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Intracellular procaryotic cells were consistently that the bacteria found in larvae might represent a found in TEM observations of pnmary oocytes and follicle transmission stage of the gill symbionts which had cells of the deep-sea seepage clam Calyptogena soyoae. been introduced into eggs in cryptic forms. Among These bacteria resembled gill endosymbionts in the following 3 polnts: dimensions and general outllne, occurrence of elecdeep-sea tZlftia pachyptila was tron-transparent vacuoles in the periplasm, and occurrence of establish syn~biosis by talung up bacteria from freeelectron-dense materials in the periplasm. Eggs which harbored bacteria were all in the vitellogenic stage. This finding suggests some restricted time schedule of the entry of bacteria into oocytes. It is hypothesized that these bacteria represent transmission stage of gill symbionts, and continuum of the symbiosis is maintained by maternal inheritance through egg infection. Calyptogena soyoae harbors symbiotic bacteria in branchial epithelia (Endow 1988), like other vesicomyld clams such as C. magnifica, C. pacifica and C. elonyata (Cavanaugh 1983, Vetter 1985). These bacteria have been found to be sulfur oxidizers and have been postulated to be important to the nutrit~on of host clams (Felbeck et al. 1981, Sakai et al. 1987). Because of their role in clam life, it is important to know the mode of establishment of the symbiosis in each new generation. In endosymbiotic associations, symbionts can be acquired by an individual host through one of 3 ways: by acquisition of microorganisms from an environmental stock (Bauer 1981), by horizontal transn~ission (from symbiotic individual to aposymbiotic individual) (Soldo et al. 1974), or by vertical transmission (from one generation to offspring) (Buchner 1965). Among chemoautotrophic symbioses, transmission of symbionts through egg infection, during a short phase of oviposition, was first reported for the shallow water oligochaetes Phallodrilus leukodermatus and P. planus by Giere & Langheld (1987). Gustafson & Reid (1988) observed in the shallow water gutless bivalve Solemya reidi that bacteria first occurred in the locomotory test cells of the larvae and then in the perivisceral cavity of post-larvae, but not in or on eggs. They hypothesized living environmental populations (Cary et al. 1989). Through transmission electron microscopic studies of Calyptogena soyoae, we provide some new information relevant to the vertical transmission of symbionts in the deep-sea seepage clam. Materials and methods. Samples of Calyptogena soyoae were collected on 3 November 1988 during Dive no. 379 of the Japanese submersible 'Shinkai 2000', from Sagami Bay off Hatsushima Island at a depth of 1160 m (34" 59.g1N, 139' 13.6'E) (see Hashimoto et al. 1989 for a general description of the seep field). Four adult specimens ranging from 113.5 to 125.5 cm in shell length were used for this study. The clams were dissected within 5 h of being brought on board and pieces of ovary were fixed in a mixed-aldehyde fixative (0.5 % paraformaldehyde and 2 % glutaraldehyde in 0.075 M cacodylate buffer at pH 7.4 containing 0.19 M sodium chloride) at 4 OC for 1.5 d followed by thorough washing with buffered sodium chloride. Post-fixation was performed in buffered osmium tetroxide (1 O/O) for 1.5 h at room temperature. Tissues were dehydrated through a graded series of ethanol followed by propylene oxide and then embedded in Epon. Ultra-thin sections were stained by uranyl acetate and lead citrate and then examined in a JEOL lOOCX transmission electron microscope. Results and discussion. Ovaries contained various stages of oocytes along with follicle cells. Like Calyptogena rnagnifica (Berg 1985), oogenesis of C. soyoae appears to be continuous. Procaryotic cells with a Gram-negative type cell envelope were found within follicular tubules. Like gill O Inter-Research/Printed in F. R Germany 0171-8630/90/0064/0309/$ 03.00 310 Mar Ecol Prog Ser 64 309-311, 1990 Fig 1 Calyptogena soyoae Bactena In oocytes (a) Bacter~urn near the basement of vitellogenic oocyte (arrow). c t connectwe tissue surrounding follicle, mt m~tochondna. L lipid droplet; Y yolk granules; scale bar = 2 pm Insert. Enlargement of the bacterium showng fine structure and enclos~ng vacuolar membrane, scale bar = 0 5 &m. ( b ) Bacter~um with electron-transparent vacuole in periplasm (arrow), scale bar = 0 S bun. (c) Electron-dense matenal deposited in periplasm (arrow), scale bar = 0.5 pm endosymbionts, a peptidoglycan layer was scarcely observed. Van Caeseele & Lees (1969) reported that the Gram-negative facultative autotroph Thiobaallus novellus showed no peptidoglycan layer when grown autotrophically. All 4 clams harbored bactena In both vitellogenic oocytes (Fig 1) and follicle cells. In most cases, follicular tubules contained several bacteria, and in one case, up to 6 bacteria were found within a single primary oocyte on a slngle ultra-thin sectlon. The bacteria inside the eggs and follicle cells were enclosed by a vacuolar membrane (Fig l a . insert) which was probably of host ongin. Some of the bactena contamed electron-transparent vacuoles In their penplasms (Fig. l b ) Electron-dense matenals were also observed in the perlplasm of some of the bactena (Fig lci. These findings show the strong resemblance of the bacteria within the follicular tubules to gill endosymbionts (Fig. 2) reflected also by their s ~ m i l a n t ~ e s in dimensi.ons and general outlines (F~gs . 1 and 2). Thls is the first observation that a host organism symbiotic with sulfur-oxidizing bacteria harbors bactena In its primary oocytes. Occurrence of bacteria in ovary accessory cells is not unusual for a symbiotic host in which transmission of symbionts takes place vertically from mother to progeny. For example, in Calandra (Curculionidae; Insecta), the bacter1.a reproduce within nutrient cells and pass into oocytes through the nutrient chord when the nutrient cells begin to secrete nour~shment into growing oocytes (Buchner 1965). In the scaphopods Dentalium dentale and D. antillarum, bactena, which are present in the space between follicle cells and oocytes and probably also in follicle cells, appear to become attached to the vegetal pole of the eggs during the very short time of spawning (Timmermans et a1 1970, Geilenkirchen et al. 1971). Occasionally bacteria were found intercellularly However, we hdve not yet found bacteria in the oocytes of the previtellogenic stage, though much work is Endow & Ohta: Bacteria in seepage cldin oocytes 31 1 Fig. 2. Calyptogena soyoae. Bacteria in bacteriocytes. (a) Bacteriocyte occupied by abundant symbiotic bacteria. b: bacteriocyte; bs: blood space; n: nucleus; S: symbiont; scale bar = 5 ,pm. (b) Transparent vacuole in the periplasm of endosymbiont (arrow); scale bar = 0.5 pm. ( C ) Electron-dense material at upper right of bacterium (arrow); scale bar = 0.5 pm needed to confirm the absence of bacteria in previtellogenic oocytes. This suggests some regulated mode of infection, especially in time of entry into oocytes. These observations strongly suggest that the bacteria found in the primary oocytes of Calyptogena soyoae represent transmission stage of gill endosymbionts. Symbiotic bacteria in C. soyoae appear to be passed vertically from mother to offspring by means of egg ~nfection. Acknowledgements. We thank the 'Shinkai 2000' operation group and the captain and crew of the tender ship 'Natsushima' of JAMSTEC, for the valuable support in carrying out the investigation. We also thank Dr T Gamo for his effort This note was presented by Professor N. D. Holland, La Jolla, California, USA in collecting Calyptogena soyoae. We are indebted to Dr K. Ohwada for helpful comments on the manuscript. This study was aided by Special Coordination Funds of the Science and Technology Agency of Japan

Occurrence of bacteria in the primary oocytes of vesicomyid clam Calyptogena soyoae