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 microM. 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Molecular Characterization of a Deep-Sea Methanotrophic Mussel Symbiont that Carries a RuBisCO Gene

In our previous investigation on the genes of 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) in deep-sea chemoautotrophic and methanotrophic endosymbioses, the gene encoding the large subunit of RuBisCO form I (cbbL) had been detected in the gill of a mussel belonging to the genus Bathymodiolus from a western Pacific back-arc hydrothermal vent. This study further examined the symbiont source of the RuBisCO cbbL gene along with the genes of 16S ribosomal RNA (16S rDNA) and particulate methane monooxygenase (EC 1.14.13.25; pmoA) and probed for the presence of the ATP sulfurylase gene (EC 2.7.7.4; sopT). The 16S rDNA sequence analysis indicated that the mussel harbors a monospecific methanotrophic Gammaproteobacterium. This was confirmed by amplification and sequencing of the methanotrophic pmoA, while thiotrophic sopT was not amplified from the same symbiotic genome DNA. Fluorescence in situ hybridization demonstrated simultaneous occurrence of the symbiont-specific 16S rDNA, cbbL and pmoA, but not sopT, in the mussel gill. This is the first molecular and visual evidence for a methanotrophic bacterial endosymbiont that bears the RuBisCO cbbL gene relevant to autotrophic CO₂ fixation.     

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.     

Association of Thioautotrophic Bacteria with Deep-Sea Sponges

We investigated microorganisms associated with a deep-sea sponge, Characella sp. (Pachastrellidae) collected at a hydrothermal vent site (686 m depth) in the Sumisu Caldera, Ogasawara Island chain, Japan, and with two sponges, Pachastrella sp. (Pachastrellidae) and an unidentified Poecilosclerida sponge, collected at an oil seep (572 m depth) in the Gulf of Mexico, using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) directed at bacterial 16S rRNA gene sequences. In the PCR-DGGE profiles, we detected a single clearly dominant band in each of the Characella sp. and the unidentified Poecilosclerida sponge. BLAST search of their sequences showed that they were most similar (>99% identity) to those of the gammaproteobacterial thioautotrophic symbionts of deep-sea bivalves from hydrothermal vents, Bathymodiolus spp. Phylogenetic analysis of the near-full length sequences of the 16S rRNA genes cloned from the unidentified Poecilosclerida sponge and Characella sp. confirmed that they were closely related to thioautotrophic symbionts. Although associations between sponges and methanotrophic bacteria have been reported previously, this is the first report of a possible stable association between sponges and thioautotrophic bacteria.     

Deep-sea mussels from a hybrid zone on the Mid-Atlantic Ridge host genetically indistinguishable symbionts

The composition and diversity of animal microbiomes is shaped by a variety of factors, many of them interacting, such as host traits, the environment, and biogeography. Hybrid zones, in which the ranges of two host species meet and hybrids are found, provide natural experiments for determining the drivers of microbiome communities, but have not been well studied in marine environments. Here, we analysed the composition of the symbiont community in two deep-sea, Bathymodiolus mussel species along their known distribution range at hydrothermal vents on the Mid-Atlantic Ridge, with a focus on the hybrid zone where they interbreed. In-depth metagenomic analyses of the sulphur-oxidising symbionts of 30 mussels from the hybrid zone, at a resolution of single nucleotide polymorphism analyses of ~2500 orthologous genes, revealed that parental and hybrid mussels (F2–F4 generation) have genetically indistinguishable symbionts. While host genetics does not appear to affect symbiont composition in these mussels, redundancy analyses showed that geographic location of the mussels on the Mid-Atlantic Ridge explained most of the symbiont genetic variability compared to the other factors. We hypothesise that geographic structuring of the free-living symbiont population plays a major role in driving the composition of the microbiome in these deep-sea mussels.Subject terms: Metagenomics, Population genetics, Symbiosis, Microbiome     

“Candidatus Thiobios zoothamnicoli,” an Ectosymbiotic Bacterium Covering the Giant Marine Ciliate Zoothamnium niveum

Zoothamnium niveum is a giant, colonial marine ciliate from sulfide-rich habitats obligatorily covered with chemoautotrophic, sulfide-oxidizing bacteria which appear as coccoid rods and rods with a series of intermediate shapes. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the ectosymbiont of Z. niveum belongs to only one pleomorphic phylotype. The Z. niveum ectosymbiont is only moderately related to previously identified groups of thiotrophic symbionts within the Gammaproteobacteria, and shows highest 16S rRNA sequence similarity with the free-living sulfur-oxidizing bacterial strain ODIII6 from shallow-water hydrothermal vents of the Mediterranean Sea (94.5%) and an endosymbiont from a deep-sea hydrothermal vent gastropod of the Indian Ocean Ridge (93.1%). A replacement of this specific ectosymbiont by a variety of other bacteria was observed only for senescent basal parts of the host colonies. The taxonomic status “Candidatus Thiobios zoothamnicoli” is proposed for the ectosymbiont of Z. niveum based on its ultrastructure, its 16S rRNA gene, the intergenic spacer region, and its partial 23S rRNA gene sequence.     

Multiple I-Type Lysozymes in the Hydrothermal Vent Mussel Bathymodiolus azoricus and Their Role in Symbiotic Plasticity

The aim of this study was first to identify lysozymes paralogs in the deep sea mussel Bathymodiolus azoricus then to measure their relative expression or activity in different tissue or conditions. B. azoricus is a bivalve that lives close to hydrothermal chimney in the Mid-Atlantic Ridge (MAR). They harbour in specialized gill cells two types of endosymbiont (gram—bacteria): sulphide oxidizing bacteria (SOX) and methanotrophic bacteria (MOX). This association is thought to be ruled by specific mechanism or actors of regulation to deal with the presence of symbiont but these mechanisms are still poorly understood. Here, we focused on the implication of lysozyme, a bactericidal enzyme, in this endosymbiosis. The relative expression of Ba-lysozymes paralogs and the global anti-microbial activity, were measured in natural population (Lucky Strike -1700m, Mid-Atlantic Ridge), and in in situ experimental conditions. B. azoricus individuals were moved away from the hydrothermal fluid to induce a loss of symbiont. Then after 6 days some mussels were brought back to the mussel bed to induce a re-acquisition of symbiotic bacteria. Results show the presence of 6 paralogs in B. azoricus. In absence of symbionts, 3 paralogs are up-regulated while others are not differentially expressed. Moreover the global activity of lysozyme is increasing with the loss of symbiont. All together these results suggest that lysozyme may play a crucial role in symbiont regulation.     

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.     

Stable-Carbon-Isotope Composition of Fatty Acids in Hydrothermal Vent Mussels Containing Methanotrophic and Thiotrophic Bacterial Endosymbionts

Fatty acid biomarker analysis coupled with gas chromatography-isotope ratio mass spectrometry was used to confirm the presence of methanotrophic and thiotrophic bacterial endosymbionts in the tissues of a hydrothermal vent mussel (Bathymodiolus sp.), collected from the Menez Gwen vent field on the mid-Atlantic ridge. Monounsaturated (n-8) fatty acids, which are diagnostic of methanotrophic bacteria, were detected in all three types of tissues examined (gill, posterior adductor, and mantle), although levels were highest in gill tissues where the bacteria were found. Stable-carbon-isotope compositions (δ-¹³C per mille relative to that of Peedee belemnite) of fatty acids for all three tissues ranged from −24.9 to −34.9‰, which encompasses the range predicted for both thiotroph- and methanotroph-based nutrition. The data suggest that these thio- and methanotrophic bacterial endosymbionts are equally important in the nutrition of the vent mussel at this particular vent site.     

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.     

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.     

"Candidatus Thiobios zoothamnicoli," an ectosymbiotic bacterium covering the giant marine ciliate Zoothamnium niveum

Zoothamnium niveum is a giant, colonial marine ciliate from sulfide-rich habitats obligatorily covered with chemoautotrophic, sulfide-oxidizing bacteria which appear as coccoid rods and rods with a series of intermediate shapes. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the ectosymbiont of Z. niveum belongs to only one pleomorphic phylotype. The Z. niveum ectosymbiont is only moderately related to previously identified groups of thiotrophic symbionts within the Gammaproteobacteria, and shows highest 16S rRNA sequence similarity with the free-living sulfur-oxidizing bacterial strain ODIII6 from shallow-water hydrothermal vents of the Mediterranean Sea (94.5%) and an endosymbiont from a deep-sea hydrothermal vent gastropod of the Indian Ocean Ridge (93.1%). A replacement of this specific ectosymbiont by a variety of other bacteria was observed only for senescent basal parts of the host colonies. The taxonomic status "Candidatus Thiobios zoothamnicoli" is proposed for the ectosymbiont of Z. niveum based on its ultrastructure, its 16S rRNA gene, the intergenic spacer region, and its partial 23S rRNA gene sequence.