High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Genomics 11 (2010): 559, doi:10.1186/1471-2164-11-559.Bathymodiolus azoricus is a deep-sea hydrothermal vent mussel found in association with large faunal communities living in chemosynthetic environments at the bottom of the sea floor near the Azores Islands. Investigation of the exceptional physiological reactions that vent mussels have adopted in their habitat, including responses to environmental microbes, remains a difficult challenge for deep-sea biologists. In an attempt to reveal genes potentially involved in the deep-sea mussel innate immunity we carried out a high-throughput sequence analysis of freshly collected B. azoricus transcriptome using gills tissues as the primary source of immune transcripts given its strategic role in filtering the surrounding waterborne potentially infectious microorganisms. Additionally, a substantial EST data set was produced and from which a comprehensive collection of genes coding for putative proteins was organized in a dedicated database, "DeepSeaVent" the first deep-sea vent animal transcriptome database based on the 454 pyrosequencing technology. A normalized cDNA library from gills tissue was sequenced in a full 454 GS-FLX run, producing 778,996 sequencing reads. Assembly of the high quality reads resulted in 75,407 contigs of which 3,071 were singletons. A total of 39,425 transcripts were conceptually translated into amino-sequences of which 22,023 matched known proteins in the NCBI non-redundant protein database, 15,839 revealed conserved protein domains through InterPro functional classification and 9,584 were assigned with Gene Ontology terms. Queries conducted within the database enabled the identification of genes putatively involved in immune and inflammatory reactions which had not been previously evidenced in the vent mussel. Their physical counterpart was confirmed by semi-quantitative quantitative Reverse-Transcription-Polymerase Chain Reactions (RT-PCR) and their RNA transcription level by quantitative PCR (qPCR) experiments. We have established the first tissue transcriptional analysis of a deep-sea hydrothermal vent animal and generated a searchable catalog of genes that provides a direct method of identifying and retrieving vast numbers of novel coding sequences which can be applied in gene expression profiling experiments from a non-conventional model organism. This provides the most comprehensive sequence resource for identifying novel genes currently available for a deep-sea vent organism, in particular, genes putatively involved in immune and inflammatory reactions in vent mussels. The characterization of the B. azoricus transcriptome will facilitate research into biological processes underlying physiological adaptations to hydrothermal vent environments and will provide a basis for expanding our understanding of genes putatively involved in adaptations processes during post-capture long term acclimatization experiments, at "sea-level" conditions, using B. azoricus as a model organism.We acknowledge the Portuguese Foundation for Science and Technology, FCT-Lisbon and the Regional Azorean Directorate for Science and Technology, DRCT-Azores, for pluri-annual and programmatic PIDDAC and FEDER funding to IMAR/DOP Research Unit #531 and the Associated Laboratory #9 (ISR-Lisboa); the Luso-American Foundation FLAD (Project L-V- 173/2006); the Biotechnology and Biomedicine Institute of the Azores (IBBA), project M.2.1.2/I/029/2008-BIODEEPSEA and the project n° FCOMP-01-0124- FEDER-007376 (ref: FCT PTDC/MAR/65991/2006-IMUNOVENT; coordinated by RB) under the auspices of the COMPETE program

Bacterial Acquisition in Juveniles of Several Broadcast Spawning Coral Species

Coral animals harbor diverse microorganisms in their tissues, including archaea, bacteria, viruses, and zooxanthellae. The extent to which coral-bacterial associations are specific and the mechanisms for their maintenance across generations in the environment are unknown. The high diversity of bacteria in adult coral colonies has made it challenging to identify species-specific patterns. Localization of bacteria in gametes and larvae of corals presents an opportunity for determining when bacterial-coral associations are initiated and whether they are dynamic throughout early development. This study focuses on the early onset of bacterial associations in the mass spawning corals Montastraea annularis, M. franksi, M. faveolata, Acropora palmata, A. cervicornis, Diploria strigosa, and A. humilis. The presence of bacteria and timing of bacterial colonization was evaluated in gametes, swimming planulae, and newly settled polyps by fluorescence in situ hybridization (FISH) using general eubacterial probes and laser-scanning confocal microscopy. The coral species investigated in this study do not appear to transmit bacteria via their gametes, and bacteria are not detectable in or on the corals until after settlement and metamorphosis. This study suggests that mass-spawning corals do not acquire, or are not colonized by, detectable numbers of bacteria until after larval settlement and development of the juvenile polyp. This timing lays the groundwork for developing and testing new hypotheses regarding general regulatory mechanisms that control bacterial colonization and infection of corals, and how interactions among bacteria and juvenile polyps influence the structure of bacterial assemblages in corals

Diversity and dynamics of bacterial communities in early life stages of the Caribbean coral Porites astreoides

In this study, we examine microbial communities of early developmental stages of the coral Porites astreoides by sequence analysis of cloned 16S rRNA genes, terminal restriction fragment length polymorphism (TRFLP), and fluorescence in situ hybridization (FISH) imaging. Bacteria are associated with the ectoderm layer in newly released planula larvae, in 4-day-old planulae, and on the newly forming mesenteries surrounding developing septa in juvenile polyps after settlement. Roseobacter clade-associated (RCA) bacteria and Marinobacter sp. are consistently detected in specimens of P. astreoides spanning three early developmental stages, two locations in the Caribbean and 3 years of collection. Multi-response permutation procedures analysis on the TRFLP results do not support significant variation in the bacterial communities associated with P. astreoides larvae across collection location, collection year or developmental stage. The results are the first evidence of vertical transmission (from parent to offspring) of bacteria in corals. The results also show that at least two groups of bacterial taxa, the RCA bacteria and Marinobacter, are consistently associated with juvenile P. astreoides against a complex background of microbial associations, indicating that some components of the microbial community are long-term associates of the corals and may impact host health and survival

Speciation in the Deep Sea: Multi-Locus Analysis of Divergence and Gene Flow between Two Hybridizing Species of Hydrothermal Vent Mussels

International audienceBackground: Reconstructing the history of divergence and gene flow between closely-related organisms has long been a difficult task of evolutionary genetics. Recently, new approaches based on the coalescence theory have been developed to test the existence of gene flow during the process of divergence. The deep sea is a motivating place to apply these new approaches. Differentiation by adaptation can be driven by the heterogeneity of the hydrothermal environment while populations should not have been strongly perturbed by climatic oscillations, the main cause of geographic isolation at the surface. Methodology/Principal Finding: Samples of DNA sequences were obtained for seven nuclear loci and a mitochondrial locus in order to conduct a multi-locus analysis of divergence and gene flow between two closely related and hybridizing species of hydrothermal vent mussels, Bathymodiolus azoricus and B. puteoserpentis. The analysis revealed that (i) the two species have started to diverge approximately 0.760 million years ago, (ii) the B. azoricus population size was 2 to 5 time greater than the B. puteoserpentis and the ancestral population and (iii) gene flow between the two species occurred over the complete species range and was mainly asymmetric, at least for the chromosomal regions studied. Conclusions/Significance: A long history of gene flow has been detected between the two Bathymodiolus species. However, it proved very difficult to conclusively distinguish secondary introgression from ongoing parapatric differentiation. As powerful as coalescence approaches could be, we are left by the fact that natural populations often deviates from standard assumptions of the underlying model. A more direct observation of the history of recombination at one of the seven loci studied suggests an initial period of allopatric differentiation during which recombination was blocked between lineages. Even in the deep sea, geographic isolation may well be a crucial promoter of speciation

Synthese sur les adaptations structurales liees a la nutrition des mollusques bivalves des sources hydrothermales profondes

A survey of the results of different ultrastructural and biochemical studies on bivalve molluscs found in dense populations on the sites of deep hydrothermal vents of the East Pacific Ridge shows that these organisms demonstrate specific ecological and biological features. The most remarkable characteristic of all species is a well-developed gill, constituting the main organ in the nutritive processes, with a majority of its cells colonized by abundant chemoautotrophic bacteria. Structural modifications related to the utilization of particulate material, as well as structural adaptations attributed to bacterial colonization of the gill, are different in the Vesicomyidae Calyptogena magnifica and the Mytilidae Bathymodiolus thermophilus and Bathymodiolus sp. These differences in the degrees of adaptation indicate several possible nutritive pathways

Fatty acid characteristics in two symbiotic gastropods from a deep hydrothermal vent of the west Pacific

Two deep-sea gastropods, Ifremeria nautilei and Alviniconcha hessleri, collected on a hydrothermal site of the North Fiji Basin (Southwestern Pacific) were analysed for polar and neutral lipids using gas-liquid chromatography/mass spectrometry. A high level of monounsaturated fatty acids (MUFAs) and a low level of omega 3 series polyunsaturated fatty acids (PUFAs) indicated that nutrition of both gastropods was related to a food web based mainly on bacterial supply. From differences in MUFA distribution between the 2 gastropods, it appeared that most of the energy requirements of A. hessleri were supplied by sulfur-oxidizing endobacteria whereas I. nautilei probably had a mixotrophic diet based on endogenous as well as exogenous bacteria. Given the relatively high level of linoleic acid, which represented from 2 to 8% of the phospholipid fatty acids, hydrothermal gastropods did not appear to be depleted in omega 6 PUFAs. It was hypothesized that they obtain linoleic acid from a pathway different to that in heterotrophic marine molluscs. In contrast to omega 6 PUFAs, both hydrothermal gastropods appeared to be depleted in omega 3 PUFAs, indicating the limited importance of photosynthesis-based food supplies. Some non-methylene-interrupted dienes, particularly 20:2 omega 9,15 which represented from 9 to 18% of the phospholipid fatty acids, may be synthesized by deep-sea symbiotic molluscs in order to restore the depleted omega 3 PUFAs considered as essential for animals. Gills of both gastropods had high levels of neutral lipids, mainly MUFAs that may have originated from degradation of endobacterial phospholipids

Bioaccumulation des métaux chez le mytilidae hydrothermal Bathymodiolus sp. de la ride médio-atlantique

The hydrothermal environment is characterised, among other things, by a high metal concentration, related to the convective sea-water circulation inside the oceanic crust and its interaction with basaltic rocks. The biological communities associated with the hydrothermal system can survive in this toxic environment owing to their ability to regulate their intracellular metal levels by excretion or accumulation of metal ions in non-toxic forms. Various detoxification processes have been previously studied within an hydrothermal organism: immobilisation and precipitation of the metal in lysosomal systems, or binding to specific and soluble ligands such as metallothioneins. The later are heat stable and characterised by a high content of cysteine. The quantification of Fe, Zn, Cu, Mn, Cd, Ag, Pa and Sr in the tissues of the vent mussel Bathymodiolus sp. indicates a high accumulation of these metals in two target organs, the gill and the digestive gland. This accumulation is in relation with high concentrations of metallothioneins, but the subcellular distribution of metals indicates a higher contribution of the insoluble compartment for detoxification processes. These results are compared with the literature data about metal bioaccumlation in Bathymodiolus thermophilus collected at the Galapagos Rift.L'environnement hydrothermal est caractérisé, entre autres, par une concentration élevée en métaux, due à la circulation convective de l'eau de mer dans la croûte océanique et à son interaction avec les roches volcaniques. Les organismes vivant à proximité des sources chaudes s'adaptent à cette richesse en métaux sous forme d'ions ou de sels en suspension, soit en maintenant constante leur teneur interne par des processus d'excrétion, soit en les accumulant sous des formes non toxiques. Différents procédés de détoxication ont été mis en évidence chez certaines espèces hydrothermales : immobilisation et précipitation du métal sous forme insoluble, dans les systèmes lysosomiaux, ou fixation à des ligands spécifiques et solubles dont les plus importants sont les métallothionéines, riches en cystéine et stables à la chaleur. Les teneurs en Fe, Zn, Cu, Mn, Cd, Ag, Ba et Sr dans les tissus de Bathymodiolus sp., espèce dont la physiologie est encore mal connue, montrent une accumulation importante dans les deux organes cibles, la branchie et la glande digestive. Cette accumulation est associée à des teneurs élevées en métallothionéines, mais la répartition des métaux montre une contribution plus importante du compartiment insoluble, dans les mécanismes de détoxication. Ces résultats sont comparés avec les données de la littérature concernant la bioaccumulation des métaux chez Bathymodiolus thermophilus des Galapagos

Rov and submersible surveys on faunal assemblages in a deep-sea canyon (rech Lacaze-duthiers, Western Mediterranean sea)

Fiala-Medioni, A. ... et. al.-- 18 pages, 12 figures, 2 tables52 years after the first explorations in the Lacaze-Duthiers Canyon and the observation of living populations of cold-water corals, recent video surveys using deployments of the ROV "Super-Achille" and the submersible "REMORA" (COMEX), have allowed the discovery of new sites colonized by cold-water coral populations (Lophelia pertusa, Madrepora oculata, Dendrophyllia cornígera and Desmophyllum dianthus). Dense populations were observed, from 250 to 535 m, either on blocks or cliffs. The coral community consisted of a rich fauna of invertebrates including numerous sponges, brachiopods, echinoderms, oysters, ascidians and bryozoans as well as vagile cephalopods and fishes. Among the 51 identified species, 4 species are protected by the French regulation (corals), 13 species are cited in European directives 79/109/EEC (Art 4) and 92/43/EEC (Annex 2), 2 species are cited in the OSPAR convention and 15 are of commercial interest. Direct observations allowed also establishing an increase of anthropogenic wastes mainly related to fishingPeer Reviewe