Metal interactions between the polychaete Branchipolynoe seepensis and the mussel Bathymodiolus azoricus from Mid-Atlantic-Ridge hydrothermal vent fields.

The vent blood-red commensal polynoid polychaete Branchipolynoe seepensis is commonly found in the pallial cavity of the vent mussel Bathymodiolus azoricus, the dominant bivalve species along the Mid-Atlantic-Ridge (MAR) and is known to be kleptoparasitic. Mussels were collected from three hydrothermal vent fields in the MAR: Menez Gwen (850 m depth, MG2, MG3 and MG4), Lucky Strike (1700 m depth, Montségur-MS and Eiffel Tower-ET) and Rainbow (2300 m depth). Polychaetes were absent in all Menez Gwen vent mussels, while the highest percentage was detected in mussels from Lucky Strike, where more than 70% of the mussels had at least one polychaete in their mantle cavity, followed by Rainbow with 33% of mussels with polychaetes. Total metal concentrations (Ag, Cd, Co, Cu, Fe, Mn, Ni and Zn) were determined in polychaetes whole body and in the mussel tissues (gills, digestive gland and mantle). To understand the possible metal interactions between symbiont and host, the activity of antioxidant defence (catalase (CAT), metallothioneins (MTs)), biotransformation enzymes (glutathione-s-transferases (GST)) activities and lipid peroxidation (LPO) were determined in polychaete whole soft tissues and in mussel tissues (gills, digestive gland and mantle). Metal concentrations in polychaetes and mussels tissues indicated that the accumulation patterns were species specific and also influenced by, and possibly dependent upon, the inter- and intra-variation of vent physico-chemistry between hydrothermal fields. Despite not detecting any strong correlations between metal and enzymes activities in polychaetes and mussels, when in presence of polychaetes, mussels presented less metal concentrations in the gills and digestive gland and lower activity of enzymatic biomarkers. This leads to infer that the polychaete plays a role on the detoxification process, and the interaction between the polychaete mussel association is probably an adaptation to metals concentrations at the vent sites.

Metatranscriptomics profile of the gill microbial community during Bathymodiolus azoricus aquarium acclimatization at atmospheric pressure.

BACKGROUND: The deep-sea mussels Bathymodiolus azoricus (Bivalvia: Mytilidae) are the dominant macrofauna subsisting at the hydrothermal vents site Menez Gwen in the Mid-Atlantic Ridge (MAR). Their adaptive success in such challenging environments is largely due to their gill symbiotic association with chemosynthetic bacteria. We examined the response of vent mussels as they adapt to sea-level environmental conditions, through an assessment of the relative abundance of host-symbiont related RNA transcripts to better understand how the gill microbiome may drive host-symbiont interactions in vent mussels during hypothetical venting inactivity. RESULTS: The metatranscriptome of B. azoricus was sequenced from gill tissues sampled at different time-points during a five-week acclimatization experiment, using Next-Generation-Sequencing. After Illumina sequencing, a total of 181,985,262 paired-end reads of 150 bp were generated with an average of 16,544,115 read per sample. Metatranscriptome analysis confirmed that experimental acclimatization in aquaria accounted for global gill transcript variation. Additionally, the analysis of 16S and 18S rRNA sequences data allowed for a comprehensive characterization of host-symbiont interactions, which included the gradual loss of gill endosymbionts and signaling pathways, associated with stress responses and energy metabolism, under experimental acclimatization. Dominant active transcripts were assigned to the following KEGG categories: "Ribosome", "Oxidative phosphorylation" and "Chaperones and folding catalysts" suggesting specific metabolic responses to physiological adaptations in aquarium environment. CONCLUSIONS: Gill metagenomics analyses highlighted microbial diversity shifts and a clear pattern of varying mRNA transcript abundancies and expression during acclimatization to aquarium conditions which indicate change in bacterial community activity. This approach holds potential for the discovery of new host-symbiont associations, evidencing new functional transcripts and a clearer picture of methane metabolism during loss of endosymbionts. Towards the end of acclimatization, we observed trends in three major functional subsystems, as evidenced by an increment of transcripts related to genetic information processes; the decrease of chaperone and folding catalysts and oxidative phosphorylation transcripts; but no change in transcripts of gluconeogenesis and co-factors-vitamins.

Vibrio diabolicus challenge in Bathymodiolus azoricus populations from Menez Gwen and Lucky Strike hydrothermal vent sites.

Menez Gwen (MG) and Lucky Strike (LS) deep-sea hydrothermal vents are located at 850 m and 1730 m depths respectively and support chemosynthesis-based ecosystems partially differing in heavy metal concentration, temperature range, and faunistic composition. The successfully adapted deep-sea vent mussel Bathymodiolus azoricus is found at both vent locations. In such inhospitable environments survival strategies rely on the establishment of bacteria-vent animal symbiosis In spite of the toxic nature of deep-sea vents, the problem of microbial threat and the need for immunity exist in B. azoricus. This study aims at investigating the immune system of B. azoricus from MG and LS populations by comparing immune gene expressions profiles using the deep-sea vent-related Vibrio diabolicus. Expression of nineteen immune genes was analyzed from gill, digestive gland and mantle tissues upon 3 h, 12 h and 24 h V. diabolicus challenges. Based on quantitative-Polymerase Chain Reaction (qPCR) significant gene expression differences were found among MG and LS populations and challenge times MG mussels revealed that gill and digestive gland gene expression levels were remarkably higher than those from LS mussels. Expression of Carcinolectin, Serpin-2, SRCR, IRGs, RTK, TLR2, NF-κB, HSP70 and Ferritin genes was greater in MG than LS mussels. In contrast, mantle tissue from LS mussels revealed the highest peak of expression at 24 h for most genes analyzed. The activation of immune signaling pathways demonstrated that gene expression profiles are distinct between the two mussel populations. These differences may possibly ensue from intrinsic immune transcriptional activities upon which host responses are modulated in presence of V. diabolicus. mRNA transcript variations were assessed during 24 h acclimatization taking into account the partial depuration to which mussels were subjected to. Additionally, gene expression differences may reflect still accountable effects from the presence of vent remaining microfluidic environments within the tissues analyzed.

Post-capture immune gene expression studies in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus acclimatized to atmospheric pressure.

Deep-sea hydrothermal vents are extreme habitats that are distributed worldwide in association with volcanic and tectonic events, resulting thus in the establishment of particular environmental conditions, in which high pressure, steep temperature gradients, and potentially toxic concentrations of sulfur, methane and heavy metals constitute driving factors for the foundation of chemosynthetic-based ecosystems. Of all the different macroorganisms found at deep-sea hydrothermal vents, the mussel Bathymodiolus azoricus is the most abundant species inhabiting the vent ecosystems from the Mid-Atlantic Ridge (MAR). In the present study, the effect of long term acclimatization at atmospheric pressure on host-symbiotic associations were studied in light of the ensuing physiological adaptations from which the immune and endosymbiont gene expressions were concomitantly quantified by means of real-time PCR. The expression of immune genes at 0 h, 12 h, 24 h, 36 h, 48 h, 72 h, 1 week and 3 weeks post-capture acclimatization was investigated and their profiles compared across the samples tested. The gene signal distribution for host immune and bacterial genes followed phasic changes in gene expression at 24 h, 1 week and 3 weeks acclimatization when compared to other time points tested during this temporal expression study. Analyses of the bacterial gene expression also suggested that both bacterial density and activity could contribute to shaping the intricate association between endosymbionts and host immune genes whose expression patterns seem to be concomitant at 1 week acclimatization. Fluorescence in situ hybridization was used to assess the distribution and prevalence of endosymbiont bacteria within gill tissues confirming the gradual loss of sulfur-oxidizing (SOX) and methane-oxidizing (MOX) bacteria during acclimatization. The present study addresses the deep-sea vent mussel B. azoricus as a model organism to study how acclimatization in aquaria and the prevalence of symbiotic bacteria are driving the expression of host immune genes. Tight associations, unseen thus far, suggest that host immune and bacterial gene expression patterns reflect distinct physiological responses over the course of acclimatization under aquarium conditions.

Comparative study of immune responses in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus and the shallow-water mussel Mytilus galloprovincialis challenged with Vibrio bacteria.

The deep-sea hydrothermal vent mussel Bathymodiolus azoricus and the continental European coast Mytilus galloprovincialis are two bivalves species living in highly distinct marine habitats. Mussels are filter-feeding animals that may accumulate rapidly bacteria from the environment. Contact with microorganism is thus inevitable during feeding processes where gill tissues assume a strategic importance at the interface between the external milieu and the internal body cavities promoting interactions with potential pathogens during normal filtration and a constant challenge to their immune system. In the present study B. azoricus and M. galloprovincialis were exposed to Vibrio alginolyticus, Vibrio anguillarum and Vibrio splendidus suspensions and to a mixture of these Vibrio suspensions, in order to ascertain the expression level of immune genes in gill samples, from both mussel species. The immune gene expressions were analyzed by means of quantitative-Polymerase Chain Reaction (qPCR). The gene expression results revealed that these bivalve species exhibit significant expression differences between 12 h and 24 h post-challenge times, and between the Vibrio strains used. V. splendidus induced the strongest gene expression level in the two bivalve species whereas the NF-κB and Aggrecan were the most significantly differentially expressed between the two mussel species. When comparing exposure times, both B. azoricus and M. galloprovincialis showed similar percentage of up-regulated genes at 12 h while a marked increased of gene expression was observed at 24 h for the majority of the immune genes in M. galloprovincialis. This contrasts with B. azoricus where the majority of the immune genes were down-regulated at 24 h. The 24 h post-challenge gene expression results clearly bring new evidence supporting time-dependent transcriptional activities resembling acute phase-like responses and different immune responses build-up in these two mussel species when challenged with Vibrio bacteria. High Pressure Liquid Chromatography (HPLC)-Electrospray ionization mass spectrometry (ESI-MS/MS) analyses resulted in different peptide sequences from B. azoricus and M. galloprovincialis gill tissues suggesting that naïve animals present differences, at the protein synthesis level, in their natural environment. B. azoricus proteins sequences, mostly of endosymbiont origin, were related to metabolic, energy production, protein synthesis processes and nutritional demands whereas in M. galloprovincialis putative protein functions were assumed to be related to structural and cellular integrity and signaling functions.

Site-related differences in gene expression and bacterial densities in the mussel Bathymodiolus azoricus from the Menez Gwen and Lucky Strike deep-sea hydrothermal vent sites.

The deep-sea hydrothermal vent mussel Bathymodiolus azoricus is a symbiont bearing bivalve that is found in great abundance at the Menez Gwen and Lucky Strike hydrothermal vent sites and in close vicinity of the Azores region near the Mid-Atlantic Ridge (MAR). The physiological relationships that vent mussels have developed with their physical and chemical environments are likely to influence global gene expression profiles providing thus the means to investigate distinct biological markers predicting the origin of Bathymodiolus sp. irrespectively of their geographical localization. Differences found at gene expression levels, and between fluorescence in situ hybridization (FISH) and 16S rRNA amplicon sequencing results provided experimental evidence for the distinction of both Menez Gwen and Lucky Strike vent mussel individuals based on bacterial and vent mussel gene expression signatures and on the constitutive distribution and relative abundance of endosymbiotic bacteria within gill tissues. Our results confirmed the presence of methanotroph endosymbionts in Menez Gwen vent mussels whereas Lucky Strike specimens seem to harbor a different bacterial morphotype when a methane monooxygenase gene specific probe was used. No qualitative differences could be visualized between Menez Gwen and Lucky Strike individuals when tested with a sulfur-oxidizing-related probe. Quantitative PCR (qPCR) studies revealed different gene expression profiles in both Menez Gwen and Lucky Strike mussel gill tissues for the immune genes selected. Genes encoding transcription factors presented noticeably low levels of fold expression whether in Menez Gwen or Lucky Strike animals whereas the genes encoding effector molecules appeared to have higher levels expression in gill tissues from Menez Gwen animals. The peptidoglycan recognition molecule encoding gene, PGRP, presented the highest level of transcriptional activity among the genes analyzed in Menez Gwen mussel gill tissues, seconded by carcinolectin and thus denoting the relevance of immune recognition molecules in early stage of the immune responses onset. Genes regarded as encoding molecules involved in signaling pathways were consistently expressed in both Menez Gwen and Lucky Strike mussel gill tissues. Remarkably, the immunity-related GTPase encoding gene demonstrated, in Lucky Strike samples, the highest level of expression among the signaling molecule encoding genes tested when expressions levels were compared between Menez Gwen and Lucky Strike animals. A differential expression analysis of bacterial genes between Menez Gwen and Lucky Strike mussels indicated a clear expression signature in the latter animal gill tissues. The bacterial community structure ensued from the 16S rRNA sequencing analyses pointed at an unpredicted conservation of endosymbiont bacterial loads between Menez Gwen and Lucky Strike samples. Taken together, our results support the hypothesis that B. azoricus exhibits different transcriptional statuses while living in distinct hydrothermal vent sites may result in distinct gene expressions because of physico-chemical and/or symbiont densities differences.

The influence of nutritional conditions on metal uptake by the mixotrophic dual symbiosis harboring vent mussel Bathymodiolus azoricus.

The vent mussel Bathymodiolus azoricus, host thioautotrophic and methanotrophic bacteria, in their gills and complementary, is able to digest suspended organic matter. But the involvement of nutritional status in metal uptake and storage remains unclear. The influence of B. azoricus physiological condition on its response to the exposure of a mixture of metals in solution is addressed. Mussels from the Menez Gwen field were exposed to 50 µgL(-1) Cd, plus 25 µgL(-1) Cu and 100 µgL(-1) Zn for 24 days. Four conditions were tested: (i) mussels harboring both bacteria but not feed, (ii) harboring only methanotrophic bacteria, (iii) without bacteria but fed during exposure and (iv) without bacteria during starvation. Unexposed mussels under the same conditions were used as controls. Eventual seasonal variations were assessed. Metal levels were quantified in subcellular fractions in gills and digestive gland. Metallothionein levels and condition indices were also quantified. Gill sections were used for fluorescence in situ hybridization (FISH) to assess the temporal distribution of symbiotic associations. Starvation damages metal homeostasis mechanisms and increase the intracellular Zn and MT levels function. There is a clear metallic competition for soluble and insoluble intracellular ligands at each condition. Seasonal variations were observed at metal uptake and storage.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Changes of gill and hemocyte-related bio-indicators during long term maintenance of the vent mussel Bathymodiolus azoricus held in aquaria at atmospheric pressure.

The deep-sea hydrothermal vent mussel Bathymodiolus azoricus has been the subject of several studies aimed at understanding the physiological adaptations that vent animals have developed in order to cope with the particular physical and chemical conditions of hydrothermal environments. In spite of reports describing successful procedures to maintain vent mussels under laboratory conditions at atmospheric pressure, few studies have described the mussel's physiological state after a long period in aquaria. In the present study, we investigate changes in mucocytes and hemocytes in B. azoricus over the course of several months after deep-sea retrieval. The visualization of granules of mucopolysaccharide or glycoprotein was made possible through their inherent auto-fluorescent property and the Alcian blue-Periodic Acid Schiff staining method. The density and distribution of droplets of mucus-like granules was observed at the ventral end of lamellae during acclimatization period. The mucus-like granules were greatly reduced after 3 months and nearly absent after 6 months of aquarium conditions. Additionally, we examined the depletion of endosymbiont bacteria from gill tissues, which typically occurs within a few weeks in sea water under laboratory conditions. The physiological state of B. azoricus after 6 months of acclimatization was also examined by means of phagocytosis assays using hemocytes. Hemocytes from mussels held in aquaria up to 6 months were still capable of phagocytosis but to a lesser extent when compared to the number of ingested yeast particles per phagocytic hemocytes from freshly collected vent mussels. We suggest that the changes in gill mucopolysaccharides and hemocyte glycoproteins, the endosymbiont abundance in gill tissues and phagocytosis are useful health criteria to assess long term maintenance of B. azoricus in aquaria. Furthermore, the laboratory set up to which vent mussels were acclimatized is an applicable system to study physiological reactions such as hemocyte immunocompetence even in the absence of the high hydrostatic pressure found at deep-sea vent sites.

Deep sea immunity: unveiling immune constituents from the hydrothermal vent mussel Bathymodiolus azoricus.

Marine molluscs are subjected to constant microbial threats in their natural habitats. As a result, they represent suitable models for the study of the molecular mechanisms that govern defense reactions in marine organisms. To understand humoral and cellular defense reactions in animals defying extreme physical and chemical conditions we set out to investigate the deep sea hydrothermal vent mussel Bathymodiolus azoricus found in abundance at the Mid-Atlantic Ridge. In the present study, hemocytes were stimulated with compounds of microbial origin and cellular morphological alterations as well as the production of superoxide assessed. Consequently, zymosan, glucan and peptidoglycan were considered as potent inducers of cellular reactions for inducing drastic cell morphology changes and high levels of superoxide production. Furthermore, we have presented for the first time in a deep sea hydrothermal vent animal, molecular evidence of the Rel-homology domain, a conserved motif present in all members of the Rel/nuclear-factor NF-kappaB family. Additionally we have demonstrated the occurrence of the antibacterial gene mytilin in Bathymodiolus azoricus gill tissues. Our results support the premise of an evolutionary conserved innate immune system in Bathymodiolus. Such system is seemingly homologous to that of Insects and other Bivalves and may involve the participation of NF-kappaB transcription factors and antibacterial genes.

Regulation of immunocompetence by different androgen metabolites in a blenny with alternative reproductive tactics.

In Parablennius parvicornis, small reproductive males with relatively low expression of secondary sexual characters (M- morphotype) parasite on the parental investment of the larger nest-holder males which have fully developed secondary sexual characters (M+ morphotype). In comparison with M+ males, M- males have relatively low levels of androgens while having high blood cell percentages of lymphocytes and antigen responsiveness. Here we test the hypothesis that androgens are a causal factor for these differences in immunocompetence between morphotypes. After drawing an initial blood sample, males received a silastic implant containing either oil only (C), or oil with testosterone (T) or 11-ketotestosterone (KT). Males were re-caught 2 weeks later for drawing of the final blood sample. KT but not T induced the development of secondary sexual characters in M- males. M- males treated with KT showed lower swimming activity than the males treated with T or C implants, suggesting that KT also mediates behavioral changes in M- males. As expected, blood cell percentages of lymphocytes, but not of granulocytes, were higher in M- males than in M+ males. Overall, lymphocyte percentages increased in the C group which might have been a response to the surgery/treatment. In concordance with the hypothesis, lymphocyte percentages were suppressed in males treated with T in comparison with controls. However, no significant change was found in KT-treated males. This suggests that androgens modulate central, morphological and immunological traits by partly independent androgen mechanisms in P. parvicornis.

Diversity and seasonal changes in the ichthyofauna of rocky tidal pools from Praia Vermelha and São Roque, Santa Catarina

Este trabalho realizou-se entre Abril de 1998 e Maio de 1999 e visou o estudo das comunidades de peixes em duas poças de maré situadas nas praias de Praia Vermelha (PV) e São Roque (SR), Santa Catarina, Brasil. A poça de PV é maior e mais próxima do sub-mareal. Os exemplares foram coletados mensalmente, durante a baixa-mar, com o anestésico quinaldina. Em PV recolheram-se 1287 peixes pertencentes a 19 espécies enquanto que em SR foram capturados 553 peixes de 11 species. Em ambas as poças as espécies dominantes foram Bathygobius soporator (Gobiidae), Abudefduf saxatilis (Pomacentridae), Parablennius pilicornis (Blenniidae), Labrisomus nuchipinnis (Labrisomidae) e Epinephelus marginatus (Serranidae). A diversidade e o número de peixes foram maiores nos meses de Verão e correspondem a uma época de recrutamento para as espécies A. saxatilis e E. marginatus.

Genetic study of Coris julis (Osteichthyes, Perciformes, Labridae) evolutionary history and dispersal abilities.

Microsatellite markers have been used to study the genetic variability of rainbow wrasse (Coris julis) Mediterranean and Atlantic populations. Differentiation tests failed to reveal any significant genetic differentiation among samples from continental Portugal and the Azores, despite more than 1800 km of geographical separation. Preliminary results tended to indicate a significant genetic differentiation among Atlantic and Mediterranean samples. It also supported the specific status of Cape Verde populations (Coris atlantica). We compare these results with previous mtDNA analyses and propose a biogeographic scenario that could explain our results.