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Notice: ©1999 Springer‐Verlag. This manuscript is a version of an article with the final publication found online at http://www.springerlink.com and may be cited as: Eckelbarger, K. J. and C. M. Young (1999) Ultrastructure of gametogenesis in a chemosynthetic mytilid bivalve ( childressi) from a bathyal, methane seep environment (northern Gulf of Mexico), Marine Biology 135: 635‐646 doi: 10.1007/s002270050664

Marine Biology (1999) 135: 635±646 Ó Springer-Verlag 1999

K. J. Eckelbarger á C. M. Young Ultrastructure of gametogenesis in a chemosynthetic mytilid bivalve (Bathymodiolus childressi ) from a bathyal, methane seep environment (northern Gulf of Mexico)

Received: 24 May 1999 / Accepted: 10 September 1999

Abstract The ultrastructural features of gametogenesis methane seep environment and presumed continuous have been described in the methane-seep mytilid bivalve availability of nutrients, reproduction parallels that of Bathymodiolus childressi Gustafson, Turner, Lutz & littoral mytilids, suggesting some phylogenetic con- Vrijenhoek, 1998 collected from the Gulf of Mexico in straints on the capacity for variability in gametogenic August 1995. This is the ®rst ultrastructural description processes in the family . of gametogenesis in any methane-seep bivalve. B. chil- dressi is gonochoric, and both the testis and ovary consist of acini surrounded by inter-acinal tissue com- posed of adipogranular cells that serve a nutrient storage Introduction function. Oocytes develop in close association with squamous follicle cells although the follicle cells do not Dense macrofaunal communities of vestimentiferan appear to play a primary role in yolk synthesis. During tubeworms and modioliform (Mytilidae) are the vitellogenic phase, biosynthesis of four types of or- associated with sites of oil and methane seepage at ganelles occurs, including lipid droplets, yolk granules, depths >500 m on the Louisiana slope of the Gulf of cortical granules, and unknown inclusions, which are Mexico (Kennicutt et al. 1985). The mussels attain a exocytosed as part of egg envelope biogenesis. Vitello- high biomass around these hydrocarbon seeps and har- genesis appears to be largely autosynthetic involving the bor methanotrophic endosymbionts in their gills. The Golgi complex and rough endoplasmic reticulum. Sperm bacterial symbionts oxidize methane and reduced car- di€erentiation resembles that reported in other mytilid bon although the mussels also derive nutrition from bivalves and includes the di€erentiation of proacroso- other sources, including ®lter feeding (reviewed by Lee mal granules and a ¯agellum during the early stages of et al. 1992; Lee and Childress 1995, 1996). Despite the spermatogenesis. Mature sperm have a cap-like acro- intense interest in the nutritional biology and physiology some, substantial subacrosomal material, a bullet- of hydrothermal vent and seep-associated mussels, very shaped nucleus, and four to ®ve mitochondria in the little information is available on reproduction (reviewed midpiece. The general features of gametogenesis closely by Tunnicli€e 1991; Mullineaux and France 1995; Tyler resemble those reported in shallow-water, seasonally and Young 1999). There is limited ultrastructural in- reproducing mytilids. Despite the relatively stable formation on the sperm, eggs, interstitial tissue, and gonoducts of an unidenti®ed species of Bathymodiolus from the East Paci®c Rise (Herry and Le Pennec 1987), Communicated by J.P. Grassle, New Brunswick and, more recently, Le Pennec and Beninger (1997) de- K.J. Eckelbarger (&) scribed features of spermatogenesis and mature sperm Darling Marine Center, University of Maine, 193 Clark's Cove Rd., Walpole, Maine 04573, USA ultrastructure in three species of Bathymodiolus from the East Paci®c Rise, the mid-Atlantic ridge, and the North e-mail: [email protected]; Fiji Basin. There are anecdotal observations on repro- Fax: +1-207-5633119 duction of deep-sea mytilids (Berg 1985; Hessler et al. K.J. Eckelbarger 1988) for B. thermophilus in the eastern Paci®c, and for School of Marine Sciences, University of Maine, Libby Hall, Orono, Maine 004469, USA an undescribed species of Bathymodiolus from the Lucky Strike vent ®eld on the Mid-Atlantic Ridge (Comtet and C.M. Young Á Division of Marine Sciences, Desbruyeres 1998). Harbor Branch Oceanographic Institution, There are no ultrastructural descriptions of the go- 5600 U.S. 1 North, Ft. Pierce, Florida 34946, USA nads or gametogenesis for mussels from methane seep 636 sites, and only limited information on deep-sea mussels, c in general. Hydrothermal vents and cold-water methane Fig. 1a±f Bathymodiolus childressi. a Light microscopic transverse seeps are created by di€erent geological processes, and section through the ovary showing developing oocytes within acini. Note oocytes in early (1), middle (2), and late (3) stages of the lack of species overlap between these distinct com- vitellogenesis. Arrowheads indicate stalk-like connection of oocyte munities suggests some fundamental ecological di€er- to ovary wall (N nucleus). Scale bar ˆ 40 lm. b Adipogranular ences (Tunnicli€e 1991). Hydrothermal vents are (ADG) cell showing lipid droplets (L), electron dense granules (*), and unpredictable and ephemeral, which implies that species glycogen (GL). Scale bar ˆ 10 lm. c Higher magni®cation of ADG living there might undergo rapid growth and reproduc- cells showing lipid (L), electron dense granules (*), and glycogen (GL). Scale bar ˆ 1.0 lm. d Previtellogenic oocyte (OC) in close association tion (Craddock et al. 1995). In contrast, methane seep with follicle cell containing extensive arrays of rough endoplasmic sites are believed to be more stable and to provide a reticulum (RER) (N nucleus; Nu nucleolus; M mitochondrion). Scale more constant source of nutrients. This suggests that bar ˆ 2.0 lm. e Follicle cell containing nucleus (N), mitochondrion selective pressures on species in these two contrasting (M), rough endoplasmic reticulum (RER), and lysosomal-like inclusion (*). Scale bar ˆ 2.0 lm. f Nuage-like granules in ooplasm habitats might result in divergent life history patterns, of previtellogenic oocyte. Scale bar ˆ 2.0 lm including di€erences in reproductive seasonality. Bathymodiolus thermophilus was the ®rst hydrother- mal vent described (Kenk and Wilson 1985), and (MacDonald et al. 1990) at 710 m depth (27°43.327¢N; at least seven additional species are now recognized from 91°16.606¢W). For ultrastructural studies, fresh gonadal tissues from male and female specimens of varying sizes were selected for vent sites (reviewed by Gustafson et al. 1998). Craddock ®xation: three females with maximum shell lengths (SL) of 83, 96, et al. (1995) identi®ed several additional modioliform and 110 mm and three males with SL of 70, 92, and 105 mm. taxa from sul®de/hydrocarbon seeps in the Gulf of Me- Tissue samples were excised and immersed for 1.5 h in cold (4 °C) xico using an allozyme survey and, recently, Gustafson primary ®xative containing 3% glutaraldehyde, 1% formaldehyde made from paraformaldehyde, 0.1 M Millonig's phosphate bu€er et al. (1998) described ®ve mussel species from this sur- (pH 7.4), 3% NaCl, and 3.5% sucrose. Tissue was then rinsed for vey. Prior to the work of Craddock et al. (1995), seep and 30 min in three changes of cold (4 °C) 0.1 M Millonig's phosphate hydrothermal vent mytilids were assumed to be closely bu€er containing 6% sucrose. The tissue was then post®xed for related but it now appears that there are no mytilid 1.5 h at room temperature in 1% OsO4 in 0.1 M Millonig's phos- species common to both habitats. In the present study, phate bu€er and dehydrated for 2 h in ascending concentrations of ethanol. The ®nal dehydrations with 100% ethanol and with two we describe the ultrastructural features of the gonad and changes of propylene oxide (2 min each) were followed by em- gametogenesis in the well-studied methane seep species bedding in Epon. Thin sections were cut with a diamond knife on a Bathymodiolus childressi, which has recently been de- Porter-Blum MT2-B ultramicrotome, stained with uranyl acetate scribed (Gustafson et al. 1998). This species has been followed by lead citrate, and examined with a Zeiss EM 10-A transmission electron microscope (TEM). referred to in the literature by various names since 1985, including Seep Mytilid 1a (Louisiana Continent Slope) and Seep Mytilid 1b (Alaminos Canyon) (reviewed by Gustafson et al. 1998). Placement of this species in the Results genus Bathymodiolus is provisional pending a more complete morphological and molecular analysis (R. Lutz General morphology of the gonad personal communication). The ®ne-scale distribution of this species on the Louisiana slope is correlated with Bathymodiolus childressi is a gonochoric species, and we methane concentration (MacDonald et al. 1989), and saw no histological evidence of hermaphroditism. We dense populations are found around brine-®lled depres- saw no signi®cant di€erence in degree of gamete devel- sions on the ocean ¯oor where brine density reaches 3.5 opment in either sex in the specimens we sampled, and times that of normal seawater (MacDonald et al. 1990a, none of the specimens were immature. The gonad is a b). The species has been recorded from a maximum depth simple structure consisting of acini surrounded by inter- of 2222 m at Alaminos Canyon in the western Gulf of acinal tissue composed of adipogranular cells. Each Mexico (Gustafson et al. 1998). Preliminary data from acinus contains developing germ cells and is bathed ex- histological sections of the gonads and analysis of pop- ternally in hemal ¯uid contained within an anastomos- ulation structure (M. Baker, P. Tyler and C. Young ing hemocoelic space. In the ovary, developing oocytes unpublished) indicate that this species reproduces sea- are also associated with follicle cells. In transverse sec- sonally, and that its recruitment is intermittent. The tion, each acinus contains peripherally arranged germ present study is the ®rst to document gametogenesis in cells surrounding a central lumen. In most acini exam- any methane seep mussel, and it o€ers new ultrastruc- ined, nearly all stages of development are represented tural details of this process in mytilids, in general. (Fig. 1a).

Adipogranular cells Materials and methods The gonad acini are surrounded by a matrix of large Live mussels (Bathymodiolus childressi Gustafson, Turner, Lutz & Vrijenhoek, 1998) were collected in August 1995 by the Johnson- pleomorphic somatic cells referred to as adipogranular Sea-Link II submersible at Green Canyon Block 232 (``brine pool'') (ADG) cells. Each cell is irregularly shaped and contains 637 638 a nucleus with a single nucleolus. The cytoplasm is c dominated by abundant glycogen particles, small (0.5 to Fig. 2a±e Bathymodiolus childressi. a Early vitellogenic oocyte con- 1.5 lm diam.) electron dense granules, and lipid droplets taining large nucleus (N), mitochondria (M), lipid droplets (L), and yolk granules (Y) (FC follicle cell; Nu nucleolus). Scale bar ˆ 4.0 lm. (2 to 9 lm diam.) (Fig. 1b, c). Transverse sections b Perinuclear region of early vitellogenic oocyte showing band of through the gonad indicate that the proportion of ADG mitochondria (M) and adjacent lipid (L) and yolk granules (Y) tissue versus germ cells appeared to be approximately (N nucleus). Scale bar ˆ 2.0 lm. c Dilated cisternae of rough the same in both the testis and ovary. endoplasmic reticulum (RER) and adjacent yolk bodies (Y). Scale bar ˆ 1.0 lm. d High magni®cation of yolk granules showing vacuole-like inclusions (arrows). Scale bar ˆ 1.5 lm. e Golgi complex (GC) and adjacent nascent yolk granules (Y). Scale bar ˆ 2.0 lm Oogenesis positioned organelles that appear to be exocytosed The inner wall of each ovarian acinus is lined with oo- during vitelline envelope formation. Lipid droplets ap- cytes in all stages of di€erentiation, including oogonia, pear ®rst (Fig. 2a, b), and they numerically dominate the previtellogenic and vitellogenic oocytes (Fig. 1a). How- ooplasm throughout vitellogenesis. They lack limiting ever, vitellogenic oocytes predominate in all the acini we membranes and reach 1.0 to 1.5 lm in diameter. The examined. Follicle cells are associated to some degree yolk-like granules appear next and eventually reach 0.5 with all oocytes but most notably with oogonia and to 1.3 lm in diameter. The cortical granules are mem- early oocytes, often completely surrounding them. Fol- brane-bounded, spherical bodies 0.5 to 1 lm in diameter licle cells are predominately squamous, but cell volume that are initially bipartite, consisting of a ®ne, granular and dimension vary. Their cell nuclei are spherical to cortical region and a more electron-dense core (Fig. 3b). oblong and contain scattered clumps of heterochromatin Following further di€erentiation, they develop a ho- and a single nucleolus (Fig. 1d, e). Their cytoplasm mogeneous electron-dense core and a distinctive cortex contains mitochondria, lysosomal-like inclusions and resembling bundles of microtubule-like structures ar- extensive, parallel arrays of rough endoplasmic reticu- ranged in parallel, depending on the plane of section lum (RER) (Fig. 1d, e). The RER cisternae are often (Fig. 3c, d). Cortical granules form a narrow band distended with a ®ne, granular product. Follicle cells are subjacent to the oolemma (Fig. 3a, g), and they are closely applied to the adjacent oocytes, and both cell comparatively less electron-dense than the yolk-like types are joined intermittently by desmosomes (not granules. During middle to late vitellogenesis, exocytotic shown). vesicles are commonly observed in the process of re- Oogonia and previtellogenic oocytes are generally leasing a ®ne ¯occulent material into the perivitelline small (5 to 7 lm diam.), spherical to oblong-shaped space (Fig. 3e, f). The contents of these vesicles closely cells. Oocytes at this stage are nearly devoid of ooplas- resemble the material occupying the extracellular space mic organelles except for pro®les of pleomorphic mito- between microvilli but the origin of the vesicles is un- chondria closely associated with the outer surface of the known. nuclear envelope. Clumps of nuage-like material re- In the largest oocytes observed (80 lm diam.), the egg sembling course granules appear in the perinuclear oo- envelope is 0.5 lm thick and consists of evenly-spaced, plasm (Fig. 1f), and associated follicle cells are closely parallel microvilli penetrating a homogeneous, electron- appressed to the oocyte oolemma (Fig. 1d). As oocyte dense vitelline layer (Fig. 3g). While the lipid droplets growth continues and vitellogenesis begins, the cells arise de novo without any apparent association with become pedunculate, with the basal region of the oocyte other organelles, the yolk-like granules appear to be extending from the acinus wall by a narrow stalk synthesized by the Golgi complexes, perhaps involving (Fig. 1a). Follicle cells at this stage are largely restricted the RER. The origin of the cortical granules is unclear to the stalked region of the oocyte. Early vitellogenic although presumably they are synthesized by the Golgi oocytes have prominent germinal vesicles with one or complexes. two nucleoli (Fig. 2a). Surrounding follicle cells contin- ue their close association with the oocyte surface (Fig. 2a). The perinuclear region of the oocyte has a Testis dense band of small mitochondria persisting into the late stages of vitellogenesis (Fig. 2b). Scattered Golgi com- The gross morphology of the testis closely resembles that plexes appear (Fig. 2e) as well as distinctive, parallel of the ovary in that it consists of branching acini within arrays of RER (Fig. 2c) which sometimes form whorled a supportive matrix of surrounding ADG cells (Fig. 4a). patterns. The Golgi complexes appear to secrete yolk The ADG cells are ultrastructurally indistinguishable granules, which are electron-dense organelles containing from those observed in the ovary and are approximately small vacuolar inclusions (Fig. 2d). Simple, unbranching as abundant. Occasionally, we observed ADG cells with microvilli project from the oocyte surface, and a wispy cytoplasm containing distinctive, membrane-bounded material appears between adjacent microvilli (Fig. 3a). organelles (Fig. 4b, c), a feature not reported previously. During the vitellogenic phase, four types of organ- The organelles were usually spherical and contained a elles are synthesized, including lipid droplets, yolk-like granular material of variable electron density. Their electron-dense granules, cortical granules, and cortically origin and function are unknown. 639 640 641 b (Fig. 5j). The distal centriole possesses a radiating per- Fig. 3a±g Bathymodiolus childressi. a Cortical region of vitellogenic icentriolar complex (Fig. 5g) connecting the posterior oocyte showing lipid droplets (L), yolk granules (Y), and narrow band region of the centriole to the adjacent plasmalemma of cortical granules (arrowheads) (MV microvilli). Scale bar ˆ 1.5 lm. b Cortical granules (CG) in early stages of di€erentiation (L lipid). (Fig. 5j). Numerous transverse sections through the Scale bar ˆ 1.0 lm. c Tangential section through cortical granule midpiece indicate that four or ®ve spherical mitochon- showing substructure of microtubule-like elements. Scale dria form a ring around the distal centriole (Fig. 5i). The bar ˆ 0.5 lm. d Cortical granule showing transverse and longitudinal length of the ¯agellum was not determined, but it pos- sections through peripheral microtubule-like elements. Scale sessed the characteristic 9+2 con®guration of microtu- bar ˆ 0.2 lm. e, f Cortical region of oocyte showing exocytosis of ¯occulent material (*) into the perivitelline space occupied by bules. microvilli (MV) (CG cortical granule). Scale bars ˆ 0.5 lm. g Cortical region of mature oocyte showing band of cortical granules (CG) and surrounding egg envelope (*). Scale bar ˆ 1.0 lm Discussion and conclusions Each acinus contains a variety of developing sperm stages which are distributed in a centripetal pattern from The reproductive biology and life histories of vent and the inner acinus wall to the lumen (Fig. 4a). Sperm- seep organisms remain poorly known (reviewed by Tyler atogonia are located nearest the inner wall, sperm- and Young 1999). The present study is the ®rst to doc- atocytes and spermatids are positioned between the ument gametogenesis in any methane seep mussel, but spermatogonia and the acinus lumen, and mature sperm recent papers have described oogenesis (Eckelbarger and are almost exclusively con®ned to the central lumen. Young 1997) and spermatogenesis (Hodgson et al. 1998) Spermatogonia are the largest germ cells (6.5 to 7.5 lm in the methane seep neritid gastropod Bathynerita diam.) and have a spacious nucleus containing a single naticoidea, a species closely associated with Bath- nucleolus and dispersed heterchromatin (Fig. 4d). Pri- ymodiolus childressi. mary spermatocytes are more numerous than sperm- Ultrastructural studies of bivalve oogenesis are sur- atogonia and are 5.0 to 6.5 lm in diameter. prisingly rare, being restricted to only a few species in Spermatocytes in several stages of meiosis were observed three families, while studies of sperm development have including the diplotene stage (Fig. 4e). The nuclei ®ll been conducted on over 80 species in 70 families (re- most of the cell and contain a ®nely di€use hetero- viewed by Eckelbarger and Davis 1996a, b). Ultra- chromatin while the cytoplasm may contain centrioles, structural studies of oogenesis in the family Mytilidae evidence of a ¯agellum, and small proacrosomal gran- are restricted to the littoral species Mytilis edulis (Al- ules (Fig. 4e). Late stage spermatocytes all have cyto- bertini 1985; Pipe 1987a, b) and an unidenti®ed, deep- plasm containing a number of proacrosomal granules sea, hydrothermal vent mussel from the East Paci®c Rise (Fig. 4f). (Herry and Le Pennec 1987). Sperm ultrastructure in Spermatids result from the division of spermatocytes mytilids has been described in nearly 30 species, the and are, in their early stages, spherical cells 2.5 lm in majority of which live in littoral habitats. Gonad mor- diameter (Fig. 5a). Their nuclei contain di€use patches phology in Bathymodiolus childressi generally mirrors of heterochromatin and a single Golgi complex is often that previously reported in other mytilids (Albertini observed in association with proacrosomal granules 1985; Pipe 1987a, b), including related species from hy- (Fig. 5b, c). Spherical mitochondria appear in a shallow drothermal vents (Herry and Le Pennec 1987; Le Pennec posterior nuclear fossa, and the proacrosomal granules and Beninger 1997). fuse to form a single, spherical acrosomal vesicle which Only fragmentary ultrastructural information is migrates to the anterior pole of the cell (Fig. 5d). available on oogenesis in deep-sea mussels (Herry and A ¯agellum and two associated centrioles appear at the Le Pennec 1987). The only comprehensive study of oo- posterior pole of the cell (Fig. 5e). Further di€erentia- genesis in any mytilid was that of Pipe (1987a) for the tion results in continued heterochromatin condensation shallow-water species Mytilus edulis. The oocytes of and anterior±posterior elongation of the nucleus both M. edulis and Bathymodiolus childressi share a (Fig. 5e). The acrosomal vesicle di€erentiates from an number of distinctive ultrastructural features including oval organelle (Fig. 5d) to a cap-like structure contain- whorls of RER, vesiculated yolk granules, and cortical ing homogeneous, electron-dense material (Fig. 5e, f). granules containing peculiar, cortically arranged, mic- The subacrosomal space between the nucleus and the rotubule-like structures. The unusual ®ne structure of acrosomal vesicle is ®lled with a ®ne granular product the cortical granules could represent a taxonomically (Fig. 5f, h). useful diagnostic feature in mytilid oocytes once many The mature spermatozoon has a conical head more species have been examined. Vitellogenesis in 3.9 lm in length from the acrosome tip to the posterior B. childressi is similar to that reported for M. edulis (Pipe midpiece. The nucleus alone is 2.6 lm long and pos- 1987a) and appears to be largely an autosynthetic pro- sesses a shallow posterior nuclear fossa into which the cess by the proteosynthetic organelles of the oocyte. proximal centriole is inserted (Fig. 5f). The proximal Autosynthetic yolk formation appears to be the domi- centriole lies within a shallow nuclear fossa oriented at nant process in most molluscs (Jong-Brink et al. 1983). 45°angle from the anterior±posterior axis of the cell On the other hand, Pipe (1987a) reported some 642 643 b hemocoelic space. In B. childressi, the interacinal spaces Fig. 4a±f Bathymodiolus childressi. a Light microscopic transverse are ®lled with pleomorphic ADG cells which presumably section through testis showing portion of testicular acinus containing serve as nutrient storage tissue for the developing ga- developing sperm (SP), and surrounding ADG cells. Scale bar ˆ 10 lm. b ADG cell with nucleus (N) and cytoplasmic metes, as has been demonstrated for some littoral species inclusions. Scale bar ˆ 2.0 lm. c Higher magni®cation of Fig. 4b (Mathieu and Lubet 1993). The large proportion of the showing nucleus (N) and cytoplasmic inclusions (arrowheads). Scale gonad dedicated to the nutrient-laden ADG cells sug- bar ˆ 1.0 lm. d Spermatogonium containing nucleus with single gests that yolk precursors stored within these cells are nucleolus (Nu). Arrowhead indicates small proacrosomal granule. transported relatively rapidly via the hemocoelic sinuses. Scale bar ˆ 1.5 lm. e Spermatocyte in diplotene stage with large nucleus (N) and centriole (C) and proacrosomal granules (arrowheads) Le Pennec and Beninger (1997) note that a second so- in cytoplasm. Scale bar ˆ 2.0 lm. f Late spermatocyte with nucleus matic storage cell, the vesicular connective (VCT) cell, is containing condensing heterochromatin and cytoplasm containing common to littoral bivalves but is absent from all small proacrosomal granules (arrowheads). Scale bar ˆ 2.0 lm Bathymodiolus species examined thus far. The signi®- cance of this absence is uncertain. endocytotic activity in the oocytes of the mussel M. It is unclear if the ovarian follicle cells in Bath- edulis, and Eckelbarger and Davis (1996a) presented ymodiolus childressi oocytes play any role in vitellogen- evidence for heterosynthetic yolk formation in the oo- esis. These cells show similarities to those described by cytes of the oyster Crassostrea virginica. Therefore, it is Pipe (1987a) in Mytilus edulis, but those of B. childressi likely that vitellogenesis in many bivalves is a combined possess considerably more rough endoplasmic reticu- auto- and heterosynthetic process. lum, suggesting a biosynthetic role. However, due to Annual reproduction in bivalves is common (Sastry their relatively small size, the lack of endocytotic activity 1979). In the specimens of Bathymodiolus childressi we along the oocyte±follicle cell interface, and their limited, sampled, vitellogenic oocytes dominated the acini sug- temporal association with oocytes, they probably do not gesting that oogenesis is synchronous and perhaps sea- play a central role in vitellogenesis. They might, how- sonal, but the lack of seasonal samples makes ever, be involved in the synthesis and transfer of some determination of reproductive periodicity currently im- precursors and/or the regulation of the ¯ow of metab- possible. Analysis of population structure suggests that olites to the oocytes as described in the oyster Crass- reproduction is probably annual (Baker, Tyler, and ostrea virginica (Eckelbarger and Davis 1996a). Follicle Young unpublished data). In addition, ultrastructural cells have been described from many bivalve ovaries, but evidence suggests that vitellogenesis is nearly identical to their ultrastructure and probable function(s) vary (re- that of the littoral species Mytilus edulis (Pipe 1987a), a viewed by Eckelbarger and Davis 1996a). In general, species with an annual reproductive period (Pipe 1987b). bivalve follicle cells appear to be far less biosynthetically It is possible that, despite the relatively stable environ- active than in the cephalopods and gastropods (e.g. ment of methane seep sites and the presumed availability Selman and Arnold 1977; Jong-Brink et al. 1983; of nutrients, reproduction follows a seasonal pattern Eckelbarger and Blades-Eckelbarger 1989). This may be typical of most littoral mytilids. This would suggest that due to the special energy-storing somatic cells often as- phylogenetic history plays an important role in the re- sociated with bivalve ovaries. productive biology of methane seep mytilids (reviewed The general features of spermatogenesis and sperm by Eckelbarger and Watling 1995). It may be signi®cant morphology in Bathymodiolus childressi are similar to that we observed no ultrastructural evidence of hetero- three species of Bathymodiolus (B. thermophilus, B. put- synthetic yolk synthesis in the oocytes of B. childressi eoserpentis, B. elongatus) from hydrothermal vent habi- manifested by receptor-mediated endocytosis. In many tats (Le Pennec and Beninger 1997) except for minor metazoans, high levels of endocytotic activity are often di€erences in organelle dimension. The latter authors associated with rapid yolk synthesis and aseasonal re- found no species-speci®c di€erences in the sperm of the production (reviewed by Eckelbarger 1994). One might three species they examined. The four Bathymodiolus expect rapid vitellogenesis and egg production in envi- species above all have mature sperm with a cap-like ronments where nutrients are abundant. On the other acrosome, substantial subacrosomal material, a bullet- hand, low levels of endocytotic activity can often be shaped nucleus, and four or ®ve mitochondria within a overlooked in electron microscopic investigations short midpiece. Unlike the sperm of many mytilids, they (Eckelbarger 1994), and it is likely that some hetero- lack an axial rod within an endonuclear channel (Gar- synthetic contribution to yolk formation does occur. In rido and Gallardo 1996; Drozdov and Reunov 1997), a comparison to other molluscs, bivalve oocytes show feature shared with other members of the subfamily signi®cantly lower levels of endocytotic activity during Modiolinae. Bivalve sperm morphology is believed to vitellogenesis and relatively rapid egg di€erentiation have useful applications for phylogenetic and taxonomic due, perhaps, to the provision from interacinal storage analysis particularly at or above the family level (re- tissues of abundant low molecular weight precursors viewed by Popham 1979; Healy 1996; Kafanov and (Eckelbarger and Davis 1996a). Drozdov 1998). Bivalve sperm are typically conservative The gonad of Bathymodiolus childressi is structurally with respect to midpiece and ¯agellum morphology, similar to other bivalves in that the acini are bathed in while the acrosome often demonstrates wide structural hemal ¯uid circulating throughout an anastomosing diversity. Many littoral mytilids (subfamily Mytilinae) 644 645 b in the present study. Ultrastructural examination of the Fig. 5a±j Bathymodiolus childressi. a Early spermatid. Scale sperm of three species of Bathymodiolus from hydro- bar ˆ 2.0 lm. b Spermatid with Golgi complex (GC) and proacro- thermal vent sites by Le Pennec and Beninger (1997) also somal granule (arrowhead). Scale bar ˆ 1.5 lm. c Cytoplasm of spermatid containing Golgi complex (GC) and newly formed failed to detect endosymbionts. In contrast, endosym- proacrosomal granules (arrowheads) (N nucleus). Scale bar ˆ 1.5 lm. biont-like inclusions have been reported from the oocytes d Spermatid with oval nucleus (N), acrosomal granule (A), a of the deep-sea vent bivalve Calyptogena soyoae (Endow mitochondrion (M) and two centrioles (CE). Scale bar ˆ 1.0 lm. and Ohta 1990), and it is suspected in other species in e Late spermatid with sac-like acrosome (A), subacrosomal material this genus (Cary and Giovannoni 1993). Also, Gustaf- (*), an elongated nucleus (N), a distal centriole (D) and two adjacent mitochondria (M). Scale bar ˆ 1.0 lm. f Mature spermatozoon with son and Reid (1988) have hypothesized that transovarial cap-like acrosome (A), extensive subacrosomal material (*), elongated transmission of symbionts occurs in Solemya reidi, and nucleus (N), proximal centriole (P) positioned in a posterior nuclear this was subsequently con®rmed by Cary (1994). fossa (arrowheads), a distal centriole (D), and adjacent mitochondria (M). Scale bar ˆ 0.5 lm. g Transverse section through the distal Acknowledgements We thank the crews of the research vessel centriole showing the pericentriolar complex of radiating arms ``Edwin Link'' and the JSL II submersible of Harbor Branch (arrowheads). Scale bar ˆ 0.4 lm. h Higher magni®cation of anterior Oceanographic Institution. This work was supported by Grant No. region of spermatozoon showing acrosomal vesicle (A), subacrosomal 9539 from the NOAA National Undersea Research Program material (*) and nucleus (N). Scale bar ˆ 0.5 lm. i Transverse section (NURP) at the University of North Carolina at Wilmington. through sperm midpiece showing distal centriole surrounded by ®ve mitochondria. Scale bar ˆ 1.0 lm. j Posterior region of spermatozoon showing nucleus (N), proximal centriole (P), distal centriole (D), ¯agellum (F), and pericentriolar complex extending to the cytoplasmic References membrane of the cell (arrowheads). Scale bar ˆ 0.4 lm Albertini L (1985) Recherches cytologiques et expe rimentales sur l' have highly elongated acrosomes (Hodgson and Bernard ovogeneÁ se chez la moule (Mytilus edulis L., mollusque bivalve). 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