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Deep ocean hydrothermal vent ecosystems face physical disturbances from naturally occurring volcanic and tectonic activities and are at increasing risk of mineral resource exploitation, raising concerns about the resilience of endemic biological communities. Following destructive events, efficient and rapidly applicable surveys of organisms are required to monitor the state, evolution and a possible return of these ecosystems to their original baseline status. In this study, we explored the environmental DNA (eDNA) approach as a tool (1) to assess biodiversity of benthic communities associated with deep-sea hard substrata and (2) tracked the recolonization dynamics of benthic invertebrate communities living on the Montségur edifice within the Lucky Strike vent field (Mid-Atlantic Ridge), after an induced disturbance that consisted of faunal clearance within experimental quadrats. Hard substratum samples were collected prior to and one year after the disturbance, for eDNA metabarcoding using one marker of the mitochondrial Cytochrome Oxidase I (COI) gene and three markers of the nuclear 18S ribosomal RNA (18S) gene. We also generated a DNA barcoding inventory that consisted of taxa physically collected from Montségur and morphologically identified. This inventory contained amplified barcodes from COI, 18S and the nuclear large subunit ribosomal RNA (28S) gene. The resulting sequence information from the COI and 18S were used for eDNA taxonomic assignment. The eDNA datasets uncovered a high diversity of metazoan OTUs, which included macro- and meiofauna common to Lucky Strike. Baseline data collected at the start of the experiment identified higher OTU richness at sites peripheral to the active edifice, as well as at inactive sites. One year following the initial disturbance, analysis of recolonization data found no statistical difference in presence/absence from baseline communities. The eDNA protocols provide a reproducible strategy to quickly assess biodiversity associated with deep sea hard substratum, enabling comparisons across various habitats. To follow recolonization dynamics at small spatial scales, however, we recommend an approach that uses both molecular and morphological-based traditional methods. Finally, we present original data on the “unseen” diversity of the fauna inhabiting the poorly studied inactive sites, locations that are targeted by commercial mining. Continued monitoring of these sites is currently ongoing and will bring new insight on recovery potential over time, with the ultimate goal of informing conservation and management decisions in relation to the protection of hydrothermal vent ecosystems.

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.

Abstract Proteomic changes in the "gill-bacteria complex" of the hydrothermal vent mussel B. azoricus exposed to cadmium in pressurized chambers ((Incubateurs Pressurises pour l'Observation en Culture d'Animaux Marins Profonds - IPOCAMP) were analyzed and compared with the non-exposed control group. 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) showed that less than 1.5% of the proteome of mussels and symbiotic bacteria were affected by a short-term (24 h) Cd exposure. Twelve proteins of the more abundant differentially expressed proteins of which six were up-regulated and six were down-regulated were excised, digested and identified by mass spectrometry. The identified proteins included structural proteins (actin/actin like proteins), metabolic proteins (calreticulin/calnexin, peptidyl-prolyl cis-trans isomerase, aminotransferase class-III, electron transfer flavoprotein, proteasome, alpha-subunit and carbonic anhydrase) and stress response proteins (chaperone protein htpG, selenium-binding protein and glutathione transferases). All differently expressed proteins are tightly connected to Cd exposure and are affected by oxidative stress. It was also demonstrated that B. azoricus was well adapted to Cd contamination therefore B. azoricus from hydrothermal vent areas may be considered a good bioindicator. Graphical abstract fx1 Highlights • Proteomics analysis of B. azoricus exposed to Cd was done by 2D-DIGE. • Identified proteins include structural, metabolic and stress-related proteins. • Cd mainly exerts toxicity in B. azoricus through oxidative stress induction and sulfhydryl-group binding. • One identified expressed protein was from the symbiotic bacteria, suggesting an important role of these symbionts in the resistance of B. azoricus. [ABSTRACT FROM AUTHOR]

In the 40 years since the discovery of the rich faunal community around hydrothermal vents, many studies have clearly shown that environmental conditions have a strong influence on species distribution in these habitats. Nevertheless, the mechanisms that determine the spatial and temporal dynamics of species’ responses to vent conditions remain elusive. Metabolic studies to assess faunal interactions with vent fluid are particularly difficult to perform in the deep sea and are generally executed in isolation ex situ . Available data mainly concern foundation species, which visually dominate these ecosystems. This work uses a modelling approach to integrate biotic and abiotic data that have been acquired through the years on Eiffel Tower, a large sulphide edifice located on the Lucky Strike vent field on the Mid-Atlantic Ridge, and particularly on its dominant species, Bathymodiolus azoricus . A carbon-flux model was built using seven state variables: the biomass of mussels and their associated thiotrophic (SOX) and methanotrophic (MOX) symbionts and the ambient concentrations of oxygen, dihydrogen sulphide, methane and (particulate and dissolved) organic carbon. Temperature of the surrounding water and mussel density were the forcing variables in the system. Results showed no statistically significant differences between predicted and observed mussel biomass and estimates of energy partitioning within the mussel were in the range of available data. Metabolic rates were generally rather low and greatly reduced by a temperature effect in the coldest samples. These low metabolic rates imply a long lifespan for B. azoricus . Simulations suggest that they would strongly hinder re-establishment and resilience of mussel biomass. However, because symbionts respond quickly to changes in vent fluid, mussels would be able to buffer strong variations in the hydrothermal fluid supply. The model showed that if mussels fed indifferently on both types of symbionts, coexistence of MOX and SOX cannot be reached, thereby likely favouring hypotheses of competition for space inside the mussel gills and/or a differential use of the production of each symbiont. Model predictions are highly dependent on current knowledge, and the results presented here highlight the need for more quantitative data on the biology of B. azoricus across different size classes, on its interactions with symbionts, and in varying environmental concentrations in its substrates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Over the past years, several studies have been dedicated to understanding the physiological ability of the vent mussel Bathymodiolus azoricus to overcome the high metal concentrations present in their surrounding hydrothermal environment. Potential deep-sea mining activities at Azores Triple junction hydrothermal vent deposits would inevitably lead to the emergence of new fluid sources close to mussel beds, with consequent emission of high metal concentrations and potential resolubilization of Cu from minerals formed during the active phase of the vent field. Copper is an essential metal playing a key role in the activation of metalloenzymes and metalloproteins responsible for important cellular metabolic processes and tissue homeostasis. However, excessive intracellular amounts of reactive Cu ions may cause irreversible damages triggering possible cell apoptosis. In the present study, B. azoricus was exposed to increasing concentrations of Cu for 96 h in conditions of temperature and hydrostatic pressure similar to those experienced at the Lucky Strike hydrothermal vent field. Specimens were kept in 1L flasks, exposed to four Cu concentrations: 0 μg/L (control), 300, 800 and 1600 μg/L and pressurized to 1750 bar. We addressed the question of how increased Cu concentration would affect the function of antioxidant defense proteins and expression of antioxidant and immune-related genes in B . azoricus . Both antioxidant enzymatic activities and gene expression were examined in gills, mantle and digestive gland tissues of exposed vent mussels. Our study reveals that stressful short-term Cu exposure has a strong effect on molecular metabolism of the hydrothermal vent mussel, especially in gill tissue. Initially, both the stress caused by unpressurization or by Cu exposure was associated with high antioxidant enzyme activities and tissue-specific transcriptional up-regulation. However, mussels exposed to increased Cu concentrations showed both antioxidant and immune-related gene suppression. Under a mining activity scenario, the release of an excess of dissolved Cu to the vent environment may cause serious changes in cellular defense mechanisms of B. azoricus . This outcome, while adding to our knowledge of Cu toxicity, highlights the potentially deleterious impacts of mining activities on the physiology of deep-sea organisms. [ABSTRACT FROM AUTHOR]

In community ecology, niche analysis is a classic tool for investigating species׳ distribution and dynamics. Components of a species׳ niche include biotic and abiotic factors. In the hydrothermal vent ecosystem, although composition and temporal variation have been investigated since these deep-sea habitats were discovered nearly 40 years ago, the roles and the factors behind the success of the dominant species of these ecosystems have yet to be fully elucidated. In the Lucky Strike vent field on the Mid-Atlantic Ridge (MAR), the dominant species is the mussel Bathymodiolus azoricus. Data on this species and its associated community were collected during four oceanographic cruises on the Eiffel Tower edifice and integrated in a novel statistical framework for niche analysis. We assessed the thermal range, density, biomass and niche similarities of B. azoricus and its associated fauna. Habitat similarities grouped mussels into three size categories: mussels with lengths ranging from 0.5 to 1.5 cm, from 1.5 to 6 cm, and mussels longer than 6 cm. These size categories were consistent with those found in previous studies based on video imagery. The three size categories featured different associated fauna. The thermal range of mussels was shown to change with organism size, with intermediate sizes having a broader thermal niche than small or large mussels. Temperature maxima seem to drive their distribution along the mixing gradient between warm hydrothermal fluids and cold seawater. B. azoricus constitutes nearly 90% of the biomass (in g dry weight /m 2 ) of the ecosystem. Mean individual weights were calculated for 39 of the 79 known taxa on Eiffel Tower and thermal ranges were obtained for all the inventoried species of this edifice. The analysis showed that temperature is a suitable variable to describe density variations among samples for 71 taxa. However, thermal conditions do not suffice to explain biomass variability. Our results provide valuable insights into mussel ecology, biotic interactions and the role of B. azoricus in the community. [ABSTRACT FROM AUTHOR]

Deep-sea environments are largely unexplored habitats where a surprising number of species may be found in large communities, thriving regardless of the darkness, extreme cold, and high pressure. Their unique geochemical features result in reducing environments rich in methane and sulfides, sustaining complex chemosynthetic ecosystems that represent one of the most surprising findings in oceans in the last 40 years. The deep-sea Lucky Strike hydrothermal vent field, located in the Mid Atlantic Ridge, is home to large vent mussel communities where Bathymodiolus azoricus represents the dominant faunal biomass, owing its survival to symbiotic associations with methylotrophic or methanotrophic and thiotrophic bacteria. The recent transcriptome sequencing and analysis of gill tissues from B. azoricus revealed a number of genes of bacterial origin, hereby analyzed to provide a functional insight into the gill microbial community. The transcripts supported a metabolically active microbiome and a variety of mechanisms and pathways, evidencing also the sulfur and methane metabolisms. Taxonomic affiliation of transcripts and 16S rRNA community profiling revealed a microbial community dominated by thiotrophic and methanotrophic endosymbionts of B. azoricus and the presence of a Sulfurovum-like epsilonbacterium.

Deep sea hydrothermal Bathymodiolus azoricus mussels from Portuguese EEZ Menez Gwen hydrothermal field possess the remarkable ability to overcome decompression and survive successfully at atmospheric pressure conditions. We investigated the potential use of antioxidant defense enzymes in mussel B. azoricus as biomarkers of oxidative stress induced by long term acclimatization to atmospheric pressure conditions. Mussels collected at Menez Gwen hydrothermal field were acclimatized for two weeks in three distinct conditions suitable of promoting physiological stress, (i) in plain seawater for concomitant endosymbiont bacteria loss, (ii) in plain seawater under metal iron exposure, (iii) constant bubbling methane and pumped sulfide for endosymbiont bacteria survival. The enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and iron storage proteins in addition to electrophoretic profiles were examined in vent mussel gills and digestive gland. Gills showed approximately 3 times more SOD specific activity than digestive glands. On the other hand, digestive glands showed approximately 6 times more CAT specific activity than gills. Iron storage proteins were identified in gill extracts from all experimental conditions mussels. However, in digestive gland extracts only fresh collected mussels and after 2 weeks in FeSO 4 showed the presence of iron storage proteins. The differences between SOD, CAT specific activities and the presence of iron storage proteins in the examined tissues reflect dissimilar metabolic and antioxidant activities, as a result of tissue specificities and acclimatization conditions influences on the organism. [ABSTRACT FROM AUTHOR]

Hydrothermal vent mussels belonging to the genus Bathymodiolus dominate communities at hydrothermal sites of the Mid-Atlantic Ridge. The mussel Bathymodiolus azoricus harbors thiotrophic and methanotrophic symbiotic bacteria in its gills and evolves in naturally highly metal contaminated environments. In the context of investigations on metal tolerance/effect in B. azoricus, we focused our work on the short-term adaptive response (15 days) of mussels to different metals exposure at a molecular level using metal concentrations chosen to mimic natural situations at three vents sites. The expression of a set of 38 genes involved in different steps of the metal uptake, detoxication and various metabolisms was analysed by qPCR. Mussels were also genotyped at 10 enzyme loci to explore the relationships among natural genetic variation and gene expression. Relation between symbiont content (both sulfur-oxidizing and methanogen bacteria) and gene expression was also analysed. Our study demonstrated the influence of metal cocktail composition and time exposure on the transcriptome regulation with a specific pattern of regulation observed for the three metal cocktail tested. We also evidenced the significant influence of some specific Pgm genotype on the global gene expression in our experimental populations and a general trend of a higher gene expression in individuals carrying a high symbiont content. [ABSTRACT FROM AUTHOR]

• This paper presents the longest daily time-series of plurisdisciplinary data within a vent assemblage. • The diffuse-flow habitat was highly stable over 7 years with small-scale variability mainly related to tidal periodicity and localised temperature anomalies. • The Bathymodiolus azoricus mussel assemblage and zoanthid patches were remarkably stable. • Microbial mats displayed heterogeneous spatio-temporal dynamics. • Our results question the assumption that vent assemblages are ephemeral and highly dynamics. Understanding scales and drivers of ecological variability is essential to a full understanding of ecosystem functioning. At remote deep-sea hydrothermal vents, infra-annual dynamics remain poorly described. This study aims to characterise the factors that drive the dynamics of a vent faunal assemblage dominated by Bathymodiolus azoricus mussels from infra-daily to monthly time steps. We analysed a 7-year time series of images and environmental data collected at a depth of 1695 m at the base of the active Eiffel Tower edifice in the Lucky Strike vent field (Mid-Atlantic Ridge). Using images acquired by the TEMPO ecological module connected to the EMSO-Azores observatory, we assessed the dynamics of key species inhabiting the faunal assemblage in relation to changes in environmental conditions monitored daily. Our results show that habitat conditions were generally stable over the 7-year period, with small-scale variability related to tidal periodicity and local temperature anomalies. Likewise, the mussel and zoanthid assemblages exhibited remarkable stability. Changes in fluid exposure and substratum instability induced decimetre-scale movements of the mussel assemblage. Microbial mats displayed infra-annual changes characterised by aperiodic growth and decline. Their development patterns could not be entirely attributed to environmental conditions, because other factors, including biotic interactions, appeared to be involved. The crab population preferentially occupied the mussel habitat, but no predation was observed. Scales of variation and driving factors were compared with those governing intertidal zones. The outcomes question the assumption that vent fauna experience extreme and highly variable conditions. On the MAR, mussel assemblages appear to experience relatively stable and mild environmental conditions compared with their coastal counterparts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The Menez Gwen hydrothermal vents, located on the flanks of a small young volcanic structure in the axial valley of the Menez Gwen seamount, are the shallowest known vent systems on the Mid-Atlantic Ridge that host chemosynthetic communities. Although visited several times by research cruises, very few images have been published of the active sites, and their spatial dimensions and morphologies remain difficult to comprehend. We visited the vents on the eastern flank of the small Menez Gwen volcano during cruises with RV Poseidon (POS402, 2010) and RV Meteor (M82/3, 2010), and used new bathymetry and imagery data to provide first detailed information on the extents, surface morphologies, spatial patterns of the hydrothermal discharge and the distribution of dominant megafauna of five active sites. The investigated sites were mostly covered by soft sediments and abundant white precipitates, and bordered by basaltic pillows. The hydrothermally-influenced areas of the sites ranged from 59 to 200m2. Geo-referenced photomosaics and video data revealed that the symbiotic mussel Bathymodiolus azoricus was the dominant species and present at all sites. Using literature data on average body sizes and biomasses of Menez Gwen B. azoricus, we estimated that the B. azoricus populations inhabiting the eastern flank sites of the small volcano range between 28,640 and 50,120 individuals with a total biomass of 50 to 380kg wet weight. Based on modeled rates of chemical consumption by the symbionts, the annual methane and sulfide consumption by B. azoricus could reach 1760molCH4 yr−1 and 11,060molH2Syr−1. We propose that the chemical consumption by B. azoricus over at the Menez Gwen sites is low compared to the natural release of methane and sulfide via venting fluids. [Copyright &y& Elsevier]

Abstract: Deep-sea hydrothermal mussels Bathymodiolus azoricus live in the mixing zone where hydrothermal fluid mixes with bottom seawater, creating large gradients in the environmental conditions and are one of the most studied hydrothermal species as a model of adaptation to extreme conditions. Thiol proteins, i.e. proteins containing a thiol or sulfhydryl group (major roles in intracellular stress defense against reactive oxygen species (ROS) and are especially susceptible to oxidation. However, they are not particularly abundant, representing a small percentage of proteins in the total proteome and therefore are difficult to study by proteomic approaches. Activated thiol sepharose (ATS) was used for the rapid and quantitative selection of proteins comprising thiol- or disulfide-containing subproteomes. This study aims to isolate thiol-containing proteins from the gills of B. azoricus collected in distinct hydrothermal vents and to study the thiol-containing subproteome as a function of site-specific susceptibility to ROS. Results show that ATS is a powerful tool to isolate the thiol-containing sub-proteome and differently-expressed protein spots showed significant differences among the three vent sites, supporting previous findings that specific environmental conditions are crucial for ROS formation and that B. azoricus have different susceptibilities to oxidative stress depending on the vent site they inhabit. [Copyright &y& Elsevier]

Deep-sea environments are largely unexplored habitats where a surprising number of species may be found in large communities, thriving regardless of the darkness, extreme cold, and high pressure. Their unique geochemical features result in reducing environments rich in methane and sulfides, sustaining complex chemosynthetic ecosystems that represent one of the most surprising findings in oceans in the last 40 years. The deep-sea Lucky Strike hydrothermal vent field, located in the Mid Atlantic Ridge, is home to large vent mussel communities where Bathymodiolus azoricus represents the dominant faunal biomass, owing its survival to symbiotic associations with methylotrophic or methanotrophic and thiotrophic bacteria. The recent transcriptome sequencing and analysis of gill tissues from B. azoricus revealed a number of genes of bacterial origin, hereby analyzed to provide a functional insight into the gill microbial community. The transcripts supported a metabolically active microbiome and a variety of mechanisms and pathways, evidencing also the sulfur and methane metabolisms. Taxonomic affiliation of transcripts and 16S rRNA community profiling revealed a microbial community dominated by thiotrophic and methanotrophic endosymbionts of B. azoricus and the presence of a Sulfurovum-like epsilonbacterium. [ABSTRACT FROM AUTHOR]

Abstract: Hydrothermal vent mussels Bathymodiolus azoricus are naturally exposed to toxic chemical species originated directly from vent chimneys. The amount of toxic elements varies significantly among vent sites along the Mid-Atlantic Ridge and B. azoricus must be able to adapt to changes in hydrothermal fluid composition, temperature and pressure. The aim of this work was to study changes in the proteome in the “gill-bacteria complex” of mussels B. azoricus from three hydrothermal vent sites with distinct environmental characteristics using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). Results showed that 31 proteins had different expression profiles among vent sites and both cluster and principal component analysis confirm a clear separation of mussels between sites. This suggests the existence of specific parameters grouping individuals from the same hydrothermal site. Protein spots of the more abundant differentially expressed proteins were excised, digested with trypsin and identified by mass spectrometry. All identified proteins (actin, ubiquinone, S-adenosylhomocysteine hydrolase, cysteine peptidases, chaperonin and catalase) have been related previously with oxidative stress conditions and are known to be affected by ROS inducing stressors, including metals. Results point out to specific adaptations at the proteome level of B. azoricus depending on the level of toxicants present in their environment. [Copyright &y& Elsevier]

Abstract: The turbulent mixing of hydrothermal hot fluid with cold seawater creates large chemical gradients at a small spatial scale that may induce variable physiological and biochemical adaptations within the vent fauna. The adaptation to such a variable environment by the vent mussel Bathymodiolus azoricus relies on a dual symbiosis hosted in the gills, and digestion of particulate organic matter. The surrounding environment not only provides the necessary energy sources and suspended organic particles for the vent mussel nutrition, but also potentially toxic compounds such as metals. Our main goal was to see if there is a relation between metal accumulation in mussel organs and the chemical characteristics of their close environment. Mussels were collected at six locations in a cold part of the Eiffel Tower fluid-seawater mixing zone, characterized by distinct chemical compositions. Metals (Cd, Cu, Fe and Zn) and metallothioneins were quantified in the gills and digestive gland. The physiological condition of the sampled mussels was also evaluated using tissues and gill indices. Our study indicates that the accumulation of metals in B. azoricus is related to their spatial distribution and linked to fine scale environmental conditions that influence the physiological status of the organism. [Copyright &y& Elsevier]

Bettencourt, R., Costa, V., Laranjo, M., Rosa, D., Pires, L., Colaço, A., Lopes, H., and Serrão Santos, R. 2011. Out of the deep sea into a land-based aquarium environment: investigating physiological adaptations in the hydrothermal vent mussel Bathymodiolus azoricus. – ICES Journal of Marine Science, 68: 357–364.Deep-sea hydrothermal vents are considered to be some of the most extreme environments in the world, yet the animals dwelling around the vent sites exhibit high productivity and must therefore deal with unusual levels of heavy metals, pH, temperature, CO2, and sulphides, in addition to environmental microbes. In an attempt to understand the physiological reactions of animals able to endure these extreme conditions, adaptation processes in the mussel Bathymodiolus azoricus maintained for long periods under laboratory conditions were investigated. Even in the absence of the characteristic high hydrostatic pressure found at deep-sea vent sites and without methane and/or sulphide supplementation, vent mussels seem to survive well in aquarium conditions. Therefore, the maintenance of live vent mussels in our laboratory is a key factor in gaining insights into their physiology, as well as into the study of evolutionary conserved molecules commonly found in other marine bivalves. With the aim of finding distinct genetic signatures in the expression of genes such as the metal-binding protein metallothionein (MT), the present work centred on cellular and humoral mechanisms in animals acclimatized to “sea-level” conditions. In addition, we also conducted experiments under hydrostatic pressure, using the hyperbaric chamber IPOCAMP to establish an in vitro experimental system in which the expression of genes that typically respond to heavy metal contaminants and oxidative stress could be studied under controlled hyperbaric pressure. We also analysed the occurrence of glycosylation in mantle and gill tissues from mussels subjected to elevated hyperbaric pressure, as well as the variation in haemocyte total counts as a result of increased pressure. Our results suggest that even after prolonged aquarium maintenance at atmospheric pressure, mussels were still able to induce the MT gene, whether or not they had been subjected to repressurization in the IPOCAMP chamber. Taken together, our results suggest that B. azoricus can be used as a model species and is particularly useful for the assessment of expression levels of critical genes, such as MT, in response to experimentally induced hydrostatic pressure. [ABSTRACT FROM PUBLISHER]

Background: The deep-sea hydrothermal vent mussel Bathymodiolus azoricus harbors thiotrophic and methanotrophic symbiotic bacteria in its gills. While the symbiotic relationship between this hydrothermal mussel and these chemoautotrophic bacteria has been described, the molecular processes involved in the cross-talking between symbionts and host, in the maintenance of the symbiois, in the influence of environmental parameters on gene expression, and in transcriptome variation across individuals remain poorly understood. In an attempt to understand how, and to what extent, this double symbiosis affects host gene expression, we used a transcriptomic approach to identify genes potentially regulated by symbiont characteristics, environmental conditions or both. This study was done on mussels from two contrasting populations. Results: Subtractive libraries allowed the identification of about 1000 genes putatively regulated by symbiosis and/ or environmental factors. Microarray analysis showed that 120 genes (3.5% of all genes) were differentially expressed between the Menez Gwen (MG) and Rainbow (Rb) vent fields. The total number of regulated genes in mussels harboring a high versus a low symbiont content did not differ significantly. With regard to the impact of symbiont content, only 1% of all genes were regulated by thiotrophic (SOX) and methanotrophic (MOX) bacteria content in MG mussels whereas 5.6% were regulated in mussels collected at Rb. MOX symbionts also impacted a higher proportion of genes than SOX in both vent fields. When host transcriptome expression was analyzed with respect to symbiont gene expression, it was related to symbiont quantity in each field. Conclusions: Our study has produced a preliminary description of a transcriptomic response in a hydrothermal vent mussel host of both thiotrophic and methanotrophic symbiotic bacteria. This model can help to identify genes involved in the maintenance of symbiosis or regulated by environmental parameters. Our results provide evidence of symbiont effect on transcriptome regulation, with differences related to type of symbiont, even though the relative percentage of genes involved remains limited. Differences observed between the vent site indicate that environment strongly influences transcriptome regulation and impacts both activity and relative abundance of each symbiont. Among all these genes, those participating in recognition, the immune system, oxidative stress, and energy metabolism constitute new promising targets for extended studies on symbiosis and the effect of environmental parameters on the symbiotic relationships in B. azoricus. [ABSTRACT FROM AUTHOR]

Abstract: Bathymodiolus azoricus, thriving at Mid-Atlantic Ridge deep vents, benefits from a symbiosis with methane- and sulphide-oxidising (MOX and SOX) bacteria, and feeds on particulate and dissolved organic matter. To investigate the temporal evolution in their nutrition adult mussels were collected from one location at the Menez Gwen vent site (817 m depth) on four occasions between 2006 and 2007 and studied using different techniques, including stable isotope analyses and FISH. Gill and mantle tissues δ13C and δ15N signatures varied by 2–3‰ during the year and these variations were linked to fluctuations in tissue condition index, C and N contents and SOX/MOX volume ratios as quantified by 3D-FISH. October and January mussels presented a particularly poor condition, possibly related with the prolonged summer period of low sea-surface primary production and/or with the stress of the transplant to acoustically retrievable cages for the October mussels, and with their reproductive state in January mussels, since they were spawning. Our results point to the possibility that May mussels benefited from a pulse of sinking sea-surface plankton material. Results underline the dependency of stable isotopic signatures on the physiological state of the mussel at the time of collection, and on the type of tissue analyzed. [ABSTRACT FROM AUTHOR]

The mussel Bathymodiolus azoricus is one of the most abundant species in the Mid-Atlantic Ridge hydrothermal vents and is continually exposed to the high-temperature venting fluids containing high metal concentrations and enriched in sulphides and methane, which constitute a potential toxic environment for marine species. The aim of this study was to assess the effects of a sub-lethal Cd concentration on the antioxidant defence system of this mussel. B. azoricus were collected at Menez Gwen vent site (37°51''N, 32°31''W) and exposed to Cd (50μgl−1) during 24 days, followed by a depuration period of six days. A battery of stress related biomarkers including antioxidant enzymes (superoxide dismutase–SOD, catalase–CAT; glutathione peroxidases–GPx), metallothioneins (MT), lipid peroxidation (LPO) and total oxyradical scavenging capacity (TOSC) were measured in the gills and mantle of B. azoricus. Cd was accumulated linearly during the exposure period in both tissues and no significant elimination occurred after the 6 days of depuration. Antioxidant enzymes activities were significantly higher in the gills. Cyt-SOD, T-GPx and Se-GPx were induced during the experiment but this was also observed in control organisms. Mit-SOD and CAT activities remained relatively unchanged. MT levels increased linearly in the gills of exposed mussels in the first 18 days of exposure. No significant differences were observed between LPO levels of control and exposed mussels. TOSC levels remained unchanged in control and exposed mussels. This suggests that although Cd is being accumulated in the tissues of exposed mussels, MT defence system is enough to detoxify the effect of Cd accumulated in the tissues. Furthermore, other factors besides the presence of Cd are influencing the antioxidant defence system in B. azoricus. [Copyright &y& Elsevier]

The article presents a study which investigated the assimilation of particulate matter in mussels Bathymodiolus azoricus at hydrothermal vents of the Azores Triple Junction on the Mid-Atlantic Ridge. It is stated that there is a contribution of photosynthetically produced organic matter to the diet of the mussels and that active suspension-feeding on particulates and dissolved organic matter could contribute to amino acid budget of the mussels. The authors discussed the organic compounds that originate from a mixture of biogenic and abiogenic processes taken from hydrothermal fluids.

Specimens of the hydrothermal mussel Bathymodiolus azoricus collected in Menez Gwen hydrothermal vent field (NE Atlantic) during 2002–2003 were examined for feeding patterns variations through three seasons. The fatty acid profile and lipid classes of the mussels were studied, together with the MODIS/AQUA-derived near-surface chlorophyll a to test the hypothesis that surface productivity might be related to the feeding patterns of this species. The lipid levels showed pronounced seasonal fluctuations with the highest values occurring in January and August. Seasonal variations in lipid classes and fatty acid composition of neutral and polar lipids in the mussels are presented. Differences in the fatty acid profile of lipid classes in different seasons suggest that the higher energy requirements in summer and winter were supplied by bacterial biomarkers ω7 MUFA (monounsaturated fatty acids), whereas ω6 PUFA (polyunsaturated fatty acids) and NMI (non-methylene-interrupted) fatty acids predominated during the spring. The MODIS/AQUA data show marked seasonal variability and an anomalous peak during January of 2003, although this cannot be directly linked to lipid composition variation. [Copyright &y& Elsevier]

Abstract: The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca2+ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components. [Copyright &y& Elsevier]

Abstract: Bathymodiolus azoricus is a mussel from vent fields in the south-west of the Azores Triple Junction (Mid-Atlantic Ridge-MAR). Experimental evidence indicates that B. azoricus is a mixotrophic organism, which obtains energy from a dual endosymbiosis and filter-feeding. Yet the relative contribution of symbiosis and filter-feeding to B. azoricus nutrition is still unclear. To address this question, we developed and individual-based model which describes sulphide and methane uptake by endosymbionts, the energy gained through microbial oxidations, the transfer of energy from endosymbionts to B. azoricus, filter-feeding of particulate organic matter (POC) by B. azoricus and the energetic wastes of the mytilid with respiration. The model accounts for size-dependent relationships obtained from empirical data. External concentrations of H2S and CH4 correspond to estimated values for the Menez Gwen vent field, maximal and minimal values measured at MAR. From in situ observed densities of B. azoricus, productivity predictions at the individual level were upscale to the mytilid population at Menez Gwen and compared to estimated values. Predicted biomass of B. azoricus and its endosymbionts show a very high fitting level with estimated values. Results suggest that the relative contribution of filter-feeding and endosymbiosis varies with B. azoricus size, with small mytilids being strongly dependent on filter-feeding, whilst larger mussels obtain a significant portion of its energy from endosymbiosis. This is related with the variation of gill weight with total weight. Results also suggest that, an individual of a certain size can potentially regulate the relative contribution of filter-feeding and endosymbiosis according to external conditions. However, large B. azoricus exhibit a higher level of nutritional flexibility than small mytilids. The relative contribution of endosymbioisis and filter-feeding to the total energy budget of B. azoricus, as well as the mytilid particulate organic matter requirements, are assessed and discussed under several scenarios. [Copyright &y& Elsevier]

Abstract: This study reports on the haemocyte responses after induction of shell regeneration in the hydrothermal mussel Bathymodiolus azoricus. Haemolymph was drawn from live mussels collected at Menez Gwen hydrothermal vent site (850 m depth) at the Mid Atlantic Ridge (MAR) and was compared with those collected following laboratory acclimatisation (1 atm and Ca-rich algal diet) and also with induced specimen for up to 30 days. Simultaneously, histological changes in mantle micro-morphology with the histochemical detection of Ca mobilisation in tissues were conducted. On the basis of light- and transmission electron microscopy, it is concluded that the physiological equipment involved in shell regeneration in the deep sea bivalve closely resembles that in littoral mytilids, a group that B. azoricus is closely related. This in spite of previously alleged molecular and cellular adaptations to extreme conditions typical at deep sea hydrothermal vents. Three types of blood cells were identified sharing various morphological similarities with those in many non-vent bivalves. Significant increase in the number of circulating haemocytes was detected from day 5 after induction shell regeneration. It is suggested that the increase may be a result of migration of haemocytes from the connective tissue, probably to the shell growth frontline. It is alleged that a first peak in haemocyte number is a non-specific immune response related wound healing, which renders changes in the pallial fluid that are favourable for CaCO3 deposition. The conspicuous presence of an unidentified, acid soluble, highly refractive structure in the haemolymph of induced mussels was detected, which may play a role in Ca nucleation. This study has set the stage for investigations underway on the influence of hydrostatic pressure on shell biomineralisation in B. azoricus subjected to post-capture hyperbaric simulations. [Copyright &y& Elsevier]

Aliphatic hydrocarbons and PAHs in the whole soft tissues of Bathymodiolus azoricus from three Mid-Atlantic Ridge hydrothermal vents (Menez-Gwen, Lucky Strike and Rainbow), and Mytilus galloprovincialis from three contaminated coastal sites in South Portugal were analysed, and its effects on the digestive gland microsomes mixed-function oxygenase system (MFO) were assessed. Aliphatic hydrocarbons levels were present in the same magnitude in both coastal and hydrothermal environments, while the UCM (unresolved complex mixture) for coastal mussels were higher than in vent mussels. In general, significantly higher PAHs concentrations were found in coastal mussels, compared to B. azoricus where low molecular weight PAHs (2–3 rings) represented the majority of PAHs contrarily to what was observed in M. galloprovincialis. The MFO components were present in both mussel species, and were detected in vent mussels for the first time. However this system seems to have different roles in species from these contrasting environments. In coastal mussels MFO responded to hydrocarbon contamination while response in hydrothermal organisms appeared to be related mainly to endogenous factors. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Specimens of Bathymodiolus azoricus were sampled along the Mid-Atlantic Ridge at the Menez Gwen, Lucky Strike and Rainbow hydrothermal fields. Individual shells (n =51), through the weight range 0.62 to 15.70g, were analyzed for their magnesium, strontium, iron, manganese, copper, zinc and cadmium concentrations. Amongst the marine molluscs the shell of B. azoricus is confirmed as being particularly impoverished in strontium (mean 943μgg−1). Trace metal concentrations in the shells decreased in the order Fe>Mn>Zn>Cu>Cd. Despite originating from trace metal rich environments mean concentrations were low (37.9, 13.2, 10.7, 1.1 and 0.7μgg−1, respectively). Irrespective of geographical origin magnesium, strontium and copper concentrations were primarily dictated by shell weight. In contrast cadmium concentrations were elevated in shells from the Rainbow field and ambient seawater chemistry imparted site specific chemical fingerprints to the shells with respect to the iron to manganese ratio. [Copyright &y& Elsevier]

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed. [ABSTRACT FROM AUTHOR]

Abstract: We previously reported evidence of increased levels of DNA damage in the hydrothermal mussel Bathymodiolus azoricus, which suggested that the species was not fully resistant to the natural toxicity of its deep-sea vent environment. In the present study, HSP70 was used as a biomarker of sub-cellular stress. Differences in HSP70 expression pattern were observed between vent sites, typified by different depths/toxicity profiles, and between different mussel tissue types. A comparison of specimens collected by remote operated vehicle (ROV) and acoustically-operated cages showed that less stress (as indicated by changes in HSP70 levels) was induced by the faster cage recovery method. Therefore alternatives to ROV collection should be considered when planning experiments involving live deep sea organisms. Significantly, a positive correlation was found between the levels of DNA strand breakage, as measured using the Comet assay, and HSP70 expression pattern; evidence was also obtained for the constitutive expression of at least one HSP isoform which was located within the cell nucleus. [Copyright &y& Elsevier]

Abstract: 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-κB 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-κB transcription factors and antibacterial genes. [Copyright &y& Elsevier]

Specimens of the mussel Bathymodiolus azoricus were collected from Menez Gwen, a relatively shallow (850 m) hydrothermal vent field on the Mid-Atlantic Ridge. Each bivalve shell (n =21) was individually cleaned by selective chemical. The residual crystal matrix of each shell was individually analysed for the concentrations of the minor elements magnesium and strontium and the trace elements iron, manganese, copper and zinc. The chemical composition of the crystal matrix is unusual. B. azoricus is identified as a species having one of the most strontium impoverished shells amongst the marine molluscs. For a bimineral species the magnesium concentration is also extraordinary low. Despite originating from a trace metal rich environment; the metal concentrations in the shells were exceptionally low. Mean concentrations of iron, manganese, copper and zinc were 20.6, 3.7, 0.6 and 9.4 μg g−1 respectively. Minor and trace element concentrations exhibited a marked intra-population variability. Copper concentrations increased and iron and zinc concentrations decreased with increasing shell weight. Due to its insensitivity to the high environmental levels of trace elements and the variability in intra-population concentrations induced by shell weight the crystal matrix of the shell of B. azoricus has little potential for use in environmental trace metal monitoring in areas contiguous to deep-sea hydrothermal vents. [Copyright &y& Elsevier]

The vent mussel Bathymodiolus azoricus is the dominant member of the Northern Mid-Atlantic Ridge (MAR) hydrothermal megafauna, and lives in an environment characterized by temporal and spatial variations in the levels of heavy metals, methane and hydrogen sulphide, substances which are known to increase reactive oxygen species levels in the tissues of exposed organisms. To evaluate the effects of two contrasting hydrothermal environments on the antioxidant defence system of this vent mussel species, a 2-week transplant experiment was carried out involving mussels collected from the relatively deep (2300 m), and chemical rich, Rainbow vent field. These were transplanted to the shallower (1700 m), and relatively less toxic, Lucky Strike vent field. To achieve this objective, levels of superoxide dismutase, catalase (CAT), total glutathione peroxidase (GPx), selenium-dependent glutathione peroxidase and lipid peroxidation (LPO) were measured in the gills and mantle tissues of resident and transplant mussels before and after the transplant experiment. With the exception of CAT, the gills of the transplanted mussels had significantly higher antioxidant enzyme activity compared with the basal levels in the donor (Rainbow) and recipient (Lucky Strike) populations; whereas the antioxidant enzyme levels in the mantle tissues of the transplants reflected the baseline levels of activity in the native Lucky Strike mussels after 2 weeks. In contrast, LPO levels were significantly higher in both tissue types in the transplants than in either the source or the recipient populations, which suggested a response to hydrostatic pressure change (note, the transplant animals were brought to the surface for transportation between the two vent fields). The fact that the Rainbow mussels survived the transplant experience indicates that B. azoricus has a very robust constitution, which enables it to cope behaviourally, physiologically and genetically with the extreme conditions found in its naturally contaminated deep-sea environment. [ABSTRACT FROM AUTHOR]

Abstract: This study aims to improve our knowledge on the reproduction cycles of invertebrates from deep-sea hydrothermal vents, which is fragmentary to date, owing to the prohibitive costs of regular time series sampling required for a complete assessment. However, new technologies such as acoustically retrievable cages and the long-term laboratory maintenance of vent species enabled sampling at relatively low costs. Reproductive patterns of the vent bivalve Bathymodiolus azoricus were studied with regard to the onset of spermatogenesis in captivity as compared to specimens from the wild. Following 1-year in our controlled laboratory set-up, spermatogenesis started in January–February, as “naturally occurs” in mussels at hydrothermal vents, which provide further supporting evidence for an annual reproduction in B. azoricus. In addition, we describe a functional methane-feeding system for the long-term maintenance of the hydrothermal mussel that represents great potential for post-capture experimental investigations. [Copyright &y& Elsevier]

Abstract: The hydrothermal vent bivalve Bathymodiolus azoricus is naturally exposed to elevated levels of trace elements (Fe, Cu and Zn) and is therefore a suitable model organism to study physiological adaptations to extreme environments. Whole shells and various shell compartments of B. azoricus from two geochemically different hydrothermal vents of the Mid- Atlantic Ridge were analysed regarding their micro-essential metal concentration, and compared with levels in their shore analogues from contaminated sites reported in the relevant literature. Concentrations of Cu and Zn in hydrothermal shells exceeded many-fold levels reported for polluted areas worldwide, and thus reveal a prominent potential of this organ to accumulate bivalent metals (average concentrations of metals in whole shells of mussels from the Lucky Strike vent site were above average 280μg g− 1 for Fe, 80μg g− 1 for Cu and 40μg g− 1 for Zn). Moreover, shell metal concentrations reflected fluid composition. In spite of higher metal concentrations in the periostracum than in the nacre, the latter has a higher metal burden because of its greater mass as compared to the thin periostracum. Additionally, metals were shown to bind reversibly to the outer surface of the shell (up to 6% of both total Cu and Zn, and 30% of total Fe from shells were recovered in an acidic solution in which the shells were soaked for 12 hours). It is concluded that shells are good indicators of environmental levels of Fe, Cu and Zn at hydrothermal vents and thus may be considered markers of putative changes in metal exposure over the mussel''s lifespan. [Copyright &y& Elsevier]

Abstract: Hydrothermal communities are built on highly specialised organisms possessing effective adaptation mechanisms to tolerate elevated levels of toxic heavy metals typical of these extreme habitats. Bioavailability and tissue compartmentalisation of micro-essential metals (Cu, Zn, and Fe) were investigated in the bivalve Bathymodiolus azoricus from three geochemically distinct hydrothermal vents (Rainbow, Lucky Strike, Menez Gwen). Additionally, in order to make inferences on the effect of biological interactions on the metal uptake, the bivalves'' endosymbiont bacteria and commensal parasite Branchipolynoe seepensis were analysed for metal bioaccumulation. Micro-essential metal concentrations in byssus threads exceeded many-fold concentrations in the gill and digestive gland, which in turn were consistently one order of magnitude above levels measured in the mantle. In spite of its high metal concentrations, the byssus is unlikely to be an active bioaccumulator. Its high surface to mass ratio and its binding sites for metals suggest a reversible adsorption of micro-essential metals in the vent mussel. Inter-site comparison showed highest Fe concentrations in tissues of mussels from the Rainbow site, whereas Zn and Cu in all tissues were highest in mussels from the Lucky Strike site, reflecting metal concentrations in the water surrounding macro-invertebrates at these vent sites. The omnipresence of the commensal parasite polychaete in gills of B. azoricus from the Lucky Strike vent field, unlike the other sites, is suggested to be an adaptation to the typically elevated Fe concentrations in the water column near mussel beds. Unprecedented Fe concentrations measured in the digestive gland of mussels from the Rainbow site (4000μg g−1, three times higher than levels in bivalves from polluted sites) call for further post-capture ecotoxicological investigations of potentially novel Fe-handling strategies. We provide the first information on the bioaccumulation potential of hydrothermal organisms by calculation of concentration factors for Cu, Zn and Fe in the gills of the bivalve host and also in its endosymbiont and/or parasites. Adaptation strategies (biochemical, physiological, morphological and life-history adaptations) of vent organisms to elevated levels of micronutrients and non-essential metallic elements are discussed. [Copyright &y& Elsevier]

Abstract: Dense macrofaunal communities of modioliform mussels are a major component of many hydrothermal vent and cold seep ecosystems. The hydrothermal vent mussel Bathymodiolus azoricus, that dominates hydrothermal vent communities near the Azores Triple Junction, can be maintained in aquaria at atmospheric pressure. Cages containing these mussels were placed over diffuse vent outlets and recovered at different times. Cages recovered in January 2003 contained mussels with ripe gonads while those recovered in July, August and November 2001 and in April 2003 did not. Mussels collected post-spawning in April 2003 spawned in the aquaria in January 2004. Young mussels recruited to the cages in April 2003. The data support a main single period of spawning and of juvenile recruitment for B. azoricus. [Copyright &y& Elsevier]

Abstract: In this study, we investigated on concentrations of trace metals (Al, Cd, Mn, Co, and Hg) in the hydrothermal bivalve Bathymodiolus azoricus, a dominant species at most vent sites along the Mid-Atlantic Ridge (MAR), and in its endosymbiont bacteria and commensal parasite Branchipolynoe seepensis. Comparison of our results with data from the literature on nonhydrothermal bivalves suggests lack of “extreme” uptake of trace metals by B. azoricus, except for Hg concentration which exceeded manyfold previously reported values. Mussels collected from three geochemically distinct vent sites, Menez Gwen, Lucky Strike, and Rainbow, along the MAR showed significant differences in tissue concentration of metals. Proportionality of metals in soft tissues of mussels reflected variation of water chemistry at different vents, which in turn conserved the order of trace metal prevalence in undiluted fluids. There were significant tissue-specific differences in trace metal compartmentalization for all metals investigated. Byssus thread contained the highest metal concentration among examined tissues, and thus it is suggested to be an important detoxification route. Size-dependent differences in metal concentrations were detected only for Hg, revealing a general trend of small mussels accumulating more metal than big mussels. Endosymbiont bacteria are shown to exclusively sequester Al from the host gill and contribute to removal of other toxic metals in mussels from Menez Gwen. The commensal parasite present in all mussels from Lucky Strike had higher tissue concentrations of Mn, Al, and Co than the host gill, unlike Cd and Hg which were considerably lower in the former, and thus its role in detoxification remains unclear. Bioaccumulation potential of vent bivalves and associated organisms are quantified as concentration factors and compared to make inferences on the putative role of the endosymbiont bacteria and the commensal parasite in detoxification of trace metals. [Copyright &y& Elsevier]

Abstract: Enzymatic defenses involved in protection from oxygen radical damage were determined in gills and mantle of Bathymodiolus azoricus collected from three contrasting Mid-Atlantic Ridge (MAR) hydrothermal vent fields (Menez-Gwen, Lucky Strike and Rainbow). The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and Se-dependent), and levels of total oxyradical scavenging capacity (TOSC), metallothioneins (MT) and lipid peroxidation (LPO) were determined in B. azoricus tissues and the impact of metal concentrations on these antioxidant systems and lipid peroxidation assessed. SOD, CAT, TOSC, MTs and LPO levels were higher in B. azoricus gills while glutathione peroxidases (total and Se-dependent) were higher in the mantle, and with the exception of CAT, were of the same order of magnitude as in other molluscs. TOSC levels from Menez-Gwen indicate that the vent environment at this site is less stressful and the formation of ROS in mussels is effectively counteracted by the antioxidant defense system. TOSC depletion indicates an elevated ROS production in molluscs at the other two vent sites. Cytosolic SOD, GPx and LPO were more relevant at Lucky Strike (Bairro Alto) where levels of essential (Cu and Zn) and toxic metals (Cd and Ag) were highest in the organisms. CAT activity and LPO were predominant at the Rainbow vent site, where an excess of Fe in mussel tissues and in vent fluids (the highest of all three vent sites) may have contributed to increased LPO. Therefore, three distinct pathways for antioxidant enzyme systems and LPO based on environmental metal speciation of MAR vent fields are proposed for Bathymodiolus gills. At Menez-Gwen, TOSC towards peroxyl and hydroxyl radicals and peroxynitrite are predominant, while at Lucky Strike cytosolic SOD activity and GPx are the main antioxidant mechanisms. Finally at Rainbow, catalase and lipid peroxidation are dominant, suggesting that resistance of mussels to metal toxicity at these vent fields decreases in the sequence Menez-Gwen>Lucky Strike and Rainbow. [Copyright &y& Elsevier]

The present study describes several features of the aquatic environment with the emphasis on the total vs. filter-passing fraction (FP) of heavy metals in microhabitats of two typical deep-sea vent organisms: the filter-feeder, symbiont-bearing Bathymodiolus and the grazer shrimps Rimicaris/Mirocaris from the Mid-Atlantic Ridge (MAR). The concentration of 10 trace elements: Al, Mn, Co, Cu, Mo, Cd, Fe, Zn, Pb and Hg was explored highlighting common and distinctive features among the five hydrothermal vent sites of the MAR: Menez Gwen, Lucky Strike, Rainbow, Saldanha, and Menez Hom that are all geo-chemically different when looking at the undiluted hydrothermal fluid composition. The drop off in the percentage of FP from total metal concentration in mussel and/or shrimp inhabited water samples (in mussel beds at Rainbow, for instance, FP fraction of Fe was below 23%, Zn 24 %, Al 65%, Cu 70%, and Mn 89%) as compared to non-inhabited areas (where 94% of the Fe, 90% of the Zn, 100% of the other metals was in the FP fraction) may indicate an influence of vent organisms on their habitat’s chemistry, which in turn may determine adaptational strategies to elevated levels of toxic heavy metals. Predominance of particulate fraction over the soluble metals, jointly with the morphological structure and elemental composition of typical particles in these vent habitats suggest a more limited metal bioavailability to vent organisms as previously thought. In addition, it is evoked that vent invertebrates may have developed highly efficient metal-handling strategies targeting particulate phase of various metals present in the mixing zones that enables their survival under these extreme conditions. [ABSTRACT FROM AUTHOR]

Abstract: Invertebrates harbouring endosymbiotic chemoautotroph bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. In these species mechanisms of symbiont transmission are likely to be key elements of dispersal strategies that remained partially unresolved because the early life stages are not available for developmental studies. To study cessation and re-establishment of symbiosis in the host gill a laboratory experiment was conducted over 45 days in a controlled set-up (LabHorta) that endeavour re-creation of the hydrothermal vent chemical environment. Our animal model was the vent bivalve Bathymodiolus azoricus from the Menez Gwen vent site of the Mid Atlantic Ridge (MAR). Animals were exposed to conditions lacking inorganic S supply for 30 days, which is vital for their symbionts, and then re-acclimatized in sulphide-supplied seawater for an additional 15 days. Gradual disappearance of bacteria from the symbiont-bearing gill cells was observed in animals kept in seawater free of dissolved sulphide for up to 30 days, and was evidenced by histological, ultrastructural observations and Polymerase Chain Reaction tests. Following re-acclimatisation in S-supplied seawater, proliferation of sulphur-bacteria in the gill bacteriocytes confirms the functionality of our sulfide-feeding system in supporting chemoautotrophic symbionts. It may also indicate a horizontal endosymbiont acquisition, i.e. from the environment to the host by means of phagocytosis-like mechanism involving special “pit-like” structures on the apical cell membrane. The present work reports the first laboratory set-up successfully used to maintain the hydrothermal vent bivalve B. azoricus for prolonged periods of time by supplying inorganic sulphur as an energy source for its bacterial endosymbionts. Survival of symbiont bacteria is a critical factor influencing the host physiology and thus the methods reported here represent great potential for future studies of host-symbiont dynamics and for post-capture experimental investigations. [Copyright &y& Elsevier]

The hydrothermal vent bivalve Bathymodiolus azoricus is naturally exposed to putatively elevated levels of mercury (Hg), exposure that dates back to the geological occurrence of vent ecosystems, and thus may have evolved evolutionary detoxification mechanisms. Therefore, it was used as a model organism in the present investigation to study the Hg-animal interaction. Mussels were exposed to inorganic Hg by daily administration of 20 µg l-1 Hg for 21 days (cumulative added concentration was 420 µg l-1, i.e. ∼2 mmol l-1) under controlled laboratory conditions, and consequent bioaccumulation and detoxification patterns were investigated, while shell gaping behaviour indicative of filtering activity was monitored. As a result of Hg exposure, significant increase in duration, as well as decline in frequency of shell gaping occurred, which did not recover to pre-exposure levels following 21 days of Hg-free treatment. An increase in the duration of open-shelled status may indicate the absence of an avoidance reaction in the vent mussel coming in contact with Hg, unlike other bivalves that normally close their shells in response to stress compounds. Alternatively, it may suggest that Hg had an inhibitory effect on the adductor muscle function that is responsible for closing the shells. As a result, elevated Hg levels were measured in the soft tissues (270±71 µg g-1 in gills, 245±52 µg g-1 in digestive glands, 93±25 µg g-1 in the mantle and 46±9 µg g-1 in the foot), in byssus threads (peak levels of 442±89 µg g-1) and in pseudofaeces (reaching levels as high as 1000 µg g-1). Overall, gills contributed 75% to the total Hg body burden followed by mantle (13%), digestive gland (7%), byssus (3%) and foot (2%). Tissue Hg levels remained elevated in mussels transferred to Hg-free seawater even after 21 days, despite the high concentrations persistently eliminated with pseudofaeces both, during and after, exposure. This potential for bioaccumulation of inorganic Hg (concentration factors reached the order of magnitude of 104) by the vent mussel, which does not seem to prevent uptake by shell closure, suggests that the main Hg-handling strategy is elimination via mucus. [ABSTRACT FROM AUTHOR]

Comet and agarose gel electrophoresis (AGE) assays were used to show that haemocytes (blood cells) and gill tissues of vent mussels, Bathymodiolus azoricus, are sensitive to hydrostatic pressure change, but can repair DNA damage induced by retrieval from 840 m to the sea surface. In contrast, animals collected from 1700 m survived for only a few days in the laboratory, which was reflected in their poor DNA quality. These findings support the hypothesis of a physiological barrier to survival around 1000–1500 m depth, which these results show affects both vent and non-vent species alike. Based on in vitro experimental exposures to hydrogen peroxide and MMC, vent mussels appear to have sensitivities to the environmental mutagens that are not significantly different from those of coastal mussels. [Copyright &y& Elsevier]