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Transmission electron micrographs show the branchial epithelium of the ascidian Ciona intestinalis to be composed of three kinds of cells: ciliated, parietal, and "dark." Absorptive processes of phagocytosis appear in ciliated and parietal cells; moreover, apocrine-like secretion is evident exclusively in the parietal cells. Finally, the occurrence of secretory cells in the branchial epithelium suggests a digestive role of the ascidian branchial sac.

Recent field observations have identified the widespread occurrence of fluid seepage through the eastern Mediterranean Sea floor in association with mud volcanism or along deep faults. Gas hydrates and methane seeps are frequently found in cold seep areas and were anticipated targets of the MEDINAUT/MEDINETH initiatives. The study presented herein has utilized a multi-disciplinary approach incorporating observations and sampling of visually selected sites by the manned submersible Nautile and by ship-based sediment coring and geophysical surveys. The study focuses on the biogeochemical and ecological processes and conditions related to methane seepage, especially the anaerobic oxidation of methane (AOM), associated with ascending fluids on Kazan mud volcano in the eastern Mediterranean. Sampling of adjacent box cores for studies on the microbiology, biomarkers, pore water and solid phase geochemistry allowed us to integrate different biogeochemical data within a spatially highly heterogeneous system. Geophysical results clearly indicate the spatial heterogeneity of mud volcano environments. Results from pore water geochemistry and modeling efforts indicate that the rate of AOM is similar to 6 mol m(-2) year(-1), which is lower than at active seep sites associated with conditions of focused flow, but greater than diffusion-dominated sites. Furthermore, under the non-focused flow conditions at Kazan mud volcano advective flow velocities are of the order of a few centimeters per year and gas hydrate formation is predicted to occur at a sediment depth of about 2 m and below. The methane flux through these sediments supports a large and diverse community of micro- and macrobiota, as demonstrated by carbon isotopic measurements on bulk organic matter, authigenic carbonates, specific biomarker compounds, and macrofaunal tissues. Because the AOM community appears to be able to completely oxidize methane at the rate it is seeping through the sediments, ultimate sinks for methane in this environment are authigenic carbonates and biomass. Significant differences in organic geochemical data between this site and those of other cold seep environments, even within the eastern Mediterranean mud volcanoes, indicate that the microbiological communities carrying out AOM varies depending on specific conditions such as methane flux and salinity. (C) 2004 Elsevier B.V. All rights reserved.

Cold seep communities are sustained by massive methane-rich fluid expulsion through mud volcanoes located at about 5000 m in the Barbados Trench. These communities, dependent on chemosynthetic processes, are dominated by a vesicomyid bivalve assumed to be a new species related to the genus Calyptogena, and by large bushes of the sponge Cladorhizidae. Both are associated with symbiotic bacteria and are indicative of methane release in seawater and sulphide production in sediments. Non-symbiotic organisms, such as large fields of filter-feeding polychaetes and high densities of meiofauna are indicative of enhanced biological production in the sediments. The spatial distribution of the bivalve populations was mapped using video observations and a computer method based on a simple calculation of the area covered by a submersible camera. The observation of clam beds of variable densities allows us to define two types of fine-scale fluid expulsion pattern: dense Calyptogena beds (up to 150 ind.m−2) are associated with“vents” with relatively high fluid discharge velocities of about 10 cm s−1 and focused by high permeability conduits, whereas dispersed clams (1–10 ind.m−2) are probably sustained only by slow, diffusive “seepages”. The distribution of the chemosynthetic zones from the centre to the edges of the volcano, highlighting the heterogeneity of the concentric zones from the centre to the edges of the volcano, highlighting the heterogeneity of the fluid expulsion pattern at the scale of the volcano. The spatial distribution of the chemosynthetic communities characterizes the fluid expulsion on several types of volcanoes: two mud volcanoes, identified as diatremes, named Atalante and Cyclope, are flat with a central lake of warm fluid mud that is devoid of life, whereas Calyptogena beds are located in the outer regions. On the two other structures, mounds shaped as cones, all activity is concentrated near the summit and seems to be related to higher flow vents than on diatremes. However, the scarceness of bivalves on the flanks of these volcanoes indicates that low flow seepages are less developed than on diatremes. The high mean clam density and the presence of large Cladorhizidae bushes on the volcano Atalante show that larger quantities of methane are emitted through this structure than on the others, and suggest a more evolved surface, favouring chemosynthetic production, than on the other diatreme, named Cyclope. The level of colonization and the spatial patterns of the chemosynthetic communities, when compared on the different volcanoes, suggest that they are at different stages of activity.

Newly discovered hydrothermal vent communities at Lucky Strike on the Mid-Atlantic Ridge (37°18'N, 32°16'W) are comprised of an invertebrate fauna sufficiently different from known vent faunas of TAG and Snake Pit to consider Lucky Strike part of a new biogeographic province. The dominant component of the fauna is a new species of mussel, and the most unusual feature of the fauna is an echinoid echinoderm, Echinus sp. An abundance of small mussels (< 5 mm) indicates a recent recruitment event at Lucky Strike, and modal analysis of length-freque ncy data indicate a discontinuous recruitment process in space and time. Copyright © 1996 Elsevier Science Ltd

The Barbados trench is characterized by large fields of volcanoes and mounds located over a distance of 30 km above the northern slope of a basement ridge corresponding to an inactive transform fault. Sediments from various locations were collected and analyzed for their lipid contents. Bacterial input to the overall biomass was estimated through the analysis of phospholipid ester-linked fatty acid (PLFA) profiles and glycerol ether lipids. Results indicated a eubacterial biomass estimated to be 109cells (g dry wt)−1. Individual fatty acid profiles revealed the presence of sulfur-oxidizing bacteria common to many deep-sea sites but also a large contribution of type I and type II methanotrophs to the eubacterial biomass. The presence of methanotrophs was further supported by the analysis of specific biomarkers of these microorganisms as well as some unusual trans fatty acid isomers. Anaerobic bacteria and presumbly sulfate reducing bacteria were also present, as well as archaebacteria and primarily methanogens, as indicated by glycerol ether lipid analysis.

Dense bush-l~ke clumps of several hundred ~ n d ~ v l d u a l s of a new specles of Cladorhiza (Demosponglae, Poeciloscler~da) were observed near methane sources in mud volcanoes 4718 to 4943 m dcep In the Barbddos Trench The sponge tissue contalns 2 maln mo~pho log~ca l types of extracellular symbiotic bacteria small rod-shaped cells and larger cocco~d cells with stacked membranes Stdble carbon ~so tope values, the presence of methanol dehydrogenase and ultrastructural observatlons all lndlcate that at least some of the syrnblonts are methdnotrophic Ultrastructural e v ~ d e n c e of ~n l r ace l l~~ la r d ~ g e s t ~ o n of thp s y n ~ b ~ o n t s and the stable C and X values suggest that thc sponge obtalns a slgnlflcdnt portion of its nutr~tlon from the symbionts Ultrdstructure of the sponqe embryo suggests d ~ r e c t ransmission throuqh qc.neratlons In brooded embryos The sponge also ma~ntrilns a carnivorous feed~ng hdblt on tin\ swlmmlng prey, as do other cladorhlzlds K E Y L\/ORDS. Methanotrophy POI-~fera . Deep-sea . Symbiosis . Cold-seep communities

Three sectors of the south Barbados prism between 1000 and 2000 m depth were explored by the French submersible Nautile. Chemosynthesis-based benthic communities were discovered on several structures affected by diapirism, including mud volcanoes, domes and an anticlinal ridge. The communities are associated with the expulsion of methane-rich fluids which is a wide-spread process in the area. These communities are dominated by large bivalves and vestimentiferans which harbour chemoautotrophic symbiotic bacteria. The symbiotic bivalves include two species of Mytilidae and one of Vesicomyidae, with dominance of a methanotrophic mussel. Cartography of the benthic communities, interpretation of thermal measurements and observation of sedimentary patterns have been used to define the life habits of each of the three species of symbiotic bivalves. Each species has a characteristic preference for different conditions of edaphic and fluid flow: the dominant methanotrophic mussel appears to require high velocity vents and hard substratum. The vesicomyids and the other species of mussel are able to take up sulfide from the sediments, and so are associated with low seepages, but also require soft sediment. The three bivalve species are assumed successively to colonize the top of a diapiric ridge, in a succession related to the temporal evolution of fluid flow and sedimentation. The composition of the bivalve assemblages, their densities and biomasses all differ between the several mud volcanoes and domes studied, and these parameters are thought to be related to the spatial and temporal variations of fluid expulsion through the structures, and the lithification processes linked to fluid expulsion. One very active dome is at present colonized by an exceptionally large and dense population of the methanotrophic mussel. In contrast, communities in another area, on the domes and volcanoes that are currently inactive, were colonized by only a few living vesicomyids and mussels, both associated with sulfur-oxydizing bacteria, and there were numerous empty shells. The densities and biomasses of symbiotic bivalves were far greater in the area studied than in a deeper mud volcano field on the same prism that had been studied previously. This is consistent with a report that methane production is greater in the southern region of this accretionary prism than in the northern. Numerous non-symbiotic organisms were observed in and around the areas of the seeps, some are endemic to the seep communities, including some gastropods and shrimps, others are either colonists or vagrants from the surrounding deep-sea floor. Filter feeders were very abundant, and some of these, like the serpulids and large sponges, may also be dependent on the chemosynthetic production. Faunistic composition of both symbiotic and non-symbiotic taxa, of the assemblages around these cold seeps, is closely related to that reported for communities living on hydrocarbon seeps in the Gulf of Mexico.

Fiala-Medioni, A. ... et. al.-- 18 pages, 12 figures, 2 tables, 52 years after the first explorations in the Lacaze-Duthiers Canyon and the observation of living populations of cold-water corals, recent video surveys using deployments of the ROV "Super-Achille" and the submersible "REMORA" (COMEX), have allowed the discovery of new sites colonized by cold-water coral populations (Lophelia pertusa, Madrepora oculata, Dendrophyllia cornígera and Desmophyllum dianthus). Dense populations were observed, from 250 to 535 m, either on blocks or cliffs. The coral community consisted of a rich fauna of invertebrates including numerous sponges, brachiopods, echinoderms, oysters, ascidians and bryozoans as well as vagile cephalopods and fishes. Among the 51 identified species, 4 species are protected by the French regulation (corals), 13 species are cited in European directives 79/109/EEC (Art 4) and 92/43/EEC (Annex 2), 2 species are cited in the OSPAR convention and 15 are of commercial interest. Direct observations allowed also establishing an increase of anthropogenic wastes mainly related to fishing

Four mud volcanoes of several kilometres diameter named Amon, Osiris, Isis, and North Alex and located above gas chimneys on the Central Nile Deep Sea Fan, were investigated for the first time with the submersible Nautile. One of the objectives was to characterize the seafloor morphology and the seepage activity across the mud volcanoes. The seepage activity was dominated by emissions of methane and heavier hydrocarbons associated with a major thermal contribution. The most active parts of the mud volcanoes were highly gas-saturated (methane concentrations in the water and in the sediments, respectively, of several hundreds of nmol/L and several mmol/L of wet sediment) and associated with significantly high thermal gradients (at 10 m below the seafloor, the recorded temperatures reached more than 40 °C). Patches of highly reduced blackish sediments, mats of sulphide-oxidizing bacteria, and precipitates of authigenic carbonate were detected, indicative of anaerobic methane consumption. The chemosynthetic fauna was, however, not very abundant, inhibited most likely by the high and vigorous fluxes, and was associated mainly with carbonate-crust-covered seafloor encountered on the southwestern flank of Amon. Mud expulsions are not very common at present and were found limited to the most active emission centres of two mud volcanoes, where slow extrusion of mud occurs. Each of the mud volcanoes is fed principally by a main narrow channel located below the most elevated areas, most commonly in the centres of the structures. The distribution, shape, and seafloor morphology of the mud volcanoes and associated seeps over the Central Nile Deep Sea Fan are clearly tectonically controlled. © 2007 Elsevier Ltd. All rights reserved.

cited By 52; International audience; Specimens of shrimp (Rimicaris exoculata) and mussel (Bathymodiolus puteoserpentis) were collected 3500 m below the ocean surface at the hydrothermal vents of the mid-Atlantic Ridge (TAG and Snake Pit sites, respectively). Arsenic, a potentially toxic element, is among the substances emitted by the hydrothermal vents. The hydrothermal vent shrimp, which are known to be a primary consumer of the primary producing chemolithoautotrophic bacteria, contained arsenic at 13 μg g-1 almost exclusively as arsenobetaine (AsB). Arsenic was present in the soft tissues of the mussel at 40 μg g-1 and the major part of the extractable arsenic species in the adductor muscle/mantle tissues and in the gill were present as dimethylarsinylriboside-derivatives (arsenosugars), while AsB was present at 16 and 3.6%, respectively, in these tissues. In spite of the absence of biosynthetically active algae, the pattern of arsenic species found in the shrimp and mussel species in the deep-sea is similar to that found in their counterparts from the ocean surface. It is concluded that the autotrophic bacteria of the hydrothermal vent ecosystem and the symbiotic bacteria harboured in the mussel species are responsible for the biosynthesis of the organoarsenicals detected in the two hydrothermal vent animal species. It is not known if the formation of the same organoarsenicals in the surface and deep sea ecosystems is primarily a detoxification process of inorganic arsenic, or whether these compounds play a physiological role.

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Since 1987, Japanese and French geologists, geophysicists, and biologists have been studying the North Fiji Basin Ridge within the framework of a joint project named STARMER (Science and Technology Agency of Japan—IFREMER of France). This ridge was first geologically, geophysically, and geochemically surveyed during the 1985 SEAPSO 3 cruise of the R/V Jean Charcot [Auzende et al., 1988]. At that time, water sampling and morphotectonic analysis indicated that the North Fiji Basin Ridge was technically and hydrothermally active. Within the STARMER project, four surface ship cruises have been conducted {Kaiyo 87-88-89 and Yokosuka 90). One significant result of these surveys is the complete mapping of the entire ridge between 14°S and 22°S (Figure 1), an area approximately 900 km long and more than 50 km wide (Sea Beam and Furuno multibeam swath-mapping systems were used). During the Kaiyo 87 cruise, the presence of active hydrothermalism (sulfide deposits, chimneys expelling shimmering water, and associated living animal colonies) was discovered through water sampling and video deep towing.

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Transmission and scanning electron microscopy of food trapping filters or “mucous films” of several Ascidians reveal the same type of extensive net with elongate rectangular meshes. The ultrastructural appearance and mesh size varies only slightly within the group. Pore sizes from 0.2 to 0.5 μm in width and from 0.5 to 2.2 μm in length with a thickness of the filaments from 10 to 40 nm are probably representative for the nets under in vivo conditions. These observations corroborate earlier experimental results which indicate a remarkably high and constant efficiency of filtration in all species kept undisturbed. Great quantities of water are evidently filtered through these nets at relatively low resistance and particles even below bacterial size (0.5 μm) are effectively trapped. Histochemical reactions indicate the presence of both mucoproteins and acidic polysaccharide components and that the filaments are probably made up of a peptide core surrounded by polysaccharides.

With the critical point method, well preserved samples of ascidian gills have been obtained for a study by scanning electron microscopy. The electron micrographs illustrate the different stages in the structural complexity of the branchial sac in four species: Clavelina lepadiformis (Muller 1776) represents the simplest stage where the gill sheet is formed by a single screen with slits; Ciona intestinalis (Linne 1767) and Phallusia mammillata (Cuvier 1815) which exhibit intermediate stages with a secondary screen superimposed on the first, thus increasing the active surface. Maximum gill extent and, consequently, a maximum contact with the water carrying particles is observed in Styela plicata (Lesueur 1823) with the formation of gill folds. Our observations also demonstrate a morphological analogy between the zones exhibiting a positive reaction to proteolytic enzymes (chymotrypsin-like): namely the concave edge of the gill papillae of Ciona and Phallusia, the languets of the dorsal lamina of Phallusia, the folds of the longitudinal bars of Styela.

Specimens of Bathymodiolus sp. were collected at 2 620 m depth during the “Biocyarise” Cruise (12°58′80″N; 13°56′60″W) in March 1984, and samples of the gill were fixed for ultrastructural observations. The gill of this hydrothermal-vent mytilid constitutes the main organ in the nutritional processes. The lamellae display abundant ciliation, normally comprised of frontal cilia, compound latero-frontal cirri and lateral cilia. At the ventral margin of each demi-branch, a longitudinal, ciliated, feeding groove is present. the lamellae are composed of numerous homorhabdic filaments connected by tufts of cilia. Each filament is made of a thin wall overlying a central lumen containing amoebocytes. Ultrastructural observations revealed the filament wall to be composed of four types of cells: (1) The ciliated cells of the frontal, latero-frontal and lateral ciliation, characterized by an abundance of mitochondria. (2) Mucous cells present to some degree among the ciliated cells, but more abundant on the distal edge and containing dense droplets of mucus. (3) Cells colonized at their apical pole by numerous bacteria enclosed in membrane-delimited clear spaces and composing the major part of the filament wall. (4) Thin ciliated cells separating the bacterial cells and characterised by a dense fringe of microvilli at their apical pole. The lumen of the filament contains amoebocytes of different morphological aspects which seem to accumulate electron-dense granules, possibly related to detoxification processes.

Water current in 4 species of ascidians (Ascidia mentula Muller, 1776; Phallusia mammillata Cuvier, 1815; Styela plicata (Lesueur, 1823) and Microcosmus sabatieri Roule, 1885) has been recorded for the first time in situ with hot film probes. The mathematical treatment of probe calibration has allowed the evaluation of the characteristics of currents (minimal, maximal and average velocities) and the calculation of pumping rates. The method utilized yields qualitative and quantitative results: Pumping is continuous, and no particular rhythm shows up in the curves obtained. The velocity of the current does not change in the course of a recording, but it can vary from one experiment to another in the same individual; these variations are related to the variability of the diameter of the cloacal siphon rather than to a change in the rhythm of the ciliary activity. The average velocities are 3.42 cm sec-1 for A. mentula, 3.92 for P. mammillata, 3.30 for S. plicata and 3.79 for M. sabatieri. The pumping rates are fairly constant for the same individual. The means, expressed as ml x h-1 x g-1 organ dry weight are 3422 for A. mentula, 2070 for P. mammillata, 4892 for S. plicata and 3084 for M. sabatieri.

Anatomical, histological and ultrastructural work on two species of Calyptogena found exclusively in association with the fluid emissions in subduction zones off Japan was done in order to elucidate their nutritional and reproductive strategies. The gill appears as the main organ of nutrition as indicated by its enlargement and profound structural modifications; its dominant cell type is the bacteriocyte which is filled with dense gram-negative bacteria. Transit of particles is observed, but the different parts of the digestive tract are greatly reduced or simplified. These results indicate, for the nutrition of these clams, the dominance of chemosynthetic processes through symbiotic relationships with the endocellular bacteria of the gill. The presence of abundant crystals of elemental sulphur in the gill and the ultrastructural features of the bacteria suggest an active sulfur oxidizing metabolism. Both species are gonochorist; the unusual size of the gonad, the permanent sexual activity probably entailing continuous spawning reflect the high fecundity of these species. The reproductive strategy of Calyptogena in the Japanese subduction zones thus appears to be similar to that of the East Pacific species C. magnifica and indeed reminiscent of other deep sea molluscs.

A combination of a direct and an indirect method has been used for the first time in the study of filter-feeding in benthic invertebrates. Experiments over 12 h periods under constant temperature, pH, light and feeding conditions have been carried out with 3 species of ascidians: Ciona intestinalis (Linne, 1767); Phallusia mammillata (Cuvier, 1815) and Styela plicata (Lesueur, 1823). In C. intestinalis and P. mammillata, no pumping rhythm has been found; the water flow was constant for 12 h, except for some brief accidental interruptions. In S. plicata, the particular rhythm of spontaneous periodical contractions recorded during the experiment were related to gamete emission. The speed of water flow, which was fairly constant during recording, varied considerably during the 12 h period; these variations were generally related to modifications in the diameter of the cloacal siphon. The average speeds, in cm sec-1, lay between 5.7 and 9.5 for C. intestinalis, 5.2 and 19.4 for P. mammillata, and between 5.3 and 10.7 for S. plicata. Variations in the pumping-rate were small, oscillating around a mean value. In ml h-1 g-1 dry weight of organs these were 5,829 to 5,982 (mean=5,906) for C. intestinalis, 6,142 to 6,592 (mean=6,312) for P. mammillata, 1,0508 to 1,1505 (mean=1,0708) for S. plicata. Filtration was continuous without any particular rhythm; filtration rates in ml h-1 g-1 dry weight of organs varied between 4,244 and 4,418 (mean=4,331) for C. intestinalis, 4,620 and 4,960 (mean=4,779) for P. mammillata, 8,482 and 9,078 (mean=8,760) for S. plicata. The curves representing pumping and filtration were clearly parallel, indicating that filtration efficiency did not vary greatly during the course of an experiment; the rates obtained were 65 to 87% (mean=74%) for C. intestinalis, 66 to 88% (mean 76%) for P. mammillata, and 73 to 90% (mean=80%) for S. plicata. The slightly higher mean value in S. plicata is probably related to the higher complexity of the branchial apparatus of this species.

In Clavelina, Ciona, Phallusia and Styela, the branchial sac, the stomach and the intestine are involved in the absorption of dissolved amino aids and glucose. Autoradiography and double countings on 14C and 3H labelled samples show that, in the first hour, the branchial sac can concentrate several hundred times the dissolved nutritive molecules from dilute solutions, whereas the digestive tract is not involved significantly. In Phallusia the hepato-pancreas has a specific activity higher than in the intestine and may be an organ of storage. In all species, the branchial sac absorbs the largest proportion of labelled material.

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Specimens of hydrothermal vent organisms, Alvinella caudata and Bathymodiolus thermophilus were collected in 1982 from 13 degree N and 103 degree W, on the East Pacific Rise at a depth of 2600 m. The elemental composition was determined for some of their soft tissues at the structural and ultrastructural levels. Two microanalytical methods were used: secondary ion mass spectrometry and X-ray spectrometry. As examples, external and digestive epithelium and lumen of the digestive tract of Alvinella caudata and the gill and digestive gland of Bathymodiolus thermophilus were investigated. Intracellular localization of the elements, performed by electron microscopy, showed that spherocrystals and lysosomes were the target organelles of metal concentration. Moreover, bacteria were shown to be capable of elemental concentration. The correlations between the metal concentrations within the vent organisms and the metal concentrations characteristic of the vent environment are examined.

The gill of Ciona intestinalis, Phallusia mammillata, Styela plicata and Microcosmus sabatieri is the seat of enzymatic activities. alpha-amylases as well as proteases secreted into the mucus might start the digestion of the filtered organic particles. As for the proteasic activity, only a chymotrypsine-like enzyme could be detected and localized.

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

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