Dual Symbiosis in a Bathymodiolus sp. Mussel from a Methane Seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA Phylogeny and Distribution of the Symbionts in Gills

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 μM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.     

Diversity, relative abundance and metabolic potential of bacterial endosymbionts in three Bathymodiolus mussel species from cold seeps in the Gulf of Mexico

Cold seeps in the Gulf of Mexico are often dominated by mussels of the genus Bathymodiolus that harbour symbiotic bacteria in their gills. In this study, we analysed symbiont diversity, abundance and metabolic potential in three mussel species from the northern Gulf of Mexico: Bathymodiolus heckerae from the West Florida Escarpment, Bathymodiolus brooksi from Atwater Valley and Alaminos Canyon, and 'Bathymodiolus' childressi, which co-occurs with B. brooksi in Alaminos Canyon. Comparative 16S rRNA sequence analysis confirmed a single methanotroph-related symbiont in 'B.' childressi and a dual symbiosis with a methanotroph- and thiotroph-related symbiont in B. brooksi. A previously unknown diversity of four co-occurring symbionts was discovered in B. heckerae: a methanotroph, two phylogenetically distinct thiotrophs and a methylotroph-related phylotype not previously described from any marine invertebrate symbiosis. A gene characteristic of methane-oxidzing bacteria, pmoA, was identified in all three mussel species confirming the methanotrophic potential of their symbionts. Stable isotope analyses of lipids and whole tissue also confirmed the importance of methanotrophy in the carbon nutrition of all of the mussels. Analyses of absolute and relative symbiont abundance in B. heckerae and B. brooksi using fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization indicated a clear dominance of methanotrophic over thiotrophic symbionts in their gill tissues. A site-dependent variability in total symbiont abundance was observed in B. brooksi, with specimens from Alaminos Canyon harbouring much lower densities than those from Atwater Valley. This shows that symbiont abundance is not species-specific but can vary considerably between populations.     

Unexpected co-occurrence of six bacterial symbionts in the gills of the cold seep mussel Idas sp. (Bivalvia: Mytilidae)

Bathymodioline mussels occur in chemosynthesis-based ecosystems such as cold seeps, hydrothermal vents and organic debris worldwide. Their key adaptation to these environments is their association with bacterial endosymbionts which ensure a chemosynthetic primary production based on the oxidation of reduced compounds such as methane and sulfide. We herein report a multiple symbiosis involving six distinct bacterial 16S rRNA phylotypes, including two belonging to groups not yet reported as symbionts in mytilids, in a small Idas mussel found on carbonate crusts in a cold seep area located north to the Nile deep-sea fan (Eastern Mediterranean). Symbionts co-occur within hosts bacteriocytes based on fluorescence in situ hybridizations, and sequencing of functional genes suggests they have the potential to perform autotrophy, and sulfide and methane oxidation. Previous studies indicated the presence of only one or two symbiont 16S rRNA phylotypes in bathymodioline mussels. Together with the recent discovery of four bacterial symbionts in the large seep species Bathymodiolus heckerae , this study shows that symbiont diversity has probably been underestimated, and questions whether the common ancestor of bathymodioline mussels was associated with multiple bacteria.     

Independent phylogenetic origins of methanotrophic and chemoautotrophic bacterial endosymbioses in marine bivalves.

The discovery of bacterium-bivalve symbioses capable of utilizing methane as a carbon and energy source indicates that the endosymbionts of hydrothermal vent and cold seep bivalves are not restricted to sulfur-oxidizing chemoautotrophic bacteria but also include methanotrophic bacteria. The phylogenetic origin of methanotrophic endosymbionts and their relationship to known symbiotic and free-living bacteria, however, have remained unexplored. In situ localization and phylogenetic analysis of a symbiont 16S rRNA gene cloned from the gills of a recently described deep-sea mussel species demonstrate that this symbiont represents a new taxon which is closely related to free-living, cultivable Type I methanotrophic bacteria. This symbiont is distinct from known chemoautotrophic symbionts. Thus, despite compelling similarities between the symbioses, chemoautotrophic and methanotrophic symbionts of marine bivalves have independent phylogenetic origins.     

Relative abundances of methane- and sulphur-oxidising symbionts in the gills of a cold seep mussel and link to their potential energy sources

Bathymodiolus mussels are key species in many deep-sea chemosynthetic ecosystems. They often harbour two types of endosymbiotic bacteria in their gills, sulphur- and methane oxidisers. These bacteria take up sulphide and methane from the environment and provide energy to their hosts, supporting some of the most prolific ecosystems in the sea. In this study, we tested whether symbiont relative abundances in Bathymodiolus gills reflect variations in the highly spatially dynamic chemical environment of cold seep mussels. Samples of Bathymodiolus aff. boomerang were obtained from two cold seeps of the deep Gulf of Guinea, REGAB (5°47.86S, 9°42.69E, 3170 m depth) and DIAPIR (6°41.58S, 10°20.94E, 2700 m depth). Relative abundances of both symbiont types were measured by means of 3D fluorescence in situ hybridisation and image analysis and compared considering the local sulphide and methane concentrations and fluxes assessed via benthic chamber incubations. Specimens inhabiting areas with highest methane content displayed higher relative abundances of methane oxidisers. The bacterial abundances correlated also with carbon stable isotope signatures in the mussel tissue, suggesting a higher contribution of methane-derived carbon to the biomass of mussels harbouring higher densities of methane-oxidising symbionts. A dynamic adaptation of abundances of methanotrophs and thiotrophs in the gill could be a key factor optimising the energy yield for the symbiotic system and could explain the success of dual symbiotic mussels at many cold seeps and hydrothermal vents of the Atlantic and Gulf of Mexico.     

Environmental acquisition of thiotrophic endosymbionts by deep-sea mussels of the genus bathymodiolus

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally "leaky" system of vertical transmission could not be excluded.     

Sulphur-oxidizing extracellular bacteria in the gills of Mytilidae associated with wood falls

Six morphotypes of small mussels (Bivalvia: Mytilidae) were found attached to naturally sunken wood collected in the Bohol Sea (Philippines). These specimens are related to the large Bathymodiolus mussels that are found worldwide at cold seeps and hydrothermal vents. In these habitats, the mytilids harbour sulphur- and methane-oxidizing endosymbionts in their gills and depend on the energy and carbon provided by the symbionts. In this study, bacteria associated with the gills of wood-associated mussels are characterized using molecular and microscopic techniques. The existence of bacteria in the lateral zone of gill filaments in all specimens is demonstrated. Comparative analyses of 16S rRNA gene and adenosine 5'-phosphosulphate (APS) reductase gene sequences indicate that the bacteria are closely related to sulphur-oxidizing endosymbionts of Bathymodiolus. FISHs using specific probes confirm that sulphur oxidizers are by far the most abundant, if not the only bacteria present. Electron micrographs displayed mostly extracellular bacteria located between microvilli at the apical surface of host gill epithelial cells all along the lateral zone of each gill filament. In some specimens, occasional occurrence of intracellular bacteria with similar morphology was noted. This study provides the first molecular evidence for the presence of possible thiotrophic symbiosis in sunken wood ecosystems. With their epibiotic bacteria, wood-associated mussels display a less integrated type of interaction than described in their seep, vent and whale fall relatives.     

Phylogenetic characterization of endosymbionts of the hydrothermal vent mussel Bathymodiolus azoricus by analysis of the 16S rRNA, pmoL, and cbbA genes

In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine shellfish Bathymodiolus azoricus, total DNA was extracted from the gills. The PCR fragments corresponding to the genes encoding 16S rRNA, ribulose-bisphosphate carboxylase (cbbL), and particulate methane monooxygenase (pmoA) were amplified, cloned, and sequenced. For the 16S rDNA genes, only one phylotype was revealed; it belonged to the cluster of Mytilidae thiotrophic symbionts within the Gammaproteobacteria. For the RuBisCO genes, two phylotypes were found, both belonging to Gammaproteobacteria. One of them was closely related to the previously known mytilid symbiont, the other, to a pogonophore symbiont, presumably a methanotrophic bacterium. One phylotype of particulate methane oxygenase genes was also revealed; this finding indicated the presence of a methanotrophic symbiont. Phylogenetic analysis of the pmoA placed this endosymbiont within the Gammaproteobacteria, in a cluster including the methanotrophic bacterial genus Methylobacter and other methanotrophic Bathymodiolus gill symbionts. These results provide evidence for the existence of two types of endosymbionts (thioautotrophic and methanotrophic) in the gills of B. azoricus and demonstrate that, apart from the phylogenetic analysis of 16S rRNA genes, parallel analysis of functional genes is essential.     

Comparative analysis of symbiont ratios and gene expression in natural populations of two Bathymodiolus mussel species

Bathymodiolus mussels associated with deep-sea hydrothermal vents and cold seeps harbor chemosynthetic endosymbiotic bacteria in bacteriocytes located in the gill epithelium. Two distinct morphotypes of γ-proteobacteria, sulfur- and methane-oxidizing, have been identified and form a dual symbiosis in B. azoricus mussels from the Mid-Atlantic Ridge and in B. aff. boomerang from cold seeps in the Gulf of Guinea. Thiotrophic bacteria (SOX) are capable of fixing CO2 in the presence of sulfide or thiosulfate and methanotrophic bacteria (MOX) use methane both as a carbon and an energy source. In this study we used quantitative real-time PCR to test whether symbiont abundance and gene expression varied between the two mussel species. Results showed that B. azoricus from two hydrothermal sites had similar ratios and gene expression pattern for both symbiont types. In B. aff. boomerang, SOX ratio and ATP sulfurylase gene expression show differences between specimens collected on the different sites. Analysis of symbiont ratios in both species indicated a clear dominance of MOX symbionts in B. aff. boomerang and SOX symbionts in B. azoricus. We also evidenced that the species from the deeper sites (B. aff. boomerang) and mussels collected from sulfur and methane rich habitats showed higher symbiont ratio suggesting that environmental parameters may have significant impacts on the symbiont ratios in Bathymodiolus mussels.     

Microbial diversity in Frenulata (Siboglinidae,Polychaeta) species from mud volcanoes in the Gulf of Cadiz (NE Atlantic)

Frenulates are a group of gutless marine annelids belonging to the Siboglinidae that are nutritionally dependent upon endosymbiotic bacteria. We have characterized the bacteria associated with several frenulate species from mud volcanoes in the Gulf of Cadiz by PCR-DGGE of bacterial 16S rRNA genes, coupled with analysis of 16S rRNA gene libraries. In addition to the primary symbiont, bacterial consortia (microflora) were found in all species analysed. Phylogenetic analyses indicate that the primary symbiont in most cases belongs to the Gammaproteobacteria and were related to thiotrophic and methanotrophic symbionts from other marine invertebrates, whereas members of the microflora were related to multiple bacterial phyla. This is the first molecular evidence of methanotrophic bacteria in at least one frenulate species. In addition, the occurrence of the same bacterial phylotype in different Frenulata species, from different depths and mud volcanoes suggests that there is no selection for specific symbionts and corroborates environmental acquisition as previously proposed for this group of siboglinids.     

A dual symbiosis shared by two mussel species, Bathymodiolus azoricus and Bathymodiolus puteoserpentis (Bivalvia: Mytilidae), from hydrothermal vents along the northern Mid-Atlantic Ridge

Bathymodiolus azoricus and Bathymodiolus puteoserpentis are symbiont-bearing mussels that dominate hydrothermal vent sites along the northern Mid-Atlantic Ridge (MAR). Both species live in symbiosis with two physiologically and phylogenetically distinct Gammaproteobacteria: a sulfur-oxidizing chemoautotroph and a methane-oxidizer. A detailed analysis of mussels collected from four MAR vent sites (Menez Gwen, Lucky Strike, Rainbow, and Logatchev) using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH) showed that the two mussel species share highly similar to identical symbiont phylotypes. FISH observations of symbiont distribution and relative abundances showed no obvious differences between the two host species. In contrast, distinct differences in relative symbiont abundances were observed between mussels from different sites, indicating that vent chemistry may influence the relative abundance of thiotrophs and methanotrophs in these dual symbioses.     

Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences.

The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis [C. R. Woese, Microbiol. Rev. 51: 221-271, 1987]). Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species.     

Genetic connectivity between north and south Mid-Atlantic Ridge chemosynthetic bivalves and their symbionts

Transform faults are geological structures that interrupt the continuity of mid-ocean ridges and can act as dispersal barriers for hydrothermal vent organisms. In the equatorial Atlantic Ocean, it has been hypothesized that long transform faults impede gene flow between the northern and the southern Mid-Atlantic Ridge (MAR) and disconnect a northern from a southern biogeographic province. To test if there is a barrier effect in the equatorial Atlantic, we examined phylogenetic relationships of chemosynthetic bivalves and their bacterial symbionts from the recently discovered southern MAR hydrothermal vents at 5°S and 9°S. We examined Bathymodiolus spp. mussels and Abyssogena southwardae clams using the mitochondrial cytochrome c oxidase subunit I (COI) gene as a phylogenetic marker for the hosts and the bacterial 16S rRNA gene as a marker for the symbionts. Bathymodiolus spp. from the two southern sites were genetically divergent from the northern MAR species B. azoricus and B. puteoserpentis but all four host lineages form a monophyletic group indicating that they radiated after divergence from their northern Atlantic sister group, the B. boomerang species complex. This suggests dispersal of Bathymodiolus species from north to south across the equatorial belt. 16S rRNA genealogies of chemoautotrophic and methanotrophic symbionts of Bathymodiolus spp. were inconsistent and did not match the host COI genealogy indicating disconnected biogeography patterns. The vesicomyid clam Abyssogena southwardae from 5°S shared an identical COI haplotype with A. southwardae from the Logatchev vent field on the northern MAR and their symbionts shared identical 16S phylotypes, suggesting gene flow across the Equator. Our results indicate genetic connectivity between the northern and southern MAR and suggest that a strict dispersal barrier does not exist.     

Environmental Acquisition of Thiotrophic Endosymbionts by Deep-Sea Mussels of the Genus Bathymodiolus

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally “leaky” system of vertical transmission could not be excluded.     

Several deep-sea mussels and their associated symbionts are able to live both on wood and on whale falls

Bathymodiolin mussels occur at hydrothermal vents and cold seeps, where they thrive thanks to symbiotic associations with chemotrophic bacteria. Closely related genera Idas and Adipicola are associated with organic falls, ecosystems that have been suggested as potential evolutionary 'stepping stones' in the colonization of deeper and more sulphide-rich environments. Such a scenario should result from specializations to given environments from species with larger ecological niches. This study provides molecular-based evidence for the existence of two mussel species found both on sunken wood and bones. Each species specifically harbours one bacterial phylotype corresponding to thioautotrophic bacteria related to other bathymodiolin symbionts. Phylogenetic patterns between hosts and symbionts are partially congruent. However, active endocytosis and occurrences of minor symbiont lineages within species which are not their usual host suggest an environmental or horizontal rather than strictly vertical transmission of symbionts. Although the bacteria are close relatives, their localization is intracellular in one mussel species and extracellular in the other, suggesting that habitat choice is independent of the symbiont localization. The variation of bacterial densities in host tissues is related to the substrate on which specimens were sampled and could explain the abilities of host species to adapt to various substrates.     

Phylogenetic characterization of endosymbionts of the hydrothermal vent mussel Bathymodiolus azoricus by analysis of the 16S rRNA, cbbL, and pmoA genes

In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine bivalve Bathymodiolus azoricus, total DNA was extracted from the gills. The PCR fragments corresponding to the genes encoding 16S rRNA, ribulose-bisphosphate carboxylase (cbbL), and particulate methane monooxygenase (pmoA) were amplified, cloned, and sequenced. For the 16S rDNA genes, only one phylotype was revealed; it belonged to the cluster of thiotrophic mytilid’s symbionts within the Gammaproteobacteria. For the RuBisCO genes, two phylotypes were found, both belonging to Gammaproteobacteria. One of them was closely related to the previously known mytilid symbiont, the other, to a pogonophore symbiont, presumably a methanotrophic bacterium. One phylotype of particulate methane oxygenase genes was also revealed; this finding indicated the presence of a methanotrophic symbiont. Phylogenetic analysis of the pmoA placed this endosymbiont within the Gammaproteobacteria, in a cluster including the methanotrophic bacterial genus Methylobacter and other methanotrophic Bathymodiolus gill symbionts. These results provide evidence for the existence of two types of endosymbionts (thioautotrophic and methanotrophic) in the gills of B. azoricus and demonstrate that, apart from the phylogenetic analysis of 16S rRNA genes, parallel analysis of functional genes is essential.     

Bacterial Endosymbioses of Gutless Tube-Dwelling Worms in Nonhydrothermal Vent Habitats

Gutless tube-dwelling worms of pogonophorans (also known as frenulates) and vestimentiferans depend on primary production of endosymbiotic bacteria. The endosymbionts include thiotrophs that oxidize sulfur for autotrophic production and methanotrophs that oxidize and assimilate methane. Although most of the pogonophoran and vestimentiferan tube worms possess single thiotrophic 16S rRNA genes (16S rDNA) related to γ-proteobacteria, some pogonohorans are known to bear single methanotroph species or even dual symbionts of thiotrophs and methanotrophs. The vestimentiferan Lamellibrachia sp. L1 shows symbiotic 16S rDNA sequences of α-, β-, γ-, and ε-proteobacteria, varying among specimens, with RuBisCO form II gene (cbbM) sequences related to β-proteobacteria. An unidentified pogonophoran from the world’s deepest cold seep, 7326-m deep in the Japan Trench, hosts a symbiotic thiotroph based on 16S rDNA with the RuBisCO form I gene (cbbL). In contrast, a shallow-water pogonophoran (Oligobrachia mashikoi) in coastal Japan Sea has a methanotrophic 16S rDNA and thiotrophic cbbL, which may suggest the feature of type X methanotrophs. These observations demonstrate that pogonophoran and vestimentiferan worms have higher plasticity in bacterial symbioses than previously suspected.     

Off-axis symbiosis found: Characterization and biogeography of bacterial symbionts of Bathymodiolus mussels from Lost City hydrothermal vents

Organisms at hydrothermal vents inhabit discontinuous chemical 'islands' along mid-ocean ridges, a scenario that may promote genetic divergence among populations. The 2003 discovery of mussels at the Lost City Hydrothermal Field provided a means of evaluating factors that govern the biogeography of symbiotic bacteria in the deep sea. The unusual chemical composition of vent fluids, the remote location, and paucity of characteristic vent macrofauna at the site, raised the question of whether microbial symbioses existed at the extraordinary Lost City. If so, how did symbiotic bacteria therein relate to those hosted by invertebrates at the closest known hydrothermal vents along the Mid-Atlantic Ridge (MAR)? To answer these questions, we performed microscopic and molecular analyses on the bacteria found within the gill tissue of Bathymodiolus mussels (Mytilidae, Bathymodiolinae) that were discovered at the Lost City. Here we show that Lost City mussels harbour chemoautotrophic and methanotrophic endosymbionts simultaneously. Furthermore, populations of the chemoautotrophic symbionts from the Lost City and two sites along the MAR are genetically distinct from each other, which suggests spatial isolation of bacteria in the deep sea. These findings provide new insights into the processes that drive diversification of bacteria and evolution of symbioses at hydrothermal vents.     

Characterization of symbiont populations in life-history stages of mussels from chemosynthetic environments

The densities of chemoautotrophic and methanotrophic symbiont morphotypes were determined in life- history stages (post-larvae, juveniles, adults) of two species of mussels (Bathymodiolus azoricus and B. heckerae) from deep-sea chemosynthetic environments (the Lucky Strike hydrothermal vent and the Blake Ridge cold seep) in the Atlantic Ocean. Both symbiont morphotypes were observed in all specimens and in the same relative proportions, regardless of life-history stage. The relative abundance of symbiont morphotypes, determined by transmission electron microscopy, was different in the two species: chemoautotrophs were dominant (13:1-18:1) in B. azoricus from the vent site; methanotrophs were dominant (2:1-3:1) in B. heckerae from the seep site. The ratio of CH4:H2S is proposed as a determinant of the relative abundance of symbiont types: where CH4:H2S is less than 1, as at the Lucky Strike site, chemoautotrophic symbionts dominate; where CH4:H2S is greater than 2, as at the seep site, methanotrophs dominate. Organic carbon and nitrogen isotopic compositions of B. azoricus (delta 13C = -30 per thousand; delta 15N = -9 per thousand) and B. heckerae (delta 13C = -56 per thousand; delta 15N = -2 per thousand) varied little among life-history stages and provided no record of a larval diet of photosynthetically derived organic material in the post-larval and juvenile stages.