h or 24-h colonies. Another factor in this experiment was orientation (up or down; Fig. 2). However, orientation in this experiment did not expose colonies to different amounts of siltation, as the apparatus was designed to shield colonies from the downward flux of participate matter. After metamorphosis (ca. 48 h), individuals were arranged in blocks and transplanted to the field. Each apparatus was removed daily and the numbers of autozooids, bifurcations, and brood chambers were counted for each colony. Because the blocks were de- signed to hold a small volume of seawater. colonies were never exposed to the air during this process. As the colo- nies grew it became difficult to score all parameters in a single day, so only bifurcations and brood chambers were counted for all colonies after day 12. The number of autozooids was counted for replicate boxes 1-15 on day 13 and for boxes 16-30 on day 14. Due to time con- straints, autozooids were not counted after day 14. The experiment was ended on day 17, because the largest colonies began to overgrow the apparatus, potentially introducing additional effects. Analysis of variance (ANOVA) was applied to zooid data from day 12 and I 0.5 m to water surface T flu 7.5 cm competition 11 cm no competition A ^ no competition competition O 0.9 kg lead ballast \ Figure 2. Experimental apparatus for assessing the effect of compe- tition and swimming duration on growth and reproduction. See text for details on placement of ancestrulae ( Y). bifurcation and brood chamber data from day 17: these were the last days the respective data were collected for all colonies. Data analysis The data were not significantly different from a normal distribution, and a square root transformation was used to remove heteroscedasticity. The data were back-trans- formed for presentation in graphs and text. Since all fac- tors were fixed, a Model I ANOVA was used. For the 130 D. E. WENDT experiment on swimming time and colony orientation a 2-way factorial ANOVA was used to identify heterogene- ity of variances within the data sets. The main effects were swimming duration and orientation. For the experi- ment on competition and swimming duration a 3-way ANOVA was used and the main effects were swimming duration, presence of a conspecific. and orientation. Non- linear regressions were done in SYSTAT using simple and general allometry models (Ebert and Russell, 1994). Results Increased duration of larval swimming significantly re- duced growth and reproduction in both experiments with B. neritina. Colony orientation, when designed to expose colonies to different amounts of siltation pressure, also affected growth: colonies facing down were significantly larger than those facing up. The proximity of a conspecific (i.e., intraspecific competition), did not significantly affect growth, although it slightly delayed the onset of reproduc- tion. Growth between experiments cannot be compared since there was a temperature difference of more than 5C between the first and second experiments. On average, colonies grew faster under warmer conditions. Effect of larral swimmini; duration and colonv orientation on t>ro\\'tli During the 14 days of this experiment, approximately 0.2 cm of sediment accumulated in the trap and on the surfaces of the plates. Larval swimming duration and col- ony orientation significantly affected growth as measured by the number of autozooids and bifurcations in a colony (Fig. 3A, B; Table I). In no case was the interaction between swimming duration and orientation significant (P = 0.53 for zooid number and P = 0.65 for bifurca- tions). On average, 1-h colonies facing down (light sil- tation load) had twice the number of autozooids and bifur- cations as 24-h hour colonies facing up; whereas 1-h colo- nies facing up had almost the same number of autozooids and bifurcations as 24-h colonies facing down (Fig. 3). Reproduction of colonies did not occur at levels high enough to warrant statistical analysis. Effect of larva/ swimming duration, colony orientation, and the presence of a conspecific on growth and reproduction Neither orientation nor development next to a conspe- cific colony significantly affected growth and reproduc- tive output as determined by ANOVA (Table II). Orienta- tion did not expose animals to different amounts of sil- tation in this experiment, because the apparatus was shielded from the downward flux of particulate matter. Colony proximity appears to have some effect on the onset of reproduction (Fig. 4). Among 1-h colonies, those in the presence of a conspecific reached 50% reproduction 1 2 h later than those without a conspecific neighbor. Like- wise, in the absence of a conspecific neighbor. 24-h colo- nies reached 50% reproduction more than 31 h later than 1-h colonies (Fig. 4). On average, both the presence of a 300- AA 250- T c o -o 200- U 5" 150" T T 1 | 100- T 50- 11 - B 30- T 25- ?-, o T "3 20- 1 U 0, * 15- o T y 10- tC 5- 0- 1 i 1 I h Down 24 h Down h Up 24 h Up h Down 24 h Down h Up 24 h Up Condition Figure 3. Number of autozooids and bifurcations in 1-h and 24-h colonies. 14 days after metamorphosis. Colonies were oriented up ("high siltation") or down ("low siltation"). Error bars = 95% confidence interval; n = ca. 50 colonies. LARVAL SWIMMING DURATION 131 Table I Results of two-way factorial ANOVA for the effect of lan-al swimming duration and colony orientation (up or down} on the number of ;ooids mill bifurcations in Bugula neritina colonies (n = 53 colonies) Measurement Source of variation d f SS MS F. P value Bifurcations Swimming duration 5.19 5.19 3.74 0.05 Orientation 11.5 1 1.5 8.28 0.006 Interaction 0.55 0.55 0.40 0.53 Residual 4 68.1 1.39 Autozooids Swimming duration 88.7 88.7 5.11 0.02 Orientation 108 108 6.22 0.01 Interaction 3.56 3.56 0.21 0.65 Residual 5 > 903 17.4 conspecific colony and increased duration of larval swim- ming delayed the onset of reproduction; differences of less than 1 2 h cannot be resolved. Longer larval swimming leads to reduced growth and reproductive output (Table II; Figs. 5, 6, and 7). For exam- ple, 14 days after metamorphosis the average number of autozoids was 113 7 (n = 50; mean 95% confidence) for 1-h colonies, compared to 74 6 (n = 49), for 24-h colonies. However, the slopes of the regression lines for autozooid number as a function of days after metamorpho- sis were statistically indistinguishable; an indication that both groups of colonies were growing at about the same rates, despite differences in the absolute number of auto- zooids at day 14 (Fig. 5). The number of brood chambers, a measure of reproductive output, was significantly lower in 24-h colonies (P < 0.001 ). The average number of brood chambers was 149 19 (n = 94: mean 95% confidence) for 1-h colonies and 76 1 1 (n = 93) for 24-h colonies. Thus. 17 days after metamorphosis. 1-h colonies had, on average, more than twice the number of brood chambers as did 24-h colonies. Furthermore, unlike the rate of produc- tion of new autozooids. which was similar in 1-h and 24-h colonies very soon after metamorphosis, the rate of brood-chamber production was significantly less in 24-h colonies for the duration of the experiment (Fig. 7). Similar- trends were observed in bifurcations (Fig. 6). The only significant interaction between main factors was between colony orientation and proximity of a con- specific (F = 7.6, P = 0.006 for brood chambers; F = 6.5, P = 0.01 for autozooids). Discussion Most empirical evidence that nonlethal larval experi- ences have long-term effects comes from observations Table II Results of three-wav factorial ANOVA for the effect of lan'iil swimming duration, colony orientation (u/> / ilnwn), and competition on the number of zooids, bifurcations, and brood chambers in Bugula neritina colonies (n = 180 colonies) Measurement Source of variation df SS MS F, P value Bifurcations Swimming duration 1 24.1 24.1 23.8 0.0001 Competition 1 1.83 1.83 1.81 0.18 Orientation 1 0.16 0.16 0.16 0.69 Interactions 4 3.03 0.76 0.08 >0.5 Residual 180 181 1.01 Autozooids Swimming duration 1 42.8 42.8 30.3 0.0001 Competition 1 0.46 0.46 0.32 0.57 Orientation 1 0.62 0.62 0.44 0.51 Interactions* 4 2.58 0.64 0.46 >0.5 Residual 174 252 1.41 Brood chambers Swimming duration 1 584 584 33.8 0.0001 Competition 1 1.51 1.51 0.09 0.77 Orientation 1 2.49 2.49 0.14 0.71 Interactions* 4 167 41.7 2.43 0.11 Residual 178 3100 17.3 For clarity, the interactions of main effects were collapsed into a single interaction term. * There was a significant interaction between orientation and competition: see Discussion for an examination of this outcome. 132 D. E. WENDT 75% - - 2-1 h competition abscnl B | h cnmpeltlion presenl A 24 h competition 25- 25%- 0%4 13 14 15 16 17 18 Days After Metamorphosis Figure 4. Onset of reproduction in Bii^nla ncritina as a function of larval swimming duration, competition, and time after metamorphosis. n = 40-50 colonies for each curve. on marine invertebrates with aplanktotrophic larvae. The influence of such experiences on postmetamorphic perfor- mance is not restricted to marine invertebrates, however. For example, the feeding history of larval reef fish affects the average diameter of tail muscle fibers, average size at settlement, and average juvenile feeding rates (McCor- 15- D Ih Colonies y = (0.149E-2 *x 3 ' 43 ) - 0.13 r =099 4> 24h Colonies y = (0.169E-2 * X 329 ) -0 18 r=099 16 Days After Metamorphosis Figure 6. Mean number of bifurcations as a function of larval swim- ming duration and time after metamorphosis in Bugnhi ncritinu. Error bars = 95% confidence interval of the means, n = ca. 90 colonies for each curve, and all nonsignificant data were pooled. Regressions were calculated using all zero values for v. mick and Molony. 1992). In amphibians, food deprivation at different periods of tadpole ontogeny can precipitate metamorphosis at smaller sizes (Audo et al., 1995). In insects, the reproductive fitness of the adult female flesh u a o s lon- 80- 60- D 1 h Colonies y = 0.485 * 10" 17CK r 2 = 0.99 * 24h Colonies y = 0.407 * 10" "-'" r : = 0.98 S. I 2 CQ 150- 100- 50- D Ih Colonies y = (0 465E-9*x" 3f> ) - I 35; r 2 = 98 * 24h Colonies y =|0 I04E-IO'X 1046 )-0.61; r 2 = 0.98 Days After Metamorphosis Figure 5. Mean number of aulo/ooids as a function of larval swim- ming duration and time after metamorphosis in Biti*ii/u neriiinu. Error bars = 95 r 'i confidence interval of the means, n = ca. W colonies for each curve, and all nonsignificant data were pooled. Regressions were calculated using all /.ero values for v. Days Alter Metamorphosis Figure 7. Mean number of brood chambers as a function of larval swimming duration and time after metamorphosis in Buguhi neriiinu colonies. Error bars = 95% confidence interval of the means, n = ca. 90 colonies for each curve, and all nonsignificant data were pooled. Regressions were calculated using all zero values for v. LARVAL SWIMMING DURATION 133 fly is inversely correlated with the amount of time the larva spends in diapause (Denlinger, 1981 ). Effect of Inn-til swimming duration on growth and reproduction In both the siltation and the competition experiments, a 24-h increase in the larval swimming period significantly reduced growth and reproduction compared to 1-h colo- nies (Tables I and II). Since growth in B. nehtiiui is an exponential process, small differences during early colony development will translate into large differences in colony size (Figs. 5 and 6). On average, 24-h colonies had half as many autozooids, bifurcations, and brood chambers as did 1 -h colonies over the same time of development. The observed difference is not a result of 24-h colonies finish- ing metamorphosis 23 h later than 1-h colonies, as experi- ments were designed so that both groups of animals fin- ished metamorphosis and began feeding at nearly the same time. Thus, the lower values observed for 24-h colo- nies cannot be attributed to a shorter period of postmeta- morphic development in fact, 24-h larvae take an addi- tional 6-8 h to metamorphose (Wendt, 1996). Overall, then, the differences observed between 1-h and 24-h colo- nies are conservative. Because 24-h colonies initially grow more slowly, they will always have fewer autozooids, bifurcations, and brood chambers at a given point in time and, assuming similar growth potentials, they will never "catch up" to their 1-h counterparts. But is there a point at which the two groups have equal rates of growth or reproduction? For growth rate, as measured by the number of auto- zooids. this happens several days after metamorphosis when the slope of the regression lines are virtually identi- cal (Fig. 5). For reproduction, however, the slopes of the regression lines appear to be different throughout the duration of the experiment (Fig. 6). indicating that. 17 days after metamorphosis, 24-h colonies have not pro- duced brood chambers at the same rate as 1-h colonies. Considering that colonies in the Indian River in the vicin- ity of the Smithsonian Marine Station at Link Port persist in the field for 5 or 6 weeks at most (L. J. Walters, pers. comm.; DEW, pers. observ.), the rate of reproductive output would be severely compromised as the larval swimming phase increases. Effect of colony orientation on growth Orientation of developing colonies significantly af- fected growth in B. neritina (Table I; Fig. 3), presumably by influencing the amount of siltation the colonies en- countered. Colonies on the upper surfaces of the plates, which experienced a higher concentration of silt particles, grew more slowly. The hypothesis that the additional par- ticles interfered with feeding is supported by evidence from colonies of the intertidal bryozoan Fliixtrellulrn /'.v- pidti. In that species, the concentration of inorganic sus- pended paniculate matter (i.e., silt) is inversely related to the number of autozooids actively feeding (Best and Thorpe, 1996). A colony with fewer feeding autozooids is likely to capture less food and consequently have less energy available for growth and reproduction. Sedimenta- tion has also been shown to be a significant source of mortality in newly settled solitary ascidians (Young and Chia. 1984). That colonies on the undersides of surfaces grow sig- nificantly larger than those on the upper surfaces suggests that facing downward is beneficial. Many invertebrate larvae are known to settle and metamorphose in environ- ments that favor the chances of adult survival (Olson. 1983; Young and Chia, 1985; Walters and Wethey. 1991; Walters. 1992; Hurlbut, 1993). In the field. B. neritina colonies are most often found growing on the undersides of objects and ledges. Whether this distribution is the result of larval behavior, differential postmetamorphic mortality, or some combination of the two has not been explicitly investigated. However, certain larval behaviors may, in part, account for the patterns observed in the field. Larvae of B. neritina settle preferentially on the undersides of plates in the field, and they also settle in shaded areas (Ryland, 1977; Table II). At the time of attachment, larvae settle such that the incipient zooid and the resultant colony face away from light (McDougall, 1943). Under natural conditions with light from above, the colony is oriented with its frontal surface and lophophore facing away from the water surface; thus, the colony is shielded from the downward flux of paniculate matter. Not all behaviors produce this result, however. Colonies of B. uviciikiria and B. neritina show a positive phototro- pism (Aymes. 1956; Schneider, 1959), which, according to the results of the current study, would retard growth, since the colonies would grow frontal surface up. Orientation did not significantly affect growth in the competition experiment, which is not surprising in that orientation in this experiment did not expose colonies to different amounts of siltation. All colonies were shielded from sedimentation, and no sediment accumulated on the surfaces where colonies were growing. One factor not controlled for in this experiment was LJV radiation. Al- though UV light has the potential to affect growth, the overall exposure for any colony in the experiment was minimal because none of the replicates were ever exposed to direct sunlight. Effect of the presence of a conspecific colony on growth find reproduction The adjacency of a conspecific colony (i.e., a potential competitor) did not significantly affect growth and repro- 134 D. E. WENDT duction (Table II). Either postmetamorphic competitive ability is not compromised by increased duration of larval swimming or the individuals did not experience a limiting resource and were not subjected, therefore, to a competi- tive situation. A priori, the former reason appears less satisfactory given that lophophore size decreases signifi- cantly as a function of larval swimming duration ( Wendt, 1996) and that smaller lophophores generate currents with lower velocities (Best and Thorpe, 1986). Both lopho- phore size and current velocity can influence interactions as colonies grow and compete for space and food (Buss. 1979). The more likely explanation is that individuals did not experience a limited supply of food. The effects of inter- and intraspecific competition in the context of in- creased duration of larval swimming remain unresolved. Interaction het\<,'een orientation and competition In the competition experiment, neither competition nor orientation as main factors had a significant effect on growth and reproduction. However, there was a signifi- cant interaction between these factors (F = 7.6, P = 0.006 for brood chambers; F = 6.5, P = .01 for autozooids), which suggests that some combination of orientation and competition may result in reduced growth and reproduc- tion. Ad hoc analysis showed that in 4 out of 5 cases, individuals that faced competition and were oriented up- ward (i.e., had a light siltation load in this experiment) had, on average, fewer zooids, brood chambers, and bifur- cations. This result indicates that competition and upward orientation acting individually were not strong enough factors to reduce growth and reproduction, but that in concert they may compromise colony fitness. Mechanisms of action The observed difference in the rates of autozooid bud- ding is probably a result of a delay in the time to first bud, because the increased larval swimming period un- doubtedly uses energy that would otherwise go to form the first autozooid. Any energetic deficiency of the ances- trula should not persist, however, so the growth rate should approach normal by the time the first several buds have formed. The difference in the quantity and produc- tion rate of brood chambers between 1-h and 24-h colo- nies is enigmatic. The difference is unlikely to result solely from the energetic deficiency caused by a length- ened period of larval swimming. One other mechanism that might play a part in producing these long-term effects is interspecific competition between bryozoans and stalked protozoans (e.g., Canchesium sp., Zootlianiiiin sp., Vorticclla sp.). Unfortunately it was impossible to exclude these pervasive interspecific competitors, which colo- nized the surfaces of colonies. Consequently, all condi- tions had a background of interspecific competition and there could be some difference in interspecific competi- tive ability of 1-h and 24-h colonies. An alternative expla- nation suggested by Pechenik ct ul. (in press) is that cer- tain gene products transcribed early in development may be needed for organogenesis and that certain environmen- tal stresses encountered in larval life may interfere with transcriptional or translational processes. Furthermore, it is well known that environmental stress can damage popu- lations of cells and even entire organs during develop- ment. In any case, it seems that the effects observed in B. neritina may not be attributed entirely to energetic causes. Effect of lan'ul dispersal ability on species evolution Taylor (1988) proposed that the major radiation of cheilostome bryozoans 150 million years ago was in part due to the evolution of nonfeeding larvae (like those of B. neritina}. which severely limited the dispersal of these species. In general, species with this type of larva (short- lived, aplanktotrophic) have lower gene flow between subpopulations and thus a greater subpopulation ge- netic structure than species with long-lived, plankto- trophic larvae (Palumbi, 1994). My results support Tay- lor's hypothesis by demonstrating that individuals of B. neritina (and probably of all bryozoans with aplankto- trophic larvae) incur substantial lethal and nonlethal costs after relatively short periods of larval swimming (hours to days). These costs limit the dispersal of bryozoans with aplanktotrophic larvae and may contribute significantly to subpopulation genetic structure. On evolutionary time scales, these costs and their consequences have probably played a central role in speciation within the Bryozoa. Acknowledgments I thank Sherry Reed, Hugh Reichardt. and Dr. Mary E. Rice, all of the Smithsonian Marine Station at Fort Pierce, Fort Pierce. Florida, for their assistance in carrying out this research. Colleen Cavanaugh. Peter Goss, Ed Seling. and Robert Woollacott (all of Harvard University) provided thoughtful discussions and comments which greatly improved the study. I thank Wendy Lynn Wendt for editing the manuscript. This research was supported by a Smithsonian Fellowship and by a Grant-in-Aid of Research from Sigma Xi to Dean E. Wendt. This paper is contribution #448 to the Smithsonian Marine Station at Fort Pierce, Fort Pierce, Florida. Literature Cited Audo, M. C., T. M. Mann, T. I,. Polk. C. M. Loudenslager. W. J. Diehl, and R. Altig. 1">V5. Food deprivation during different peri- ods of tadpole (Hylti fluysuscelix) ontogeny affects metamorphic performance differently. Oecologia 103: 518-522. LARVAL SWIMMING DURATION 135 Aymes, V. 1956. Croissance phototropique chez les Bryozoaires du genre Bugula. C. R. Hebd. Seances Acad. Sci. 242: 1237-1238. Best, M. A., and J. A. Thorpe. 1986. Effects of food concentration on feeding current velocity in six species of marine Bryozoa. Mar. Bin/. 93: 255-262. Best, M. A., and J. A. Thorpe. 1996. The effect of suspended panicu- late matter (silt) on the feeding activity of the intertidal ctenostomate bryozoan Fliitrellidra hispida (Fabricius). Pp. 39-45 in Bryo-oa in Space and Time, D. P. Gordon, A. M. Smith, and J. A. Grant-Mackie, eds. Colorographic International. Wellington, New Zealand. Buss, L. W. 1979. Bryozoan overgrowth interaction the interdepen- dence of competition for space and food. Nature 281: 475 \11 . Chia, F.-S. 1989. Differential larval settlement of benthic marine in- vertebrates. Pp. 3-12 in Reproduction, Genetics, and Distribution of Marine Organisms, J. S. Ryland and P. A. Tyler, eds. Olsen & Olsen. Fredensborg. Denmark. Crisp, D. J. 1984. Overview of research on marine invertebrate larvae. 1940-1980. Pp. 103- 125 in Marine Biodeterioration: An Interdisci- plinary Study, J. D. Costlow and R. C. Tipper, eds. Naval Institute Press. Annapolis, Maryland. Denlinger, D. L. 1981. Basis for a skewed sex ratio in diapause- destined flesh flies. Evolution 34: 1247-1248. Ebert, T. A., and M. P. Russell. 1994. Allometry and model 11 non- linear regression. /. Theor. Biol. 168: 367-372. Hadfield, M. G. 1978. Metamorphosis in marine molluscan larvae: an analysis of stimulus and response. Pp. 165-175 in Settlement and Metamorphosis of Marine Invertebrate Larvae, F.-S. Chia and M. E. Rice, eds. Elsevier North-Holland, New York. Havenhand, J. N. 1995. Evolutionary ecology of larval types. Pp. 79- 122 in Ecology of Marine Invertebrate Lan-ae, L. R. McEdward, ed. CRC Press. Boca Raton. Florida. Highsmith. R. C., and R. B. Emlet. 1986. Delayed metamorphosis: effect on growth and survival of juvenile sand dollars (Echinoidea: Clypeasteroida). Bull. Mar. Sci. 39: 347-361. Hunter, E., and N. Eusetani. 1996. Studies of the effects of larval swimming time on settlement, metamorphosis, and post-larval devel- opment of Bitgu/a neritina (Cheilostomatida). Pp. 139-148 in Bryo- zoa in Space and Time, D. P. Gordon, A. M. Smith, and J. A. Grant- Mackie, eds. Colorographic International, Wellington, New Zealand. Hurlbut, C. J. 1993. The adaptive value of larval behavior of a colo- nial ascidian. Mar. Biol. 115: 253-262. McCormick, M. I., and B. W. Molony. 1992. Effects of feeding his- tory on the growth characteristics of a reef fish at settlement. Mar. Biol. 114: 165-173. McDougall, K. D. 1943. Sessile marine invertebrates at Beaufort, North Carolina. Ecol. Monogr. 13: 321-374. Morgan, S. G. 1995. Life and death in the plankton: larval mortality and adaptation. Pp. 279-321 in Ecology of Marine Invertebrate Lamie. L. R. McEdward, ed. CRC Press, Boca Raton, Florida. Olson, R. R. 1983. Ascidian-Prochloron symbiosis: the role of larval photoadaptation in midday larval release and settlement. Biol. Bull. 165: 221-240. Orellena. M. R., and J. M. Cancino. 1991. The effects of delaying larval settlement on metamorphosis and early colonial growth in Celleporella hyalina (Bryozoa: Cheilostomata). Pp. 309-316 in Bry- ozoa Living and Fossil, P.P. Bigley. ed. Bull. Soc. Sci. Nat. Quest. Fr., Mem. HS1. Saint-Herblain, Nantes, France. Nielsen, C. 1981. On morphology and reproduction of Hippodiplosiu inscu/pta and Fenestrulina ma/usii (Bryozoa, Cheilostomata). Ophe- lia 20: 91-125. Palumbi, S. R. 1994. Genetic divergence, reproductive isolation, and marine speciation. Annu. Rev. Ecol. Syst. 25: 547-572. Pawlik, J. R. 1992. Chemical ecology of marine invertebrates. Oceanogi: Mar. Biol. Annu. Rev. 30: 273-335. Pechenik. J. A. 1990. Delayed metamorphosis of benthic marine in- vertebrates: Does it occur 1 ? Is there a price to pay? Ophelia 32: 63- 94. Pechenik, J. A., and T. R. Cerulli. 1991. Influence of delayed meta- morphosis on survival, growth, and reproduction of the polychaete Capitella sp. I. J. Exp. Mar. Biol. Ecol. 151: 17-27. Pechenik, J. A., and \.. S. Eyster. 1989. Influence of delayed meta- morphosis on the growth and metabolism of young Crepidula forni- cata (Gastropoda) juveniles. Biol. Bull. 176: 14-24. Pechenik, J. A., D. Rittschof, and A. R. Schmidt. 1993. Influence of delayed metamorphosis on survival and growth of juvenile barnacles Balwnis ampltitrite. Mar. Biol. 115: 287-294. Pechenik, J. A., J. N. Jarret. and D. E. Wendt. Metamorphosis is not a new beginning. BioScience (in press). Roughgarden, J.. S. Gaines, and H. Possingham. 1988. Recruitment dynamics in complex life cycles. Science 241: 1460-1466. KIIIIII ill. S. S. 1990. Natural mortality of marine invertebrate larvae. Ophelia 32: 163-198. Ryland, J. S. 1977. Taxes and tropisms of bryozoans. Pp. 411-436 in Biology of Bryozoans, R. M. Woollacott and R. L. Zimmer, eds. Academic Press, New York. Scheltema, R. S. 1974. Biological interactions determining larval set- tlement of marine invertebrates. Thalassia Jugosl. 10: 263-296. Schneider, D. 1959. Der Aufhau der BgK/a-tierstocke und seine Beeinflussung durch Aussenfaktoren. Biol. Zentbl. 78: 250-283. Strathmann, R. R. 1985. Feeding and nonfeeding larval development and life-history evolution in marine invertebrates. Annu. Rev. Ecol. Syst. 16: 339-361. Strathmann, R. R. 1993. Hypotheses on the origins of invertebrate larvae. AIIIIII. Rev. Ecol. Syst. 24: 89-117. Taylor, P. D. 1988. Major radiation of cheilostome bryozoans: trig- gered by the evolution of a new larval type? Hist. Biol. 1: 45-64. Vance, R. R. 1973. On reproductive strategies in marine bottom inver- tebrates. Am. Nat. 107: 353-361. Walters, L. J. 1992. Post-settlement success of the arborescent bryo- zoan Bugula neritina (L.): the importance of structural complexity. J. Exp. Mar. Biol. Ecol. 164: 55-71. Walters, L. J., and D. S. Wethey. 1991. Settlement, refuges, and adult body form in colonial marine invertebrates: a field experiment. Biol. Bull. 180: I 1 2-1 IS. Wendt, D. E. 1996. Effect of larval swimming duration on success of metamorphosis and size of the ancestrular lophophore in Bugula neritina (Bryozoa). Biol. Bull. 191: 224-233. Wendt, D. E., and R. M. Woollacott. 1995. Induction of larval settle- ment by KC1 in three species of Bitgula (Bryozoa). Invertebr. Biol. 114: 345-351. Woollacott, R. M., J. A. Pechenik. and K. M. Imbalzano. 1989. Ef- fects of duration of larval swimming period on early colony develop- ment in Bitgula stolonifera (Bryozoa: Cheilostomata). Mar. Biol. 102: 57-63. Wray, G. A. 1995. Evolution of larvae and developmental modes. Pp. 413 J48 in Ecology of Marine Invertebrate Lan'ae, L. R. McEdward, ed. CRC Press. Boca Raton. Florida. Young, C. M., and F.-S. Chia. 1981. Laboratory evidence for delay of larval settlement in response to a dominant competitor. Int. ./. Invertehr. Reprod. 3: 221-226. Young, C. M., and F.-S. Chia. 1984. Microhabitat-associated vari- ability in survival and growth of subtidal solitary ascidians during the first 21 days after settlement. Mar. Biol. 81: 61-68. Young, C. M., and F.-S. Chia. 1985. An experimental test of shadow response function in ascidian tadpoles. J. Exp. Mar. Biol. Ecol. 85: 165-175. Reference: Biol. Bull. 195: 136-144. (October. 1998) Structural Strengthening of Urchin Skeletons by Collagenous Sutural Ligaments OLAF ELLERS 1 , AMY S. JOHNSON 2 *, AND PHILIP E. MOBERG' 1 Section of Evolution and Ecology, Division of Biological Sciences. University of California. One Shields Ave., Davis, California 95616; ~* Biology Department, Bowdoin College, Brunswick, Maine 0401 1; and "Marine Biolog\ Research Division, Scripps Institution of Oceanograph\, University of California, San Diego, La Jolla, California 92093-0202 Abstract. Sea urchin skeletons are strengthened by flex- ible collagenous ligaments that bind together rigid calcite plates at sutures. Whole skeletons without ligaments (re- moved by bleaching) broke at lower apically applied forces than did intact, fresh skeletons. In addition, in three-point bending tests on excised plate combinations, sutural liga- ments strengthened sutures but not plates. The degree of sutural strengthening by ligaments depended on sutural position; in tensile tests, ambital and adapical sutures were strengthened more than adoral sutures. Adapical sutures, which grow fastest, were also the loosest, suggesting that strengthening by ligaments is associated with growth. In fed, growing urchins, sutures overall were looser than in unfed urchins. Looseness was demonstrated visually and by vibration analysis: bleached skeletons of unfed urchins rang at characteristic frequencies, indicating that sound traveled across tightly fitting sutures; skeletons of fed ur- chins damped vibrations, indicating loss of vibrational en- ergy across looser sutures. Furthermore, bleached skeletons of fed urchins broke at lower apically applied forces than bleached skeletons of unfed urchins, indicating that the sutures of fed urchins had been held together relatively loosely by sutural ligaments. Thus, the apparently rigid dome-like skeleton of urchins sometimes transforms into a flexible, jointed membrane as sutures loosen and become flexible during growth. Introduction Animal skeletons commonly consist of rigid ossicles connected by flexible ligaments. In this design, rigid ele- Received 30 January 1998; accepted 12 June 1998. * To whom correspondence should he addressed. E-mail: ajohnsonCff 1 polar.bowdoin.edu ments resist compression and bending, whereas flexible elements resist tension and allow flexion. Vertebrate limb joints and echinoderm skeletons are examples of this de- sign. Echinoderm skeletons are composed of calcite ossi- cles and collagenous connective tissues in a ratio that differs among taxa (Moss and Meehan, 1967). In sea cucumbers, for instance, collagenous tissues predominate, whereas in most sea urchins, calcite plates predominate. In the current study, we investigate the structural role of the collagenous ligaments that bind together the calcite plates in urchin skeletons. The perforated calcite plates are attached to each other at sutures by ligaments (Fig. 1 ) that wrap around calcite rods (trabeculae), thus sewing together adjacent plates (Moss and Meehan, 1967). In addition, trabeculae project from one plate into holes in the adjacent plates, thus interlocking the plates. Ligaments are present to various degrees depending on the species and on the position of a suture on an urchin (Tel ford, 1985a). For instance, meridional (= vertical or radial or zig zag) sutures tend to have more ligaments than do circumferential (= hori- zontal or hoop) sutures. Also, some regions of the skele- ton have relatively many sutures and smaller plates. For instance, the ambulacra! regions (where the tube foot rows are located), are composed of many small plates (one plate per tube foot). In contrast, the interambulacral re- gions have larger plates and fewer sutures. The skeletal structure is completed by the peristomial membrane, which is attached at the oral margin. This membrane is a tough but flexible collagenous tissue (sometimes with calcite ossicle inclusions) that bridges the gap between the Aristotle's lantern (or jaw) and the skeleton (Wilkie etal., 1993). Urchin skeletons grow both by the accretion of calcite 136 STRENGTHENING OF URCHIN SKELETONS 137 collagenous sutural ligaments meridional suture peristomial membrane Aristotle's Lantern Figure 1. Schematic of interambulacral suture geometry and sutural ligament attachment. Interambulacral. meridional, and circumferential sutures are illustrated. Ligaments bind together adjacent plates (magni- fied inset of cross-section of a suture). at the edges and faces of the plates and by the addition of new plates at the apex (Deutler. 1926). Initially apical, new plates gradually migrate adorally during growth. Skeletal plates may show seasonal or yearly growth rings that reflect changes in growth rate (Gage. 1991. 1992a. b) or spurts of growth (Pearse and Pearse, 1975). Urchins may also shrink under conditions of lowered food avail- ability or overcrowding, as observed, for example, in Diu- dema antillamin (Levitan, 1988), and in Strongylocentro- tus purpuratus (Ebert, 1967). In Heliocidaris erythro- grcinuuu. shrinkage is associated with diminution of sutural gaps (Constable, 1993). That sutural ligaments might strengthen the skeleton was suggested in a taphonomic study in which decom- posed urchin skeletons disarticulated at sutures and broke at lower forces than did undecomposed urchin skeletons (Kidwell and Baumiller, 1990). On the basis of histologi- cal and morphological evidence, sutural ligaments were interpreted as "stress-breakers" that evenly distribute stresses and thus contribute to the structural integrity of echinoid skeletons (Moss and Meehun. 1967). In other theoretical and experimental studies of the structural me- chanics of echinoid skeletons (Telford, 1985b; Dafni. 1986. 1988: Baron. 1991a. b; Ellers. 1993; Philippi and Nachtigall, 1996), the possible structural role of the su- tural ligaments has not been a focus. Nor has a possible structural role for the peristomial membrane been tested. Although this membrane is not flexurally stiff relative to the calcite plates, it might provide tensile reinforcement to the skeleton, as do steel tensile reinforcement rods across the span of concrete bridge arches. To determine the relative contributions of skeletal com- ponents (sutural ligaments, peristomial membrane, and calcite plates) to the structural strength of urchin skele- tons, we measured breaking forces of entire skeletons and excised portions of skeletons of 5. purpuratus, with soft tissues either present or removed. To evaluate possible sutural changes during growth, we used breaking and vibration tests as well as visual inspection to compare the looseness of sutures in fed, growing specimens of S. purpuratus with that in unfed, nongrowing specimens. Materials and Methods Testing device A motor-driven device and force beam were used to apply a load at a rate of 200 //m s~ ' to various specimens until they broke. The force signal was amplified, digitized (12-bit. 100 Hz), and stored in a computer. Error in the force measurements was <0.1 N. Prior to breaking tests, the height and diameter of all urchins were measured to 0. 1 mm with calipers. Crushing of whole skeletons Strongylocentrotus purpnnitus was collected subtidally at Bodega Bay. California. Urchins were assigned at random to three groups, each prepared for crushing tests in a different way: (i) live unaltered, i.e., with peristomial membrane and sutural ligaments intact: (ii) peristomial membrane and Aris- totle's lantern removed, i.e., with sutural ligaments intact; and (iii) bleached and air-dried, i.e., with no soft tissues intact. The bleaching chemical was 5.25% sodium hypo- chlorite. Sufficient bleach was added until all soft tissues had been dissolved and washed away. The prepared skele- tons were crushed along the oral-aboral axis. Three-point breakage of plates aiul meridional sutures S. purpuratus was collected intertidally at Pillar Point. California. Urchins were kept in a 12C recirculating seawa- ter system for less than a week prior to mechanical tests. Pieces of skeleton consisting of various combinations of plates and sutures were prepared for mechanical testing as follows. From each urchin, two homonomous. ambital. interambulacral plates were cut out using a Dremel tool. 138 O. ELLERS ET AL. As a control, one plate was bleached overnight prior to testing, and the other plate was tested to breakage while fresh (held for less than 1 h in 12C seawater prior to testing). In addition, two sets of homonomous, three-plate combinations, joined to each other at a meridional, ambi- tal, interambulacral suture were cut out. Again, one set was bleached overnight prior to testing and the other was tested to breakage while fresh. For mechanical testing, each test piece was placed on the base platform, which was coated lightly with mineral oil to reduce friction. Each test piece formed a shallow arch and, as the piece was driven into the platen to which the load cell was attached, force was applied by a cylindri- cal rod (diameter 1 mm) to the apex of the arch, till break- age occurred. Thickness and width at the broken section were measured to 0.1 mm using calipers. Breaking forces were compared to determine if there was a signifi- cant reduction in breaking force after bleaching. Because of the complex geometry of the beams, we compared breaking forces of homonomous specimens paired for size rather than comparing estimated stresses. Tensile tests of circumferential .futures S. fniipiimtits was obtained from a subtidal population near Bodega Bay. One group was bleached and air-dried before testing; the other group was tested fresh. Groups of interambulacral plates were excised from three regions on the skeleton: adapical, ambital, and adoral. The ambital region was defined to be four plates wide, centered at the widest diameter of the urchin. The adapical region was defined as starting three plates above the uppermost ambi- tal plate. The adoral region was defined as starting two plates below the lowest ambital plate. For each tensile test, the strength of a circumferential suture was measured. Each test piece was excised and consisted of a column of four plates. The test piece was attached to the testing device using strands of copper wire that were looped through holes ( 1 .2 mm in diameter) drilled in the test piece. The holes in the test piece were located in the middle of the plates immediately adjacent to the suture being tested. Thickness and length of the cross-sectional area being broken were measured to 0.1 mm using calipers. These measurements were used to calculate a nominal stress; nominal because the porous nature of the plates and sutures makes it impractical to measure an actual cross-sectional area. 5 g per urchin per day), untiltered seawater, and normal room light. The starved group was given no seaweed, fil- tered incoming water, and reduced light, which reduced algal growth on the aquarium walls. At the start of the experiment, all urchins were marked with a tetracycline label as in Ebert (1982). After one year the urchins were sacrificed and bleached and the tetracycline label was in- spected. Skeletal diameter (0.5 mm) and weight (0.1 g) were measured, and the sutures were visually inspected. To quantify sutural looseness, vibrational properties of the skeletons were measured as follows. A 3.5-g weight was hung on the end of a string attached to a crossbeam hanging above a rubber-lined box of dimensions 28 by 18 by 12 cm. An urchin skeleton's vibrational properties were tested by placing the skeleton in the box and posi- tioning the cross-beam so that the weight hung vertically down to a point just 2 mm from the ambitus. The weight was then lifted to a 30 angle to the vertical and released. The weight swung and hit the skeleton, causing a sound that was recorded with a microphone and analyzed with spectral and cross-correlation procedures. The pattern of frequencies at which a particular urchin vibrated was ana- lyzed statistically with respect to diet, urchin diameter, and skeletal strength under apical loading. Results Crushing of whole skeletons Removal of the peristomial membrane had no effect on diameter-specific breaking forces of skeletons with intact sutural ligaments (Fig. 2). A simultaneous regres- soo-i 400- o 300- o 200- 1 00 * Ligaments * intact Ligaments removed Diet and sutural ,t,',v S. purpuratus was collected subtidally near Bodega Bay and kept for one year in flow-through seawater aquaria at Bodega Marine Laboratory. Urchins were assigned ran- domly to one of two diet treatments. The fed group received seaweed ad libitum ( 1 80 kg over 1 year, or an average of Diameter (cm) Figure 2. Breaking force of Strongylocentrotus /mr/mi'iints skele- tons as a function of diameter. Bleached skeletons (open triangles) broke at much lower forces than did fresh skeletons with ligaments intact (solid circles). Removing the peristomial membrane from fresh intact skeletons (solid squares) did not decrease the strength of the skeleton relative to intact skeletons. STRENGTHENING OF URCHIN SKELETONS 139 a o LL O) !5 re 0) m plates sutures Preparation Figure 3. In three-point bending, bleached (hatched) meridional sutures broke at significantly lower forces than did fresh (solid) meridional sutures, whereas bleaching did not affect the breaking force of plates. Asterisk indi- cates significant difference. Error bars indicate one standard error. sion with two slopes and two intercepts did not explain significantly more variation than did a single slope with a single intercept (P = 0.5. Ftest, d.f. = 1,44: regression with dummy variables; Weisberg. 1980). For all skeletons with intact ligaments: breaking force = 79 + 55 diame- ter, where diameter is in centimeters and force is in new- tons; the intercept was not significantly different from zero, but the slope was significantly different from zero (P < 0.001, F test. d.f. = 1,45, r = 0.42). In contrast, with the exception of two high outliers, bleached skeletons had much lower breaking strength than skeletons with intact ligaments (Fig. 2). When data from all urchins with intact ligaments were pooled and the two outliers were excluded, simultaneous regression of force on diameter showed that a model with two slopes and two intercepts for the intact versus removed ligaments explained significantly more variation than did a model with one slope and two intercepts (P < 0.05, F test, d.f. = 1,66: regression with dummy variables; Weisberg, 1980). Further, among bleached urchins, there was no significant variation of breaking force with diameter; nei- ther intercept nor slope were significantly different from zero (P = 0.1, F test. d.f. = 1,21). Three-point breakage of plates and meridional sutures The urchins ranged in diameter from 4.1 to 7.3cm, with only one being more than 5.3 cm. Over this limited size range, breaking force of test pieces was independent of urchin diameter, height, or nominal cross-sectional area (plate or suture thickness multiplied by plate or suture width) (linear regressions, probabilities of slope equal to zero ranging between 0.17 and 0.90). Fresh sutures broke at a higher mean force than did bleached sutures (P < 0.05, Mann-Whitney U test, 13 pairs of sutures), whereas fresh and bleached plates broke at similar forces (P = 0.5, Mann-Whitney U test, 14 pairs of plates) (Fig. 3). (We used the nonparametric U test instead of a paired t test to compare bleached and fresh specimens because the differences between the breakage forces of the paired sutures were not normally distributed: Shapiro-Wilk IV test. P < 0.003). Tensile tests of circumferential sutures The nominal breaking stress of circumferential sutures (Fig. 4) was significantly different (2-way ANOVA) by position of the suture on the skeleton (P < 0.0001. d.f. = 2,95) and in bleached versus fresh sutures (P < 0.0001. df = 1,95); furthermore, there was an interaction between position and bleaching (P < 0.0001, d.f. = 2.95). Group- ing all positions, the 54 bleached sutures had a lower nominal breaking stress than the 47 fresh sutures (P < 0.0001, Mann-Whitney U test). Also, at each position bleached sutures had a lower nominal breaking stress than fresh sutures (all P < 0.05. Mann-Whitney U tests). 2000 4000 6000 8000 10000 frequency Figure 7. Characteristic frequency spectra of sounds emitted by vibrating, bleached urchin skeletons. Fed urchins ring or ping like a bell when struck; a skeleton-specific set of discrete frequencies of vibration are detectable (solid line). In contrast, unfed urchins emit a dull "thud"; no discrete frequencies are identifiable (dotted line). For bleached skeletons of unfed urchins, the frequency spectrum is similar to that typical of three-dimensional vibrating objects such as drums or rectangular blocks (Kinsler el al, 1 982). The lowest of the identifiable fre- quency peaks is correlated with urchin diameter (P < O.OOOl. correlation = -0.8 1. r = 0.66, n =-- 100. fre- quency = -940 diameter + 1. 1 X 10 J . with diameter in centimeters and frequency in hertz). The negative correla- tion between frequency and diameter indicates that the size of the urchin skeleton determines some of the modes of vibration, and thus that vibrations are transmitted across sutures and throughout the skeleton. In contrast, bleached skeletons of fed urchins had no identifiable fre- quency peaks, thus indicating that vibrational energy was lost at loose sutures and that the structure as a whole did not vibrate. Skeletal vibrations can be used as a measure of sutural looseness. The cross-correlation between the sound pro- duced by a bleached skeleton and the predicted ringing frequency is a measure of looseness. The predicted ring- ing frequency is calculated using the empirical linear rela- tionship between frequency and diameter given above. Bleached skeletons of 9 fed urchins that produced the qualitatively dullest sounds had lower cross-correlations (P < O.OOl, U test) and lower breaking forces (P < 0.001, U test) than did 25 randomly chosen skeletons of fed urchins (Fig. 8). The 25 randomly chosen fed urchin skel- etons had lower cross-correlations (P < 0.01. U tests) and lower breaking forces (P < 0.01, U tests) than did 25 randomly chosen unfed skeletons. Within the randomly 142 O. ELLERS ET AL e O u> I a m TJ 0) .C O m a> CO 120 100- O dull-sounding fed random fed random unfed Category Figure 8. The qualitatively dullest sounding of the fed urchin skele- tons had lower mean breaking force and cross-correlation than did ran- domly chosen fed urchin skeletons. Randomly chosen unfed urchin skeletons have significantly higher mean breaking force and cross-corre- lation than do randomly chosen fed urchin skeletons. The relationship between breaking force and cross-correlation indicate that loose sutures weaken bleached skeletons. Crosses indicate significant differences in cross-correlation, asterisks indicate significant differences in bleached breaking force. chosen fed skeletons, breaking force was significantly correlated with the cross-correlation (P ^ 0.01, / = 0.51, r = 0.26, n = 25); in contrast, there was no significant correlation between breaking force and cross-correlation for randomly chosen unfed urchin skeletons. Discussion Sutural ligaments strengthen urchin skeletons under a variety of loadings. Under apical loading, whole skeletons broke at higher, size-specific forces when ligaments were intact than when they were removed by bleaching (Fig. 2). In three-point bending, meridional interambulacral su- tures with ligaments broke at higher forces than did paired sutures without ligaments (Fig. 3). And in circumferential interambulacral sutures with ligaments, sutures broke at higher nominal stresses than did sutures without ligaments (Fig. 4). The consistency of skeletal strengthening by liga- ments, despite differences in imposed stresses, suggests that sutural ligaments reinforce urchin skeletons under natural loads as varied as crushing forces from crab claws, apical or lateral forces from waves, and forces generated when an urchin wedges itself in a crack. The importance of sutural ligaments in strengthening an urchin's skeleton depended on urchin size. Smaller Strongylocentrotus pitrpitrutiis were strengthened less by ligaments than were larger ones (Fig. 2). Size-specific strengthening may be important across taxa as well: the echinoid Echinocyamus pusillus, which is the only species reported to lack ligaments, is a very small echinoid (Tel- ford. I985b) growing to a typical maximum size of only about 1 cm in diameter. Sutural ligaments were present in all eight other regular and irregular echinoid genera surveyed (Moss and Meehan. 1967; Telford, 1985a), and are present (pers. obs.) in four Strongylocentrotidae (S. purpiimtits, S. droebachiensis, S. fronciscanus, and Allo- centrotits fragillis), all of which grow to larger sizes. The degree of structural strengthening by sutural liga- ments depends on the position of a suture. The faster growing ambital and adapical sutures (Deutler. 1926) were strengthened more than slower growing adoral su- tures (Fig. 4). The faster growing adapical sutures also had the largest sutural gaps (Fig. 5), and larger gaps are associated with more and longer ligaments (Telford, 1985a). Such differences in composition and properties of different skeletal regions can influence deformation, stress distribution, and breakage. For instance, interambu- lacral areas were slightly stiffen than ambulacra! areas in Echinus esciileniits, and that difference influenced defor- mations and bending stresses calculated using finite ele- ment analysis (Philippi and Nachtigall. 1996). The lower stiffness of ambulacra compared to interambulacra of E. esculentus might be attributable to the difference in the relative proportion of sutures and plates because ambula- cral areas have a higher proportion of sutures. The mate- rial properties of different portions of the skeleton may generally depend on the relative area covered by sutures, especially gaping sutures with relatively more and longer ligaments. Not only do the faster growing sutures (adapical and ambital) gape more than the slower growing (adoral) su- tures on an individual urchin, but sutures on faster grow- ing urchins are looser than corresponding sutures on non- growing urchins (Figs. 5-8). Similarly, in S. francis- cumis. fed urchins develop looser sutures than starved urchins (pers. obs.). The relationship between sutural looseness and growth has potential implications for interpretation of the tapho- nomy of fossil urchins and other echinoderms. Disarticu- lated skeletons have often been used to imply the species composition, biodiversity, and relative abundance of fos- sil assemblages of echinoderm species (e.g.: Gordon and Donovan, 1992; Nebelsick, 1996). In an attempt to iden- tify taphonomic biases in such studies, the species-spe- cific tendency to disarticulate has been estimated (Greenstein, 1991) and possible biases interpreted (Greenstein, 1992). Disarticulation times dependent on temperature have also been demonstrated and their tapho- nomic consequences explored (Kidwell and Baumiller. 1990). Our study adds another variable to these tempera- ture- and species-specific effects. Specifically, the ten- dency to disarticulate depends on the growth status of a given urchin at the time of fossilization. Since looseness STRENGTHENING OF URCHIN SKELETONS 143 is growth-rate dependent, taphonomic biases likely exist between faster and slower growing age classes as well as between faster and slower growing taxa. In some urchins, differences in sutural gaping due to diet have been attributed to shrinkage of the starved ur- chins (Ebert, 1967; Levitan. 1988; Constable, 1993). but in S. droebachiensis we have observed that sutural gaping was due mainly to expansion of sutures in the fed group (Johnson, A. S., Bowdoin College, et ui, unpubl. data). In that study, we additionally observed that as fed urchins grew, the viscoelastic material properties of the sutures changed. Those changes were probably due to changes both in the degree of interlocking of trabeculae and in the material properties of the sutural ligaments. Similarly, material property changes in ligaments have been ob- served in the continuously growing teeth of sand dollars (Ellers and Telford. 1996) and sea urchins (Birenheidc and Motokawa, 1996; Birenheide. et /., 1996), in which the teeth are held in place on the jaw by dental ligaments. These dental ligaments soften periodically and enable a tiny muscle to advance the teeth (Ellers and Telford. 1997; Telford and Ellers, 1997). Thus, material property changes and rearrangements of ligaments occur during growth of echinoids. Such viscoelastic properties of ligaments may be im- portant in modeling the mechanical behavior of an urchin skeleton. They are important for three reasons. Firstly, as we demonstrated in the current study, the ligaments have a predominant role in providing structural strength, at least during times of growth. Secondly, as we demon- strated here, the gaping of the sutures varies during growth, and therefore the relative contribution of the calcite trabeculae and the ligaments to structural strength also varies during growth. Thirdly, the material properties of the ligaments may change during growth, or possibly over behavioral time spans. Therefore, time-dependent material behaviors such as creep and stress-relaxation must be considered in modeling the mechanics of urchin skeletons. Previous mechanical finite-element models have only considered more conventional measures such as stiffness (Baron, 1991b; Philippi and Nachtigall, 1996). Time-dependent properties of sutural ligaments could change very rapidly if they were mutable collagenous tissue (MCT) (Wilkie. 1996). MCT is a special tissue, known only in echinoderms, that is innervated and that can change material properties over behavioral time spans. Alternatively, sutural ligaments may change mate- rial properties over developmental time spans by reor- ganizing connective tissue constituents as is common in vertebrate connective tissues, e.g., the cervix in late preg- nancy (Winn et al.. 1993). The flexibility of sutural joints resulting from sutural gaping and the predominant role of ligaments in growing urchins also has consequences for determining the shape of urchin skeletons. The shape of a sea urchin's skeleton is closely approximated by a mathematical mechanical model that is more conventionally used to describe the shape of water droplets (Ellers, 1993). This model as- sumes that the droplet's surface, or urchin's skeleton, has zero flexural stiffness. That assumption seems initially unreasonable given that calcite plates are flexurally stiff, but the looseness of sutures during growth provides the flexibility assumed by the model. An urchin's skeleton, with its combination of flexurally stiff plates and more flexible sutures, thus behaves as a flexible jointed mem- brane during times of growth. The calculation of urchin shapes (Ellers. 1993) also assumes that an urchin's skeleton behaves like a thin membrane in which bending stresses are negligible. A shell is conventionally considered "thin" if the ratio of the radius of curvature to the thickness is greater than 20. In E. excitlentns that ratio is less than 20, and accordingly a finite-element model was used to calculate bending stresses in the skeleton of that species (Philippi and Nach- tigall. 1996). Many urchins, however, have much thinner shells and correspondingly higher ratios, in which a thin membrane model is appropriate. Some urchins, such as Allocentrotus fragillis, have extremely thin shells. Others, such as the diadematoid urchins Astropyga pulvinata and Cliaetodiadeimi /Hilliiliiiiu. are sufficiently thin-walled and loose-sutured that their skeletons deform by bending when touched lightly. Finally, the "soft-tested" echino- thurioid urchins have numerous small, thin imbricate plates imbedded in a meshwork of collagenous ligaments and can deform and contort their skeleton flexibly. (They can even adjust their shape to fit in right angles and cor- ners of an aquarium, pers. obs.) They have internal sheets of radial muscles (Tsuchiya and Amemiya, 1977) that control the bending of their flexible, thin-walled, membra- nous skeleton. Thus, urchin skeletons can be viewed statically as a continuum of flexibility and thickness, from relatively rigid and thick-walled (e.g., Echinus) to quite flexible and thin-walled (e.g.. echinothurioids). All urchin skeletons can be better understood dynamically as flexible, jointed membranes with varying degrees of flexibility and sutural looseness depending on growth stage. Acknowledgments Thanks to two anonymous reviewers who provided helpful suggestions; S. Keen, H. Leddy, and E. Pearson who critically read the manuscript; and S. Franklin and B. Lindsay who assisted with the experiments. This re- search was supported by grants from the University of California: Agricultural Research Station Grant #5134-H and a Bodega Marine Lab Travel Grant both to O. Ellers: as well as a President's Undergraduate Fellowship and a Howard Hughes SHARP Fellowship, both to P. Moberg. 144 O. ELLERS ET AL. Literature Cited Baron, C. 1991a. What functional morphology cannot explain, a model of sea urchin growth and a discussion of the role of morpho- genetic explanations in evolutionary biology. Pp. 471-488 in The Unity of Evolutionary Biology, Vo/s. 1 & 2: Fourth International Congress of Systematic ami Evolutionary Biology. College Park, Maryland, 1990, E. C. Dudley, ed. Dioscorides Press, Portland, Ore- gon. Baron, C. J. 1991b. The structural mechanics and morphogenesis of extant regular echinoids having rigid tests. Ph.D. thesis. University of California at Berkeley. Birenheide. R., and T. Motokawa. 1996. To he stiff or to be soft the dilemma of the echinoid tooth ligament. I. Morphology. Dial. Bull. 190: 218-230. Birenheide, R., A. Tsuchi, and T. Motokawa. 1996. To be stiff or to be soft the dilemma of the echinoid tooth ligament. II. Mechanical properties. Biol. Bull. 190: 231-236. Constable, A. J. 1993. The role of sutures in shrinking of the test in Heliocidaris eryllirogrumma (Echinoidea: Echinometridae). Mar. Biol. 117: 423-430. Dafni, J. 1986. A biomechanical model for the morphogenesis of regular echinoid tests. Paleobiology 12: 143-160. Dafni, J. 1988. A biomechanical approach to the ontongeny and phy- logeny of echinoids. Pp. 175-188 in Ecliinodenn Phytogeny and Evolutionary Biologv, C. R. C. Paul and A. B. Smith, eds. Clarendon Press, Oxford. Deutler, F. 1926. Uber die Wachstum des Seeigelskeletts. Zoo/. .//>. Abt. Anal. Ontog. Tier. 48: 119-201). Ebert, T. A. 1967. Negative growth and longevity in the purple sea- urchin Strongylocentrotus purpuratiis (Stimpson). Science 157: 557-558. Ebert, T. A. 1982. Longevity, life history, and relative body wall size in sea urchins. Ecol. Monogr. 52: 353-394. Ellers, O. 1993. A mechanical model of growth in sea urchins: predic- tions of shape and a developmental morphospace. Proc. R. Soc. Loml. B 254: 123-129. Ellers, O., and M. Telford. 1996. Advancement mechanics of grow- ing teeth in sand dollars (Echinodermata. Echinoidea): a role for mutable collagenous tissue. Prof. R. Soc. Loud. B 263: 39 14. Ellers, O.. and M. Telford. 1997. Muscles advance the teeth in sand dollars and other sea urchins. Proc. R. Soc. Loiul. B 264: 1525- 1530. Gage, J. 1). 1991. Skeletal growth zones as age-markers in the sea urchin Psammechinus miliaris. Mar. Biol. 110: 217-228. Gage, J. D. 1992a. Growth hands in the sea urchin Echinus esculen- tus: results from tetracycline-mark recapture. J. Mar. Biol. A.\xoc. UK 72: 257-260. Gage, J. D. 1992b. Natural growth bands and growth variability in the sea urchin Echinus esculentits results from tetracycline tag- ging. Mar. Biol. 114: 607-616. Gordon, C. M., and S. K. Donovan. 1992. Disarticulated echinoid ossicles in paleoecology and taphonomy; the last interglacial Fal- mouth formation of Jamaica. Palaios 7: 157-166. Greenstein, B. J. 1991. An integrated study of echinoid taphonomy: predictions for the fossil record of four echinoid families. Pa/aios 6: 519-540. Greenstein, B. J. 1992. Taphonomic bias and the evolutionary history of family Cidaridae (Echinodermata; Echinoidea). Paleobiology 18: 50-79. Kid well, S. M., and T. Baumiller. 1990. Experimental disintegration of regular echinoids: roles of temperature, oxygen, and decay thresh- olds. Paleohiologv 16: 247-271. Kinsler, L. E., A. R. Frey, A. B. Coppens, and J. V. Sanders. 1982. Fundamentals of Acoustics, 3rd Ed. John Wiley, New York. Levitan, D. R. 1988. Density-dependent size regulation and negative growth in the sea urchin Diadema antillarum Philippi. Oecologia 76: 627-629. Moss, M. L., and M. M. Meehan. 1967. Sutural connective tissues in the test of an echinoid Arhacia punctulata. Acta Anal. 66: 279- 304. Nebelsiek, J. H. 1996. Biodiversity of shallow-water red sea echi- noids: implications for the fossil record. J. Mar. Biol. Ass. UK 76: 185-194. Pearse, J. S.. and V. B. Pearse. 1975. Growth zones in echinoid skel- eton. Am. Zoo/. 15: 731-753. Philippi, U., and VV. Nachtigall. 1996. Functional morphology of regular echinoid tests (Echinodermata, Echinoida): a finite element study. Zootnorphologv 116: 3550. Telford, M. 1985a. Domes, arches and urchins: the skeletal architec- ture of echinoids (Echinodermata). Zoomorphology 1105: 1 14-124. Telford, M. 1985b. Structural analysis of the test of Echinocyamus pusi/lus (O. F. Muller). Pp. 353-360 in Fifth International Ec/iiuo- Jerm Conference. Ga/wav, Ireland, 1984, B. F. Keegan and B. D. S. O'Connor, eds. Balkema, Rotterdam. Telford, M., and O. Ellers. 1997. Tooth advancement muscles in the sand dollar, Echinarachnius parma. Invert. Biol. 116: 255-261. Tsuchiya. T., and S. Amemiya. 1977. Studies on the radial muscle of an echinothuriid sea-urchin, Asthenosoma I. Mechanical re- sponses to electrical stimulation and drugs. Coinp. Biochem. Physiol. 57: 69-73. Weisberg, S. 1980. Applied Linear Regression. John Wiley. New York. VVilkie, I. C. 1996. Mutable collagenous tissues: extracellular matrix as mechano-effector. Pp. 61-102 in Ecliinoderm Studies 5. M. Jan- goux and J. M. Lawrence, eds. Balkema, Rotterdam. Wilkie, I. C., M. D. C. Carnevali, and F. Andrietti. 1993. Variable tensility of the peristomial membrane of the sea-urchin Paracentro- tus In'idiis (Lamarck). Coinp. Biochem. Physiol. 105A: 493-501. \\inn, R. J.. M. B. O'day-Bowman. and O. D. Sherwood. 1993. Hor monal control of the cervix in pregnant gilts IV. Relaxin promotes changes in the histological characteristics of the cervix that are asso- ciated with cervical softening during late pregnancy in gilts. Endocri- nology 133: 121-128. Reference: Biol. Bull. 195: 1-45-155. (October, 1998) Efferent Mechanisms of Discharging Cnidae: II. A Nematocyst Release Response in the Sea Anemone Tentacle GLYNE U. THORINGTON AND DAVID A. HESSINGER Department of Physiology ami Pharmacology, School of Medicine. Lonui Limlti University. Loma Linda. California 92350 Abstract. Feeding behavior in cnidarians is a sequence of coordinated responses beginning with nematocyst dis- charge. The nematocyst response produces prey capture by envenomating prey and attaching prey to the tentacle. The strength of attachment of discharged nematocysts to the tentacle is termed intrinsic adherence and is calculated from measurements of adhesive force. Following prey capture, the feeding response involves movement of the tentacles toward the mouth and mouth opening. For inges- tion to occur, nematocysts attaching the prey to the tenta- cles must be released from the tentacle. A nematocyst release response has been proposed, but never docu- mented nor measured. Our criterion for a nematocyst re- lease response is that the intrinsic adherence of discharged nematocysts must decrease to zero. The unit of nemato- cyst discharge in sea anemone tentacles is the cnidocyte/ supporting cell complex (CSCC). The nematocyst re- sponse includes nematocysts discharged from Type C CSCCs by physical contact alone and nematocysts dis- charged from the more numerous Type B CSCCs that require both chemosensitization and physical contact. We identify two prey-derived substances, N-acetylneuraminic acid (NANA) and glycine, both of which chemosensitize nematocyst discharge from Type B CSCCs at low concen- trations. At higher concentrations NANA stimulates the release response of Type Cs, and glycine stimulates the release response of Type Bs. Received 21 April 1498; accepted 15 July 1998. E-mail: dhessingerCs'ccmail. llu.edu Abbreviations: CSCC, cnidocyte/supporting cell complex; D-600, 2- methoxyverapamil; i m , intrinsic adherence of nematocysts; NANA. N- acetylneuraminic acid; S,, tentacle stickiness. Introduction Cnidarians are obligate predators. Prey captured by cnidarians are attached to tentacles by discharged nemato- cysts. The discharged nematocysts, with attached prey, must be released in order for ingestion to proceed (Ewer, 1947). It is not known how the discharged nematocysts are released from the tentacles, and it is not known whether such release is under physiological control. In this paper, we hypothesize a "nematocyst release re- sponse." Using methods we have developed to measure the strength of attachment of discharged nematocysts to the tentacles of sea anemones (Thorington and Hessinger. 1996), we experimentally test and measure the nemato- cyst release response. We have coined the term "afferent mechanisms" of cnida discharge to refer to those processes acting to or toward the undischarged cnida to regulate or initiate dis- charge, and to distinguish them from mechanisms acting out of or from the discharged cnida's effector functions, which we have termed "efferent mechanisms" (Thoring- ton and Hessinger, 1996). Cnidae are the eversible secre- tory products of specialized cells called cnidocytes. The three known classes of cnidae are nematocysts, spirocysts, and ptychocysts (Mariscal, 1974). We have defined and measured an efferent mechanism termed "tentacle adher- ence." Tentacle adherence indicates how tightly the tenta- cle holds the capsules of discharged cnidae and is a mea- sure of how tightly targets, such as captured prey, are retained on the tentacles. The force required to remove an average discharged spirocyst or nematocyst from tenta- cles is the "intrinsic adherence." The intrinsic adherence is calculated from measurements of adhesive force and of the numbers of nematocyst discharged. 145 146 G. U. THOR1NGTON AND D. A. HESSINGER We measure adhesive force directly by using a sensitive force-transducer. The adhesive force is the applied force needed to separate a target from the tentacle (Thorington and Hessinger, 1988a). Specifically, adhesive force, as measured from sea anemone tentacles, is the sum of con- tributions arising from the stickiness of the tentacle mucus to the target (S, ) and the product of the number of cnidae (mastigophore nematocysts, n m , and spirocysts, nj dis- charging onto the target and their intrinsic adherence (mastigophore nematocysts, i m . and spirocysts, ij (Thor- ington and Hessinger, 1996): Adhesive force = S, + (n m ) (i m ) + (nj (ij (Equation 1) Adhesive force and intrinsic adherence may be expressed in units of micronewtons (/vN) or in hybrid units of milli- gram-force (mgf). since adhesive force is measured with- out a significant acceleration component and, thereby, does not involve Newton's Second Law (Miller, 1959). We measured the intrinsic adherence of discharged spirocysts (i s ) and of nematocysts (i m ) in the sea anemone Aiptasia /nil/iiUi (Thorington and Hessinger, 1996) and found that the values of i s are consistently very low rela- tive to the values of i m . We concluded that the values of i, are too low to significantly contribute to the measure- ment of adhesive force or to participate in the retention of struggling prey on feeding tentacles. Thus, equation 1 may be simplified: Adhesive force = S, + (n m ! (Equation 2) In anemone tentacles, each cnidocyte is surrounded by two or more supporting cells. The supporting cells possess chemoreceptors (Watson and Hessinger, 1988) and mech- anoreceptors (Watson and Hessinger. 1991) that detect prey and trigger nematocyst discharge. The cnidocyte, surrounded as it is by two or more supporting cells, consti- tutes a cnidocyte/supporting cell complex (CSCC), which we contend is the functional and morphological unit for triggering nematocyst discharge in the sea anemone tenta- cle. Two of the three known types of CSCC in sea anemone tentacles are germane to the present study: Types B and C (Thorington and Hessinger. 1990). Some of the CSCCs in anemone tentacles can be made to discharge by me- chanical stimulation alone; others require conjoint me- chanical and chemical stimulation. We term the former (CSCCs that respond to mechanical stimulation alone) Type C CSCCs; and the latter (which require both chemi- cal and mechanical stimulation) Type B CSCCs. Sensiti/- ing chemoreceptors for N-acetylated sugars (e.g., N-ace- tylneuraminic acid. NAN A) and for certain amino com- pounds (e.g., glycine, alanine, and proline) have thus far been identified. Stimulation of the chemoreceptors predis- poses contact-sensitive mechanoreceptors to respond to con- tact mechanical stimuli that trigger discharge (Thoring- ton and Hessinger. 1988a; 1990). Type C CSCCs, which are present in lower numbers than Type Bs. do not require chemosensitization but discharge in response to mechani- cal contact alone. In addition to proposing the nematocyst release re- sponse, we hypothesize that the response is controlled by prey-derived chemicals, as are nematocyst-mediated prey capture (Thorington and Hessinger. 1988b) and the subse- quent feeding response (Lindsted, 1971; Lenhoff and Heagy. 1977). Together, our hypotheses predict that cer- tain prey-derived chemicals will lower the intrinsic adher- ence of discharged nematocysts. Using methoxyverapamil (D-600) to selectively inhibit discharge from Type Bs. we show that (i) NAN A inhibits the intrinsic adherence of nematocysts discharged from Type C CSCCs and. therefore, controls the release re- sponse of Type Cs; and (ii) glycine inhibits the intrinsic adherence of nematocysts discharged from Type B CSCCs and. therefore, controls the release response of Type Bs. We conclude that a nematocyst release response exists in sea anemones and that it is controlled by prey- derived chemicals that also control prey capture. Materials and Methods Maintenance of sea anemones Monoclonal sea anemones (A. pallida, Carolina strain) were maintained individually in glass finger bowls con- taining natural seawater at 24 1C as previously de- scribed (Hessinger and Hessinger, 1981; Thorington and Hessinger, 1988a). Briefly, anemones were fed daily with freshly hatched brine shrimp nauplii (Anemia salina) and washed 4-6 h after feeding (Hessinger and Hessinger, 1981). Anemones were maintained on a 12/12-h photo- period using white fluorescent lights at an intensity of 5.5 klux (66 /uEs~'irT : ). Animals were starved for 72 h prior to experiments. Experimental animals and test solutions Filtered, natural seawater was obtained from Kerckoff Marine Laboratory of California Institute of Technology at Corona del Mar, California. Animals of same size were starved 72 h prior to experimentation and kept under con- stant fluorescent light at 4.5 klux (54 /vEs 'm : ) during the last 48 h of starvation. Exposure to continuous light enhanced the uniformity of anemone behavior and cnido- cyte responsiveness. Just prior to use, the animals were gently rinsed to remove soluble waste, and the medium was replaced with test solutions. Unless otherwise stated, animals were permitted to adapt to the change of medium tor 10 min before cnidocyte responsiveness was mea- NEMATOCYST RELEASE RESPONSE 147 sured. N-acetylneuraminic acid (NAN A), glycine, and methoxyverapami] (D-600) were obtained from Sigma Chemical Co., St. Louis, Missouri. Solutions or D-600 were made up fresh on the day of the experiment and protected from light. All test solutions were prepared in artificial seawater (ASW) adjusted to pH 7.62 with 1 N HCI or NaOH. The ASW consisted of 423 mM NaCl, 10 mM KC1. 24 mM MgCI 2 , 25 mM MgSO 4 , 10 mM CaCl 2 , and 1.2 mM NaHCO 3 . Calcium-free artificial seawater (Ca-free ASW) was prepared with the same components as ASW except that calcium chloride was omitted, the NaCl concentration was increased to 438 mM, and EGTA (ethyleneglycoltetraacetic acid) was added to a final con- centration of 1 mM; and then the final pH was adjusted to 7.62. Assays of cnidocyte responsiveness Physical contact of a tentacle with a gelatin-coated probe triggers discharge of local nematocysts and spiro- cysts, and adherence of the tentacle to the probe (Thoring- ton and Hessinger, 1988b, 1990). Four parameters were measured to analyze nematocyst-mediated adhesive force: total adhesive force; number of discharged nematocysts; number of discharged spirocysts; and adhesive force in tentacles in which nematocyst and spirocyst discharge had been inhibited by pretreatment with formaldehyde to measure tentacle stickiness. The methods have been described in detail previously (Thorington and Hessinger. 1990). The number of cnidae on the probes is a direct measure of the number of cnidae discharged. Measurement of adhesive force. Cnida-mediated adhe- sive force was measured as previously described (Thor- ington and Hessinger, 1988a). This technique involves using small gelatin-coated nylon beads of defined diame- ter attached to a strain gauge by means of a fine stainless steel shaft. The gel-coated bead is made to contact the distal third of a primary tentacle on an anemone in a finger bowl containing the test solution. The discharge of cnidae initiated by contact of the probe with the tentacle causes the tubules of everting cnidae to either adhere to or penetrate the gelatin surface. Withdrawing the probe from the tentacle causes the discharged cnidae to exert an opposing force on the probe: this force is measured with a gravimetrically calibrated force-transducer con- nected to a potentiometric recorder. The force necessary to separate the probe from the tentacle is called the adhe- sive force and is expressed in hybrid units of milligram- force (mgf). It is an aggregate measure of the "inherent" stickiness of the tentacle plus the nematocyst-mediated adhesive force. Counting discharged nematocysts. After adhesive force measurements, the same probes are processed for count- ing nematocysts as detailed previously (Geibel ct til.. 1988). Briefly, the gelatin coating of the probes is enzy- matically digested to release the nematocysts of the dis- charged mastigophores. The highly refractive mastigo- phores, which are resistant to proteolysis, are then counted with an inverted microscope from the flat bottoms of microtiter wells. Counting discharged spirocysts. To determine the num- ber of discharged spirocysts, we used an indirect, solid- state enzyme-linked lectin sorbant assay (ELLSA), the details of which have been published (Thorington and Hessinger. 1990). In principle, the assay is based upon the high affinity of conjugated N-acetylated sugars to the everted tubules of discharged spirocysts on the surface of the test probes. The assay involves use of a microtiter- plate spectrophotometer for colorimetric determination of bound peroxidase activity after the sequential treatment of test probes with solutions of asialomucin and Viciu villosa lectin/peroxidase conjugate. Collection tinil analysis of data Individual animals were tested at each concentration of sensitizer. Twelve probes (one per tentacle) were used on each animal to determine adhesive force and to count discharged nematocysts or spirocysts. Daily experimental means were calculated from these experiments. Replicate experiments were carried out on three different days. Each data point represents the mean of the three daily experi- mental means, and the range bar represents the standard error of the mean. Results Selective discharge of cnidae from Type C cnidocyte/supporting cell complex To test our hypotheses that a nematocyst release re- sponse exists and is under the control of prey-derived chemicals, we ask the research question: Do known che- mosensitizers of nematocyst discharge lower, in a dose- dependent manner, the values of i m for nematocysts dis- charged from either Type B or Type C CSCCs? To answer this question we proposed to determine the intrinsic ad- herence (i m values) for nematocysts discharged from Type B and from Type C CSCCs as a function of chemosensi- tizer concentration. We selectively blocked chemosensi- tized discharge of nematocysts from Type B CSCCs by using 2-methoxyverapamil (D-600) (Thorington and Hes- singer, 1992; Watson and Hessinger, 1994b), a diphe- nylalkylamine inhibitor of vertebrate L-type calcium channels (Spedding and Paoletti, 1992). With discharge from Type B CSCCs blocked, we directly determined the effect of chemosensitizers on the intrinsic adherence of nematocysts discharged from Type Cs. By subtracting the responses of the Type Cs from the combined responses 14S G. U. THORINGTON AND D A. HESSINGER of Type B and Type C CSCCs (in the presence of chemo- sensitizer, but the absence of D-600). we calculated the intrinsic adherence of nematocysts from the Type Bs. D-600 inhibits discharge from Type B CSCCs. D-600 potently and dose-dependently inhibits nematocyst dis- charge from NANA-sensitized Type Bs (Fig. 1; open cir- cles). D-600 also inhibits glycine-sensitized nematocyst discharge (Fig. 1; open squares), but less potently and over a wider range of D-600 concentrations. In the ab- sence of chemosensitizers, mechanical contact elicits dis- charge only from Type C CSCCs (Thorington and Hes- singer. 1990). D-600 has no detectable effect on nemato- cyst discharge from Type Cs at any tested dose (Fig. 1; closed circles). The half-inhibitory doses (IC 5I1 ) for D- 600 on NANA- and glycine-sensitized discharge is below 10~ lh M for NANA and 10 |s M for glycine, and the minimal doses that maximally inhibit (IC ]m ) sensitized discharge are about 10 "' M for NANA and 10 " M for glycine. Thus, D-600 blocks nematocyst discharge from Type B CSCCs, but not from Type Cs. D-600 lowers tentacle stickiness (S,). We tested the effects of D-600 on tentacle stickiness (Table I) by incu- bating anemones at room temperature in ASW and in Ca- free ASW with and without D-600. After 20 min we added formalin to 10% for 5 min to measure S, with 300 -14 -10 log D-600 Cone., M Figure I. Effects of metho.xyverapamil (D-600) on nematocyst dis- charge in the presence and absence of N-acetylneuraminic acid (NANA) or glycine. The number of nematocysts discharged from sea anemone tentacles onto probes at different molar concentrations of D-600 are indicated on the ordinate and abscissa, respectively, for anemones pre- exposed for 10 min to D-600 in seawater and then for 10 mm more to D-600 with 1.8 x 1C)" 5 M NANA in seawater (open circles; n = 31). or D-600 with 10 ' M glycine in seawater (open squares; n = 13), or D-600 in seawaier (closed circles; n = 33). Data points are the mean standard ermi of the mean. adhesive force probes in the absence of any cnida dis- charge. The value of S, is not altered by 10% formalin (Thorington and Hessinger, 1996). As expected, neither nematocysts nor spirocysts are discharged in 10% forma- lin (Table I). The mean value of S, in ASW is 36.6 0.9 mgf (358.7 8.8 //N; Table I), which is in close agreement with the value of 34.6 0.7 mgf (339. 1 6.9 //N) that we previously reported for similar experimental conditions (Thorington and Hessinger. 1996). The values of S, are decreased 44% from ASW controls by 10""' M D-600 in ASW (P < 0.001 ) and 49% from ASW controls by 10 s M D-600 in ASW (P < 0.005). In Ca-free ASW. the mean value of S, is 31.3 0.9 mgf (306.7 8.8 //N, a value 15% lower (P < 0.02) than in ASW containing 10 mM Ca :H ; the combination of Ca-free ASW and 10""' M D-600 causes a decrease of 55% from ASW (P < 0.001: Table I). Thus. D-600 significantly and dose-de- pendently lowers tentacle stickiness (S,). Effects of N-acetylneuraminic acid on intrinsic adherence The combined dose-responses of Type B and Type C CSCCs to NANA for nematocyst discharge, spirocyst discharge, and adhesive force are characteristically bipha- sic (Fig. 2A-C. open circles; Thorington and Hessinger, 1990). In the presence of 10 "' M D-600, the dose-re- sponses of Type Cs to NANA for nematocyst and spiro- cyst discharge are level and at control levels, showing no significant changes (Fig. 2A and B. closed circles). Thus, discharge from Type B CSCCs to NANA is totally inhib- ited by 10"'" M D-600, whereas discharge from Type Cs is unaffected. NANA lowers /',' . Nematocyst-mediated adhesive force is calculated by subtracting tentacle stickiness (S,) from adhesive force. To obtain nematocyst-mediated adhesive force in dose-responses to NANA. we subtracted from adhesive force measurements the appropriate value of S, from Table I depending upon whether D-600 was or was not present during the experimental measurements. Ne- matocyst-mediated adhesive force from Type Cs steadily declines as the NANA concentration increases (Fig. 2C, closed circles). Since the numbers of nematocysts and spirocysts discharging from Type Cs do not significantly decrease (Fig. 2A and B, closed circles), the decline in adhesive force is probably due to a decrease in the value of i m from the Type C CSCCs (i.e., i,,, c ). We calculated the i,,, values of nematocysts discharging from Type Cs (i.e.. i,,, L ) across a wide range of NANA concentrations ( 10 '" to 10"' M; Fig. 2D, closed circles) by using the values for nematocyst discharge and nemato- cyst-mediated adhesive force of Type C CSCCs in Equa- tion 2. The values of i,,, c decrease with increasing levels of NANA, from about 0.20 mgf (1.96 /yN) in ASW to NEMATOCYST RELEASE RESPONSE 149 Table I Cnida-independenl adhesive force (tentacle stickiness) Treatment Nematocysts'' (nj Spirocysts h (nj Stickiness (mgfr (S,) Pretreatment time = 90 min. ASW (without formalin) 1 ' 68.6 + 0.7 136.7 + 6.0 60.09 + i.oir ASW 0.30 0.10 0.91 0.35 36.55 0.86 Ca-free ASW e 0.20 0.12 3.45 2.0 31.25 0.88 Pretreatment time = 70 min. ASW + 10'"' M D-600" 0.22 0.15 0.54 0.14 20.44 1.0 ASW + 10-* M D-600 1 0.31 0.02 0.34 0.25 18.49 0.21 Ca-free ASW + D-600 1 0.50 0.28 0.30 0.21 16.55 2.0 Note: All anemones were pretreated with artificial seawater (ASW) at room temperature (23 1C) for either 70 or 90 min. If the treatment took place in Ca-free ASW, the pretreatment seawater was also Ca-free. After treatment, the anemones were contacted with test probes. Number of individuals (in = 30 for all treatments except ASW without formalin, for which n = 34. J Mean number of discharged nematocysts SEM b Mean number of discharged spirocysts SEM. 1 Mean stickiness value measured as adhesive force, mgf SEM. J Pretreatment only; no further treatment. e Values include stickiness plus contributions from discharged nematocysts and spirocysts. ' Pretreatment + exposure to 10% formalin in ASW for 5 nun. f Pretreatment + exposure to 10% formalin in Ca-free ASW for 5 min. h Pretreatment + exposure to ASW containing 10 "' M D-600 for 15 min, then exposure to 10% formalin in ASW for 5 min. 1 Pretreatment + exposure to ASW containing 10 * M D-600 for 15 min, then exposure to 10% formalin in ASW for 5 min. 1 Pretreatment + exposure to Ca-free ASW containing 10 '" M D-600 for 15 min, then exposure to 10% formalin in Ca-free ASW for 5 min. near zero at 10 4 M NANA. The concentration of NANA that half-inhibits i m c is approximately 10~ 12 M NANA. Thus, NANA dose-dependently lowers the intrinsic ad- herence of nematocysts discharged from Type Cs. NANA biphasicallv modulates /'/'. The dose-responses for discharge and adhesive force from Type Bs (Fig. 2A- C, closed squares) are calculated by subtracting the dis- charge (or adhesive force) of Type Cs in D-600 (closed circles) from the discharge (or adhesive force) of both Type Bs and Cs in the absence of D-600 (open circles). The calculated Type B dose-responses of both mastigo- phore and spirocyst discharge are narrow and biphasic. Maximum discharge (E nMX ) of nematocysts and spirocysts from Type Bs is about two times and one time, respec- tively, that of Type C ASW controls, suggesting that Type Bs and Type Cs coexist in the tentacle in ratios of 2: 1 and 1:1 for nematocyst-bearing and spirocyst-bearing CSCCs, respectively. The concentration at which maximal dis- charge occurs (ECmn) is about 10~ 5 M NANA, and the concentration at which half-maximum discharge occurs (K (1 , ) is about 10 h M NANA. The NANA dose-response of nematocyst-mediated adhesive force from Type Bs is broadly biphasic, with an ECmn of 10"^ M NANA and a Ko.s of about 10""' M NANA (Fig. 2C, open circles). Using the values for nematocyst discharge and nemato- cyst-mediated adhesive force of Type B CSCCs in Equa- tion 2, we calculate the i m values of nematocysts discharg- ing from Type Bs (i.e., i m h ) across a wide range of NANA concentrations (Fig. 2D. closed squares). The i m b dose- response to NANA is biphasic. with maximum i m b values of about 0.8 mgf (7.8 /;N) occurring at 10" "'M NANA and the K n5 occurring at about 10~ 12 M NANA. The peak of the i m h dose-response is about five orders of magnitude lower than the peak of the dose-response of Type B nema- tocyst discharge (Fig. 2A). We conclude that NANA dose-dependently modulates a biphasic change in the in- trinsic adherence of nematocysts discharging from Type B CSCCs. but does not reduce the value of i m b to zero. Effects of glycine on intrinsic adherence Glycine suppresses discharge from Type Cs. The mini- mum dose of D-600 that totally inhibits glycine-sensitized nematocyst discharge from Type Bs is 10~ h M (Fig. 1). To minimize possible side effects from such a relatively high dose, we elected to use 10 * M D-600 rather than 10~ e M. The dose-responses to glycine for nematocyst discharge, spirocyst discharge, and adhesive force are characteristically biphasic (Fig. 3A-C; Thorington and Hessinger, 1990). In 10 K M D-600 the dose-responses to glycine for discharge of both nematocysts and spirocysts and for adhesive force do not exceed those of controls (Fig. 3A-C, closed circles). Thus, 10"* M D-600 appears to inhibit cnida discharge from Type B CSCCs in re- sponse to glycine. 150 G. U. THORINGTON AND D. A. HESS1NGER 5 100 - -14 -10 -6 log NANA Cone., M 14 -10 -6 log NANA Cone.. M -14 -10 -6 log NANA Cone., M 14 -10 log NANA Cone., M Figure 2. Afferent and efferent cnida dose-responses to N-acetylneuraminic acid (NANA). Combined cnida responses from Type B plus Type C cnidocyte/supporting cell complexes (CSCCs) are measured in seawater (open circles). Cnida responses from Type Cs only are measured in the presence of 10"'" M D- 600 (closed circles). Calculated responses from Type Bs (closed squares) are obtained by subtracting Type C responses from combined Type B plus Type C responses. Values express the mean of three experiments. Each experiment consists of replicate probes for each NANA concentration; each probe and each tentacle is used only once. Vertical bars represent standard errors of the mean and are applied to all measured means, but not to calculated means. (A) Effect of NANA on nematocyst discharge (;; = 34). (B) Effect of NANA on spirocyst discharge (;; = 35). (C) Effect of NANA on nematocyst-mediated adhesive force (n = 36). (D) Effect of NANA on calculated intrinsic adherence. On the other hand, in the presence of 10 s M D-600. we observe about a 50<7r decrease in both nematocyst and spirocyst discharge from Type Cs at the lowest tested doses of glycine (/.<'., 10 "' M glycine). Nematocyst dis- charge gradually recovers to control levels of discharge at about 10~ 7 M glycine and then declines again at higher concentrations. Spirocyst discharge recovers at lower con- centrations of glycine and reaches control levels at 10 " M glycine before it, like nematocyst discharge, declines again at higher concentrations. Thus, low concentrations of glycine suppress both nematocyst and spirocyst dis- charge from Type C CSCCs. Glycine modulates /,'. Nematocyst-mediated adhesive force from Type Cs remains constant (between 12 and 1 5 mgf or 1 1 8 and 1 47 //N ) at all tested glycine concentra- tions except 10 7 M glycine, at which concentration the adhesive force increases to 20 mgf (196 /vN; Fig. 3C: closed circles). Using Equation 2, we calculated the i m values of nematocysts discharging from Type Cs (i.e.. i,,," ) across the range of tested glycine concentrations ( 10""' to 10 4 M; Fig. 3D, closed circles). The values of i m c are relatively high (about 0.6 mgf/nematocyst or 5.9 /jN/ne- matocyst) in seawater controls and in 10 "' to 10 i: M glycine. But between 10 "' and 1 7 M glycine, the values of i m " decrease by about one-half (to about 0.3 mgf/nema- tocyst or 2.9 /jN/nematocyst) and then increase (to about 0.4 mgf/nematocyst or 3.9 //N/nematocyst) at 10"" M and higher concentrations. Overall, there appears to be a NEMATOCYST RELEASE RESPONSE 151 -14 -10 -6 log Glycine Cone., M -10 log Glycine Cone., M -14 -10 -6 log Glycine Cone., M 14 -10 -6 log Glycine Cone., M Figure 3. Afferent and efferent cnida dose-responses to glycine. Combined responses from Type B plus Type C cnidocyte/supporting cell complexes (CSCCs) are measured in seawater (open circles). Cnida responses from Type Cs only are measured in the presence of 1CT S M D-600 in seawater (closed circles). Calculated responses from Type Bs (closed squares) are obtained by subtracting Type C responses from combined Type B plus Type C responses. Values express the mean of duplicate experiments. Each experi- ment consists of replicate probes for each glycine concentration; each probe and each tentacle is used only once. Vertical bars represent standard error of the mean and are applied to all measured means, but not to calculated means. (A) Effect of glycine on nematocyst discharge (n = 15). (B) Effect of glycine on spirocyst discharge (n = 15). (C) Effect of glycine on nematocyst-mediated adhesive force (n = 14). (D) Effect of glycine on calculated intrinsic adherence. downward trend in i m L with increasing glycine concentra- tions. Glycine suppresses /,''. In contrast to NANA. in which NANA-sensitized discharge of nematocysts from Type Bs spans a narrow range of NANA concentrations (Fig. 2A. closed squares), glycine-sensitized discharge of nemato- cysts spans a wide range of glycine concentrat