Jean-Claude Caprais

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

ABSTRACT 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.

Biology and ecology of the “Pompeii worm” (Alvinella pompejana Desbruyères and Laubier), a normal dweller of an extreme deep-sea environment: A synthesis of current knowledge and recent developments

Abstract Alvinella pompejana, the “Pompeii worm” lives on active hydrothermal edifices at deep-sea vents of the East Pacific Rise. The physical and chemical patterns of its microhabitat were determined from temperature probe measurements, temperature time series, and on-board and shore-based chemical analyses based on discrete sampling (pH, H2S, CO2, CH4, S2O2-3, Ca, Mg, Cu, Cd, Zn). The microhabitat is characterised by high temporal and microscale spatial variability, with temperature values in the range of 20°–45°C at the immediate periphery of tubes but reaching higher, still undetermined, values inside the tubes. The difference observed between in vitro temperature limits for the stability of biomolecules and metabolic rates, and suggested in situ conditions seems to indicate a significant protective role of biological interfaces (tubes and cuticle). Temporal instability possibly also plays an important role in the ability for these worms to colonise such an extreme habitat. The functional role of dominant epibiotic bacteria is discussed in the light of recent biochemical and molecular data: the tube-worm–bacteria system can be considered as a symbiotic entity where carbon is probably metabolised and recycled. Sulphide detoxification occurs by oxidation at the gill level and possibly at the intracellular haemoglobin level. Heavy metals, ingested or absorbed, are trapped in spherocrystals and bound to metallothionein-like proteins. Anatomical, physiological and molecular adaptations to hypoxia allow the worm to successfully colonise the chimneys. A. pompejana lives in an ephemeral environment and must reproduce and disperse accordingly. It is a gonochoric species that displays a pseucopulatory behaviour allowing transfer of sperm to female spermathecae, thus avoiding dispersion of the gametes. The size of the oocytes suggests a lecithotrophic or benthic development. The population size structure is polymodal, indicating discontinuous recruitment. Population genetics data indicate the occurrence of a microscale level of population differentiation that does not increase with increasing geographical distances, thus suggesting the occurrence of a metapopulation-like system and/or the possibility that enzyme loci evolve under stabilising selective driving forces inherent to the vents’ highly variable conditions.

Contrasted sulphide chemistries in the environment of 13°N EPR vent fauna

Abstract To improve our understanding of the environmental constraints exerted on vent fauna, we investigated sulphide chemistry in the habitats of Riftia pachyptila and Alvinella pompejana , at the Genesis and Elsa EPR 13°N sites. Temperature, pH and sulphide measurement series were acquired in situ, around the organisms, from the submersible Nautile. Hot fluid samples were also collected to evaluate end-member composition at these sites. Under the assumption of conservative mixing, pH, total sulphide concentration and sulphide speciation gradients in relation to temperature were modelled. From the comparison of measured and calculated concentrations, deviation from conservative behaviour was highlighted for total sulphide versus temperature. While the observed sulphide depletion around tubeworms suggests significant subsurface removal or biological consumption, the apparent sulphide enrichment in the alvinellid environment may reveal either conductive cooling of diffusing fluids or a secondary sulphide source. The calculated sulphide speciation appears to be contrasted at the two sites studied. Because of the low iron content in Genesis fluid, iron sulphide would not constitute a dominant sulphide species and the toxic H 2 S form would be predominant in the mixing zone. By contrast, iron is expected to play a dominant role in sulphide speciation at the Elsa site where the end-member is iron rich. With respect to sulphide, the conditions encountered in the different habitats considered in this study are strongly contrasted. A low fluid flux was observed in the R. pachyptila habitat, contrasting with previous ideas, and suggests that sulphide availability could be a major limiting factor. Particularly, the bioavailable HS − form is expected to vary weakly along the mixing gradient. In contrast, sulphide in the A. pompejana environment is shown to be particularly high, about one order of magnitude higher than observed for other Eastern Pacific alvinellids. At Genesis, because of the acidic pH and low iron conditions encountered, exposure to high levels of toxic sulphide is expected. A. pompejana thus appears to be particularly tolerant to such toxic conditions, but, as previously suggested, less severe conditions may also be found when iron is rich enough in the medium to dominate sulphide chemistry.

Archaeal and anaerobic methane oxidizer communities in the Sonora Margin cold seeps, Guaymas Basin (Gulf of California)

Cold seeps, located along the Sonora Margin transform fault in the Guaymas Basin, were extensively explored during the ‘BIG’ cruise in June 2010. They present a seafloor mosaic pattern consisting of different faunal assemblages and microbial mats. To investigate this mostly unknown cold and hydrocarbon-rich environment, geochemical and microbiological surveys of the sediments underlying two microbial mats and a surrounding macrofaunal habitat were analyzed in detail. The geochemical measurements suggest biogenic methane production and local advective sulfate-rich fluxes in the sediments. The distributions of archaeal communities, particularly those involved in the methane cycle, were investigated at different depths (surface to 18 cm below the sea floor (cmbsf)) using complementary molecular approaches, such as Automated method of Ribosomal Intergenic Spacer Analysis (ARISA), 16S rRNA libraries, fluorescence in situ hybridization and quantitative polymerase chain reaction with new specific primer sets targeting methanogenic and anaerobic methanotrophic lineages. Molecular results indicate that metabolically active archaeal communities were dominated by known clades of anaerobic methane oxidizers (archaeal anaerobic methanotroph (ANME)-1, -2 and -3), including a novel ‘ANME-2c Sonora’ lineage. ANME-2c were found to be dominant, metabolically active and physically associated with syntrophic Bacteria in sulfate-rich shallow sediment layers. In contrast, ANME-1 were more prevalent in the deepest sediment samples and presented a versatile behavior in terms of syntrophic association, depending on the sulfate concentration. ANME-3 were concentrated in small aggregates without bacterial partners in a restricted sediment horizon below the first centimetres. These niche specificities and syntrophic behaviors, depending on biological surface assemblages and environmental availability of electron donors, acceptors and carbon substrates, suggest that ANME could support alternative metabolic pathways than syntrophic anaerobic oxidation of methane.

Presence and diversity of anammox bacteria in cold hydrocarbon-rich seeps and hydrothermal vent sediments of the Guaymas Basin

Hydrothermally active sediments are highly productive, chemosynthetic areas which are characterized by the rapid turnover of particulate organic matter under extreme conditions in which ammonia is liberated. These systems might be suitable habitats for anaerobic ammonium oxidizing (anammox) bacteria but this has not been investigated in detail. Here we report the diversity and abundance of anammox bacteria in sediments that seep cold hydrocarbon-rich fluids and hydrothermal vent areas of the Guaymas Basin in the Cortés Sea using the unique functional anammox marker gene, hydrazine synthase (hzsA). All clones retrieved were closely associated to the “Candidatus Scalindua” genus. Phylogenetic analysis revealed two distinct clusters of hzsA sequences (Ca. Scalindua hzsA cluster I and II). Comparison of individual sequences from both clusters showed that several of these sequences had a similarity as low as 76% on nucleotide level. Based on the analysis of this phylomarker, a very high interspecies diversity within the marine anammox group is apparent. Absolute numbers of anammox bacteria in the sediments samples were determined by amplification of a 257 bp fragment of the hszA gene in a qPCR assay. The results indicate that numbers of anammox bacteria are generally higher in cold hydrocarbon-rich sediments compared to the vent areas and the reference zone. Ladderanes, lipids unique to anammox bacteria were also detected in several of the sediment samples corroborating the hzsA analysis. Due to the high concentrations of reduced sulfur compounds and its potential impact on the cycling of nitrogen we aimed to get an indication about the key players in the oxidation of sulfide in the Guaymas Basin sediments using the alpha subunit of the adenosine-5′-phosphosulfate (APS) reductase (aprA). Amplification of the aprA gene revealed a high number of gammaproteobacterial aprA genes covering the two sulfur-oxidizing bacteria aprA lineages as well as sulfate-reducers.

Microbial communities associated with benthic faunal assemblages at cold seep sediments of the Sonora Margin, Guaymas Basin

The Sonora Margin cold seeps present a seafloor mosaic pattern consisting of different faunal assemblages and microbial mats. To better understand if sedimentary microbial communities reflect this patchy distribution, all major habitats were investigated using four complementary approaches: 16S rRNA 454 pyrosequencing, quantitative polymerase chain reaction, fluorescence in situ hybridization and geochemistry analyses. This study reveals that sediments populated by different surface assemblages show distinct porewater geochemistry features and are associated with distinct microbial communities. In the sediments underlying the microbial mat and the surrounding macrofauna, microbial communities were dominated by anaerobic methane oxidizers (archaeal anaerobic methanotroph ANME) and sulfate-reducing Deltaproteobacteria. In contrast, sediment-associated microbial communities underlying the megafauna habitats (vesicomyids and siboglinids) were characterized by a lower biomass and important proportions of the Marine Benthic Group D (MBG-D), Chloroflexi as well as filamentous Gammaproteobacteria and Deltaproteobacteria. Together, geochemical and microbial surveys indicate that porewater methane concentrations play an important role in the microbial community structure and subsequently in the establishment of the surface colonizers. Furthermore, presence and activity of the surface colonizers influence the underlying microbial communities probably because of modification of energy source availabilities.

Bacterial communities and syntrophic associations involved in anaerobic oxidation of methane process of the Sonora Margin cold seeps, Guaymas Basin

SUMMARY The Sonora Margin cold seeps present on the seafloor a patchiness pattern of white microbial mats surrounded by polychaete and gastropod beds. These surface assemblages are fuelled by abundant organic inputs sedimenting from the water column and upward-flowing seep fluids. Elevated microbial density was observed in the underlying sediments. A previous study on the same samples identified anaerobic oxidation of methane (AOM) as the potential dominant archaeal process in these Sonora Margin sediments, probably catalysed by three clades of archaeal anaerobic methanotrophs (ANME-1, ANME-2 and ANME-3) associated with bacterial syntrophs. In this study, molecular surveys and microscopic observations investigating the diversity of Bacteria involved in AOM process, as well as the environmental parameters affecting the composition and the morphologies of AOM consortia in the Sonora Margin sediments were carried out. Two groups of Bacteria were identified within the AOM consortia, the Desulfosarcina/Desulfococcus SEEP SRB-1a group and a Desulfobulbus-related group. These bacteria showed different niche distributions, association specificities and consortia architectures, depending on sediment surface communities, geochemical parameters and ANME-associated phylogeny. Therefore, the syntrophic AOM process appears to depend on sulphate-reducing bacteria with different ecological niches and/or metabolisms, in a biofilm-like organic matrix.

Analysis of dissolved gases by headspace sampling gas chromatography with column and detector switching. Preliminary results

This preliminary study presents a new method for the determination of dissolved gases in sea-water. After headspace extraction, gaseous compounds are separated by GC on a dual column adsorption system. The individual species are thereafter eluted to three different detectors (thermal conductivity, flame ionization and FPD) using a second switching valve. The analysis is quantitative for methane and carbon dioxide with limits of determination of 0.1 and 50 µmol l–1, respectively, and corresponding standard deviations of 4 and 7%, and has been applied to deep sea hydrothermal samples. Further optimization is necessary to allow the quantitative determination of hydrogen sulfide, nitrogen, oxygen and carbon monoxide.

Structural and functional properties of hemocyanin from Cyanagraea praedator, a deep-sea hydrothermal vent crab.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen‐binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer‐specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P50 varies between 4 and 10 Torr at pH 7.5, 15°C) and a large Bohr coefficient (Δ log P50/ΔpH ∼ −1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5°C and 35°C (ΔH = 1.16 kJ/mol1 5–35°C). Dialysis of native hemolymph elicited a significant increase in Hc‐O2 affinity (ΔP50 = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment. Proteins 2001;45:351–359.

Phylogenetic and Functional Diversity of Microbial Communities Associated with Subsurface Sediments of the Sonora Margin, Guaymas Basin

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.