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Notice: ©1988 Springer. This manuscript is an author version with the final publication available at http://www.springerlink.com and may be cited as: Gustafson, R. G. & Reid, R. G. B. (1988). Association of bacteria with larvae of the gutless protobranch bivalve Solemya reidi (Cryptodonta: Solemyidae). Marine Biology, 97(3), 389‐401. doi:10.1007/BF00397769

(JY \,.0 Marine Biology 97,389-401 (1988) Marine ,,,.~"o~,~"'"" on li!einOe

,. Association ofbacteria with larvae ofthe gutless protobranch bivalve Solemya reidi (Cryptodonta: Solemyidae)

R. G. Gustafson ':":' and R. G. B. Reid

Department of Biology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada

Abstract animals (Cavanaugh et al. 1981, Felbeck et al. 1981, 1983a, Cavanaugh 1983, 1985, Southward 1986). Rod-shaped bacteria were consistently observed by trans Chemoautotrophic symbionts have been localized both mission electron microscopy in the locomotory test of lar biochemically and ultrastructurally: in trophosome tissue vae and in the perivisceral cavity of post-larvae of Solemya of pogonophorans (Cavanaugh et al. 1981, Felbeck 1981, reidi, a gutless protobranch bivalve known to possess intra Southward et al. 1981, 1986, Southward 1982); in the sub cellular chemoautotrophic bacterial symbionts in the adult cuticular space of gutless marine oligochaetes of the genus gill. Bacteria develop within granular vesicles in the larval Phallodrilus (Felbeck et al. 1983b); and in the gills of bi test, where they either remain to be ingested at metamor valves of the family Lucinidae (Fisher and Hand 1984, phosis, or are released into the space separating the test Dando et al. 1985, 1986, Schweimanns and Felbeck 1985, and embryo, to be subsequently ingested through the lar Reid and Brand 1986), of the genera Solemya (Cavanaugh val mouth. In either case, bacteria lie within the periviscer 1983, Felbeck 1983, Felbeck et al. 1983a), Calyptogena al cavity following metamorphosis. Bacteria were not seen (Felbeck etal. 1981, Fiala-Medioni and Metivier 1986), either in or on gametes or in gills of juveniles. It is Thyasira (Dando and Southward 1986, Reid and Brand hypothesized that these bacteria represent a transmission 1986, Southward 1986), and in the vent mytilid Bathy stage of the gill symbionts present in adult S. reidi and are modiolus (Le Pennec and Hily 1984, Fiala-Medioni et al. not evident in gametes or gills of juveniles due to cryptic 1986). The question of whether symbionts are transmitted packaging within granular vesicles. Perpetuation of this through the sexual cycle of the host or are acquired from symbiosis would therefore be assured through vertical other sources has been determined only for the extracellu transmission, as is typical ofother marine invertebrate-bac lar marine oligochaete symbiosis (Giere and Langheld teria endosymbioses. 1987). Since the gutless protobranch bivalve Solemya reidi (Reid 1980, Reid and Bernard 1980), is used in studies of the association between chemoautotrophic endosymbionts and host animals from sulfide-rich habitats (Felbeck 1983, Felbeck et al. 1983a, Hand and Somero 1983, Powell and Introduction Somero 1985, 1986, Fisher and Childress 1986), it is impor tant to know the life-cycle of this symbiosis. In marine habitats where oxygen and reduced-sulfur com Sofemya reidi is found from Southern California to pounds are simultaneously available, such as beneath log southern Alaska at depths of 40 to 600 m (Bernard 1980) booming grounds, near sewage outfalls, in seagrass beds, near sewage outfalls (Felbeck 1983, Felbeck et al. 1983a) hypoxic marine basins, and around deep-sea hydrothermal and in the Pacific Northwest beneath log-booming grounds vents, certain invertebrate species maintain intracellular (Reid 1980). Transmission of endosymbionts in inverte chemoautotrophic bacteria which aid in oxidation of re brate-microorganism symbioses proceeds in one of three duced-sulfur compounds and provide energy to the host ways: by vertical transmission (transfer from parent to off spring, which may include incorporation in or on the egg * Harbor Branch Oceanographic Institution Contribution of the host); by horizontal transmission (involving the No. 602 spread of symbionts between contemporary hosts); or by ** Present address: Division of Applied Biology, Harbor Branch reinfection of the new host generation from an en Oceanographic Institution, 5600 Old Dixie Highway, Fort Pierce, Florida 34946, USA vironmental stock of microorganisms (Andrewes 1957). 390 R. G. Gustafson and R. G. B.Reid: Bacteria-larvae association

The present study provides evidence concerning the mech (Costerton 1979), in that they have a central region con anism by which chemoautotrophic endosymbionts found taining fibrillar material (chromatin) and a condensed "cy in adult gills ofS. reidi are transmitted to host offspring. toplasm". Most inclusions in ripe eggs of S. reidi can be identified as lipid spheres, yolk spheres, mucus containing granules, mitochondria, or multivesicular bodies (Figs. 4, Materials and methods 6).

Adult Solemya reidi were collected from an average depth of 40 m with a Van Veen grab in the vicinity of Log-Boom Sperm ultrastructure ing Grounds Nos. 27 and 29 in Alberni Inlet on the west coast of Vancouver Island, British Columbia, Canada The spermatozoa of Solemya reidi are of the primitive type (Latitude 49° 12'N; Longitude 124°49'W). Spawning and (Franzen 1983, Anderson and Personne 1976), with four larval culture conditions have been described (Gustafson rounded mitochondria in the midpiece, a tail flagellum, and Reid 1986). Pericalymma larvae (see Gustafson and and an elongated conical nucleus surmounted by a cap Reid 1986) and post-larval specimens were cultivated shaped acrosome (Figs. 7, 8). No bacterial cells were seen through metamorphosis at Day 6 until 42 d after fertili in the testes. zation. Adult gill. ovary, testis, fertilized eggs, embryonic stages, larvae, and juveniles were prepared for transmis Bacteria - larvae association sion electron microscopy (lEM), and scanning electron mi croscopy (SEM), by fixation in a solution of 2% glutaralde Membrane-bounded, oval or rounded, intracytoplasmic hyde, 0.2 M phosphate buffer, and 0.14 M NaCI at pH 7.3; granular vesicles measuring up to 4,um in longest dimen followed by three rinses with a solution of0.2 M phosphate sion are present in the basal regions of the larval test of buffer and 0.28 M NaCI; and post-fixation in 2% OsO. in Solemya reidi, adjacent to the space separating the test and

1.25% NaHC03 . Specimens were dehydrated in alcohol. definitive tissues (Fig. 9). These granular vesicles possess a Further preparation of specimens for microscopy has been homogeneous granular matrix and are common to all test described earlier (Gustafson and Reid 1986). cells. On about the fourth day of development, particles re sembling prokaryotic cells were seen within the vesicular matrix (Figs. 10, II). An electron-lucent nuclear region is Results apparent in these particles and each vesicle is surrounded by a single-unit membrane (Fig. 12). By the fifth day of Bacteria in adult Solemya reidi development, definite prokaryotic cells are present within many of the vesicles (Figs. 13-16) while rod-shaped, Bacteriocytes, cells containing bacteria, are located in the Gram-negative, bacteria, measuring up to 1.4,um long by proximal portion of the gill lamellae and alternate regu 0.75 ,urn wide, lie free within the space between the test and larly with intercalary cells which lack symbionts (Fig. I). definitive epithelia of the embryo (Figs. 17, 18) in configu Rod-shaped unicellular bacteria, ranging in length from rations suggestive of their having been released from the 1.7 to 5,um (as determined from lEM micrographs), line test (Fig. 17). The presence of a distinct non-membrane the distal aspect of each bacteriocyte in close contact with bounded nuclear region, the absence of internal membrane the lateral surface of the gill lamellae and the mantle-cavity bounded organelles, and the presence of a tripartite cell environment. A microvillar layer, derived from adjacent wall typical of Gram-negative bacteria are the criteria used intercalary cells, covers the external surface of the bac to classify these particles as bacteria (Costerton 1979). teriocytes. A few mitochondria are present in each bac Initially, the vesicular matrix is a homogeneous granu teriocyte; however, many more occur in the intercalary lar substance (Figs. 9-11), but as bacteria undergo trans cells. The bacteria are intracellular; each bacterium is sur formation, numerous membranes and small vesicles de rounded by what appears to be a host-derived membrane velop within the matrix (Fig. 14). Large electron-lucent (Figs. 1-3). vacuoles are present in granular vesicles in which bacteria are forming (Figs. 13, 17). During synthesis of the putative bacterial cell walls, a Oocyte inclusions large amount of secondary membrane is formed and sur rounds the transforming bacteria (Figs. 14, 15). A non Ovarian oocytes, artificially stripped ova and early cleav membrane-bounded electron-lucent fibrous region is pres age stages were examined with TEM for evidence of sym ent in the center of these bacterial particles. bionts. No typical bacterial forms were present (Figs. 4-6). Following metamorphosis, which includes ingestion of However, an extensive survey of inclusions in ovarian eggs portions of the test (Gustafson and Reid 1986, 1988), many of Solemya reidi revealed several structures of doubtful rod-shaped bacteria, measuring up to 1.3,um long and identity. Unidentified inclusions ("u" in Figs. 4 and 5) bear 0.75,um wide were observed in the perivisceral cavity superficial resemblance to prokaryotic cells seen by lEM along with debris from the ingested test cells (Figs. 19-21). v-

....

.... .~ ~

-.f mc

Figs. 1-3. So/emya reidi. I: Adult gill filamen t showing alternate arrangeme nt of bacteri ocytes (be) and intercalary cells (scale bar = 5 ,urn). 2: Bacteriocyte, showing basally located nucl eus. microvilli derived from intercalary cells. and symbio nts arra nge d j ust below bacteriocyte externa l surface (scale bar= 2.5 ,urn). 3: Symbi onts in periph ery of bacteriocyte: arrows indic ate host-d erived membrane sur rounding each symbio nt (scale bar= 1.0,um). bs: blood spac e: m: mitochond ria: mc: mantl e cavity: mv: microvilli: nb : nucleus of bac teriocyte: ni: nucleus of intercalary cell: s: symbionts. All TEM s 392 R. G. G ustafso n and R. G. B. Reid : Bacteria-larvae association

Figs. 4-8. Solemya reidi. 4: Porti on of vitellogenic oocyte in ova ry (scale bar= 2.5,um). 5: Unidentified inclu sion in cytoplasm of ova rian oocyte (scale bar= 1.0,um) . 6: Assemblage of inclusions in ripe ovarian oocy te (scale bar= 1.0,um). 7: Longitudinal section of mature spermatozo an within testes (scale bar = 2.0 ,urn). 8: Transverse sec tion through mid piece and tail f1a gel lum of mature spermatozo an (scale bar=0.5,um). a: acro som e: fb: fibril lar mucous granule: gv: germinal ves icle: I: lipid sphere: m: mito chond rion: mb : multivesicular body : mp: mid piece: mv : microvilli: nu : nu cleus : tf: tail flagellum : u: unid ent i fied inclu sion : v: vesicle : yb: bipartite yo lk body : yd : dense granular yolk body. All TEM s R.G. G ustafson and R.G. B. Reid: Bacteria-larvae association 393

Figs. 9-12. Solemya reidi. 9: In tracytopl asmic. granular vesicles in test cell of 3d pericalymrna larva: arrows delineate basal plasma memb rane of test cell (scale bar= 1.0 ,um). 10: Intr acyto plasmic. granular vesicles in basal portion of test cell in 4 d peri calyrnma larva: ar rows indicate bacteria-like particles (scale bar =1.0,um). 11: detail of in complete bacteria-like part icle undergoing transform ation within the test of a 5d pericalymm a larva (scale bar= 0.25,um). 12: Unit membrane (arrowed ) which en closes each intracytoplasmic. granular vesicle (scale bar = 0.25,um). gv: granular vesicles; m: mitochond rion ; pm : plasma mem bra ne; sp: space separating test and definitive tissues; vm: vesicle matrix. All TEMs

The bacter ia a re in gested a lo ng wi th th e test a t m et amo r granula r vesi cles observed in the test of Solemya reidi co n phosis, a nd pass down th e esophagus (Fig. 20) to reside ta ine d transforming bact eri a on the fifth a nd six th d ays of within th e peri visceral cavity . Some of th ese bacte ria m a y develo pmen t (Fig. 16). The bacteria present in so me of the be co nta mi na nts, but are m ost lik ely derived fro m bacterial gra nu lar vesicles remain in the test ce lls without undergo transformat ion occurring in th e test ce lls. Not a ll of th e ing bacteri al transformation. 394 R. G. G ustafso n and R. G. B. Reid : Bacteria-larvae asso cia tio n sp

••' 0.,> , va -, <" Figs. 13-15. Solemya reidi. 13: Bacteri al morphogenesis occ urring in gra nular vesicles with in basal section of test cell in a 5 d peri calymma larva ; incomplete forms (arrowed) are associat ed with vacuoles, sugges ting that depl etion of the matrix has occ ur red (sca le bar= 1.0 ,urn). 14: Detail of bacterium (arrowed) undergoin g transformation with in test cell of a 5 d pericalymma larva ; mu ch mem brane and man y sma ll vesicles (v) surround the tran sforming bacterium (scale bar= 1.0,um). 15: Det ail of an incomplete bacterium in test cell of a 5 d pericalymma larv a ; portions of bacteri al cell wall and cell membran e are visible (ar rowed) (sca le bar= 0.25 ,urn). m: mitochondrion ; nr : nuclear region; sp: space sepa rating test and definitive tissues; va: vacuole; vm: vesicle mat rix. All TE Ms R. G. Gustafso n and R. G. B. Reid: Bacteria-lar vae associa tion 395

sp ®

Figs. 16, 17. So lemya reidi. 16: Intracytopl asmic, gra nular vesicle (gv) in basal par t of test cell in a 5d pericalymm a larva (scale bar= 1.0,um). 17: Bacterial mo rphogenesis within basal portion of test cell in a 5d pericalymma la rva; a complete Gr am-negative bac teri um (x) is present within space (sp) sepa rating test and definitive tissues ; arrows show incomplete bacteria (scale bar= 1.0,um). m: mi tochon drio n; pm : plasma membran e; vm: vesicle mat rix; va: vacuole. Both TEMs Figs. 18-20. Solemya reidi. 18: Bacterium. free within the space (sp) sepa ra ting lest (tc) and d efinitive tissu e, (d e) In a 5 d perica lyrnma larv a : a rro w, indicate G ram -nega tive-type cell wall o f bact erium (scal e ba r= 0.5 11m). 19: Pro karyo tic cell" (arrow ed) a nd cellula r debris of lest (tc ) within peri viscer al ca vity of a post-metam orphic 7 d juvenil e (scale bar = 2.5 .um). 20: Po rtion of eso phagus (e ) and pertv isceral cav ity (p v) in a post-met am orphic 7 d Juv enil e: peri viscer al cavi ty is packed wuh pro ka ryot ic ce lls (a rrow ed) a nd remnan t tevt mat er ial (tc) (scale bar = l0 .um) . c: cilia : I: lip id sphere: m: mitochon dri on : nr : nucle a r region : nu: nu cleu s. All TEM , R. G. Gustafson and R. G. B. Reid: Bacteria-larvae association 397

Bacteria were common within the perivisceral cavity was observed. These authors all concluded that Rickett during and immediately after metamorphosis (Figs. 22, siella possesses a developmental cycle similar to Chla 23). However, no bacteria were detected in this mass of in mydia. However, other investigators reported the oc gested test cells following the third day after their engulf currence of an additional stage in this complex life cycle. ment, although the ingested test cells remain intact until at Namely. formation of rod-shaped cells out of a rickettsio least the 42nd day after fertilization (Gustafson and Reid genic stroma material (Huger and Krieg 1967. Gotz 1972, 1988). Kellen et al. 1972. Browning et al. 1982. Federici 1982). A microvillar layer extends over the surface of the gill The "morphogenesis ofcellular organisms from a noncellu bud cells, some of which possess cilia (Fig. 24). An exami lar ground substance" was first reported by Huger and nation of the fine-structure of the gill-bud cells at 18 d Krieg (1967) as an ancillary means of reproduction in shows that they contain several unidentified granular in Rickettsiella. This process. as it occurs in Rickettsiella. has clusions. Bacteria were not found in the gill-buds: however. been termed "in situ transformation" (Huger and Krieg many granular vesicles. reminiscent of those seen in the 1967). "de novo synthesis", "multiple cell division" (Gotz larval test. are abundant (Fig. 25). Phagocytic hemocytes 1972), and "self assembly" (Browning et al. 1982, Federici are common within the central blood space of the gill-buds 1982). Rosenberg and Kordova (1962) and Kordova et al. (Fig. 26). (1965) have reported TEM evidence indicating de novo for mation of bacteria. from granular condensed material in Coxiella burneti and Rickettsia prowazeki, respectively. In these last two studies, cells in tissue culture were infected Discussion with pathogens and a period of time ensued when no bac terial cells could be demonstrated in the cytoplasm or nu The following hypothesis concerning symbiont transmis clcus. sion in Solemya reidi is proposed. External surfaces and in Intracellular infections involving Rickettsiae. ternal contents of oocytes have no bacteria. This would be Chlamydiae, and Mycoplasmas in branchial and digestive expected if symbionts are packaged in cryptic form within tract epithelium are not unusual in adult marine bivalves the egg, as they are in the larval test. Bacteria were never (Lauckner 1983. Elston and Peacock 1984, Elston 1986). seen in thin sections of testes. It is conceivable that dense However. the possibility that bacteria observed in larvae of vesicles consisting of symbiont bacterioplasm move from Solemya reidi were pathogenic is remote, since no mor the ova to the basal part of the test cells as a result of segre phologically observable cellular or subcellular host re gation of blastomere contents during cleavage. It is in the sponse was evident and similar phenomena were seen in test cells that transformation or self-assembly of some of many specimens and in several larval cultures raised the bacteria takes place. Symbionts are then either ingested months apart. Concurrent culture and examination of the directly through the larval mouth or, prior to metamor test in pericalymma larvae of the nuculoid protobranch bi phosis, are discharged into the space between the test and valve A cila castrensis. which does not possess the gill sym definitive embryonic tissues where they may be drawn biosis. failed to reveal bacteria or granular vesicles of the towards the mouth by the densely ciliated oral region, and type seen in S. reidi. A cellular host response to a chla subsequently ingested. In some cases the bacteria do not mydia-like infection is lacking in Mercenaria mercenaria undergo transformation in the test, but may be ingested (Meyers 1979), while Siliqua patula. Tapes japonica, and when still within the test cells. Patinopecten yessoensis exhibit a weak or non-existent host When the dorsal and lateral walls of the stomach dis response to infection with rickettsiales-Iike bacteria (Elston sociate at metamorphosis in Solemya reidi (Gustafson and and Peacock 1984, Elston 1986). Reid 1986. 1988), portions of the ingested larval test and associated bacteria come to lie within the perivisceral cav ity in close contact with potential hemocytes. These herno Transmission in other invertebrate endosymbioses cytes could engulf the bacteria and provide a means of transport through the circulatory system to the developing Since the initial report of chemoautotrophic bacterial sym gill. Elston (l980a, b) observed phagocytic coelomocytes bionts in Solemya velum (Cavanaugh 1980), the list of bi within the perivisceral cavity of larval oysters. Crassostrea valves known to possess similar intracellular gill-bacteria virginica and C. gigas. Similar phagocytes occur within gill associations has expanded to include members of four buds ofjuvenile S. reidi (Fig. 26). families; the Solernyidae. the Mytilidae, the Lucinidae and The appearance of intracytoplasmic, granular vesicles the Vesicomyidae (see Southward 1986 or Reid in press for in test cells and the presumed transformation of bacteria complete listing). Sulfur-oxidizing symbiotic bacteria in the from their granular matrices is not unique. In studies ofthe Thyasiridae occur extracellularly beneath a thin gill bac fine-structure of the obligately intracellular bacterial genus teriocyte cuticle (Reid and Brand 1986. Southward 1986). Rickettsiella (Weiss 1974) in a cockchafer (Devauchelle A methane-based gill symbiosis exists between an un et al. 1971. 1972). an isopod (Louis et al. 1977), a spider described mytilid and intracellular methanotrophic bac (Morel 1977). and an amphipod (Larsson 1982). no evi teria from hydrocarbon seeps in the Gulf of Mexico dence of transformation of bacteria from a granular matrix (Childress et al. 1986).

R.G. Gustafson and R. G. B. Reid: Bacteria -la rvae associa tion 399

Fig~ . 25. 26. So lemva reidi. 25: Gill bud cells in an 18d Juveni le: urudenti fied cell incl usions are arrowed (scale bar = 2,5 11m ). 26: Phagocyuc hemo cyte conuu nrng unidentifi ed inclusion s

" (ar rowed) within central blood space (b ~ ) 01' gill bud in an 18 d Juvenile (scale bar= 1.0 ,um). I: lipid sphere; m: mitochondri on; me: mantl e cavity; mv: microvilli; nu: nucleus. Both TEMs

.. Figs. 21-24. So!emya reidi. 21: Prokaryotic cells (arrowed) in per ivisceral cavity of dissected post-metamorp hic 7 d ju venile. SEM (scale bar =2.5 ,urn). 22: Test cells (tc) and remn ants of test cirri (cr). as well as prokaryotic cells (arrowed) within perivisceral cavity of a post metamorphic j uvenile. TEM (scale bar = 2.5 ,urn). 23: Ingested test ma terial (tc) and bacteria (arrowe d) within perivisceral cavity ofa post metamorphi c 7 d juvenile. TEM(scale bar = 5 ,urn). 24: Cross-sectio n through a single gill bud of an 18 d ju venile . TEM (scale bar = 10 ,urn). c: gill cilia; rna: mantl e; me: mantl e cavity; mv : microvilli; nu : nucleus 400 R. G. Gustafson and R. G. B. Reid: Bacteria-larvae association

The structural and physiological relationships between Cavanaugh, C. M. (1980). Symbiosis ofchemoautotrophic bacteria intracellular bacteria and both vent and non-vent pogono and marine invertebrates. BioI. Bull. mar. bioI. Lab .. Woods Hole 159: p. 457 phorans have been described (Cavanaugh et al. 1981, Cavanaugh, C. M. (1983). Symbiotic chemoautotrophic bacteria in Southward 1982, Southward et al. 1986, Schmaljohann and marine invertebrates from sulfide-rich habitats. Nature, Lond. Flugel 1987). However, transmission mechanisms have not 302: 58-61 been investigated in detail in any pogonophoran-bacteria Cavanaugh, C. M. (1985). Symbioses of chemoautotrophic bac symbiosis. TEM examination of eggs of the vent pogono teria and marine invertebrates from hydrothermal vents and reducing sediments. Bull. bioI. Soc. Wash. 6: 373-388 phoran Riftia pachyptila did not reveal bacterial symbionts Cavanaugh, C. M., Gardiner, S. L., Jones, M. L., Jannasch, H. W., (Cavanaugh et al. 1981). However, juveniles of R. pa Waterbury, J. B. (1981). Prokaryotic cells in the hydrothermal chyptila as small as 1.44 mm long by 0.33 mm wide have a vent tuhe worm Riftia pachyptila: possible chemoautotrophic trophosome identical to that of adults (Cavanaugh et al. symhionts. Science, N.Y. 213: 340-342 Childress, J. J.. Fisher, C. R., Brooks, J. M., Kennicutt II, M. 1981). c. Bidigare, R., Andersen, A. E. (1986). A methanotrophic marine Vertical transmission of chemoautotrophic symbionts molluscan (Bivalvia, Mytilidae) symhiosis: mussels fueled by located extracellularly beneath the cuticle in the gutless gas. Science, NY 233: 1306-1308 oligochaetes Phallodrilus leukodermatus and P. planus oc Costerton, J. W. (1979). The role of electron microscopy in the curs when eggs are infected at oviposition from bacteria ex elucidation of bacterial structure and function. A. Rev. Microbiol. 33: 459-479 truded from the adult genital pad. Bacteria enter the polar Dando, P. R., Southward, A. J. (1986). Chemoautotrophy in bi end of the eggs through "entrance passages" into the valve molluscs of the genus Thyasira. J. mar. bioI. Ass. U.K. 66: ooplasm (Giere and Langheld 1987). 915-929 Within the marine environment, intracellular symbiotic Dando. P. R., Southward, A. J.. Southward, E. C. (1986). Chemoautotrophic symbionts in the gills of the bivalve mollusc bacteria are found associated with tunicates (Buchner Lucinoma horealis and the sediment chemistry of its habitat. 1965), sponges (Vacelet and Donadey 1977, Wilkinson Proc. R. Soc. (Ser. B) 227: 227-247 1978), and wood-boring bivalves of the family Teredinidae Dando, P. R., Southward, A. J., Southward, E. c. Terwilliger, N. (Felbeck et al. 1983 a). Luminescent bacteria in tunicates B.. Terwilliger, R. C. (1985). Sulphur-oxidising bacteria and (pyrosomes and salps) are transmitted to the offspring via haemoglobin in gills of the bivalve mollusc Myrtea spinifera. Mar. Ecol. Prog. Ser. 23: 85-98 "spores". which are carried in the bloodstream from paired Devauchelle, G., Meynadier, G., Vago, C. (1972). Etude ultra luminous organs to follicle cells surrounding the single egg structurale du cycle de multiplication de Rickettsiella melolon of each individual tunicate (Pierantoni 1921, Buchner thae (Krieg), Philip, dans les hemocytes de son hote. J. Ul 1965). An efficient vertical transmission mechanism has trastruct. Res. 38: 134-148 Dcvauchelle, G., Vago, C, Meynadier, G. (1971). Ultrastructure been documented for bacterial transfer in sponges, wherein comparee de Rickellsiella melolonthae (Rickettsiales Wol bacteria-containing follicle cells are carried along when the bachiae) et de l'agent de la Iymphogranulomatose venerienne eggs are shed (Gallissian and Vacelet 1976, Levi and Levi (Rickettsia1es Chlamydiaceae). C. r. hebd. Seanc, Acad. Sci., 1976). Nothing is known concerning transmission of bac Paris 272: 2972-2974 teria associated with the gland of Deshayes in teredinid bi Elston, R. (1980a). Functional anatomy, histology and ul trastructure of the soft tissues of the larval American oyster, valves (Felbeck et al. 1983a). Crassostrea virginica. Proc. natn. Shellfish. Ass. 70:65-93 Acknowledgements. We thank boat captains D. Horn, C. Dillen, Elston, R. (1980 b). Functional morphology of the coelomocytes of and S. Tueit for their able assistance in collecting Solemya reidi; the larval oyster (Crassostrea virginica and Crassostrea gigas). B. Gustafson and D. Brand for assistance and perseverance in the J. mar. bioI. Ass. U.K. 60: 947-957 field on many foul weather days: and Dr. R. D. Burke of the De Elston, R. (1986). Occurrence of branchial rickettsiales-like in partment of Biology, University of Victoria, for use of his facilities fections in two bivalve molluscs, Tapes japonica and Patinopec for culturing larvae. Special thanks go to D. Brand for preparation ten yessoensis, with comments on their significance. J. Fish of the photographic plates. This research was supported by a Uni Dis. 9: 69-71 versity of Victoria Graduate Fellowship to the first author and an Elston, R. A., Peacock, M. G. (1984). A rickettsiales-like infection operating grant from the National Research Council of Canada to in the Pacific razor clam. Siliqua patula. J. Invertebr. Path. 44: the second author. 84-96 Federici. B. A. (1982). A new type of insect pathogen in larvae of the clover cutworm, Scotogramma trifolii. J. Invertebr. Path. Literature cited 40:41-54 Felbeck, H. (1981). Chemoautotrophic potential of the hydro Anderson. W. A.. Personne. P. (1976). The molluscan sper thermal vent tube worm Riftia pachyptila Jones (Vestimen matozoan: dynamic aspects of its structure and function. Am. tifera). Science, N.Y. 213: 336-338 Zoo!' 16: 293-313 Felbeck, H. (1983). Sulfide oxidation and carbon fixation by the Andrewes, C. 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