Paul R. Dando

Robotic ocean vehicles for marine science applications: the European ASIMOV project

The key objective of the ASIMOV project is the development and integration of advanced technological systems to achieve coordinated operation of an Autonomous Surface Craft (ASC) and an Autonomous Underwater Vehicle (AUV) while ensuring a fast communication link between the two vehicles. The ASC/AUV ensemble is being used to study the extent of shallow water hydrothermalism and to determine the patterns of community diversity at vents in the D. Joao de Castro (DJC) bank in the Azores.

Hydrothermalism in the Mediterranean Sea

Abstract Hydrothermalism in the Mediterranean Sea results from the collision of the African and European plates, with the subduction of the oceanic part of the African plate below Europe. High heat flows in the resulting volcanic arcs and back-arc extensional areas have set-up hydrothermal convection systems. Most of the known hydrothermal sites are in shallow coastal waters,

Chemoautotrophic Symbionts in the Gills of the Bivalve Mollusc Lucinoma borealis and the Sediment Chemistry of its Habitat

Lucinoma borealis has enlarged gills, which contain numerous prokaryotes in specialized cells (bacteriocytes) in the subfilamentar region. The gills also contain high concentrations of elemental sulphur and of a c-type cytochrome. Homogenates of gill tissue show ribulosebisphosphate carboxylase and phosphoribulokinase activity; they also show activity for adenylylsulphate reductase, an enzyme concerned in the oxidation of sulphur, and will phosphorylate ADP on the addition of sulphite or sulphide. Fixation of bicarbonate by gill tissue from starved animals is enhanced in the presence of 100 μm sulphide. The sediment in which the animals live contains 1–6 μg atoms per litre of dissolved iron and hence there is very little dissolved sulphide, 200 nM, or less (80 nmol dm-3 sediment). Thiosulphate concentrations are also low, 300 nM, or less (120 nmol dm-3 sediment). In contrast, there are acid-labile sulphide concentrations up to 14 mmol dm-3 and elemental sulphur concentrations up to 4 mg atom per cubic decimetre of sediment. The mean sulphate reduction rate in the sediment varied seasonally with temperature over the range 1640–4920 nmol sulphate reduced per hour per cubic decimetre. L. borealis was usually found below the region of maximum sulphate reduction. Hydrogen, methane and carbon monoxide concentrations were all 160 nmol dm-3, or less. Despite the low levels of dissolved sulphide the association between prokaryote and host appears to be able to exploit this habitat by the oxidation of reduced sulphur species; ways in which the bivalve may be able to extract these from the sediment are discussed. The bivalves may obtain half their carbon from the autotrophic prokaryotes.

Hydrothermal studies in the Aegean Sea

The aims of the Aegean Hydrothermal Fluxes and Biological Production project were to estimate the fluxes of fluids, chemicals, heat and bacteria from hydrothermal vents, establish the controls on venting dynamics, measure the productivity in the region of the vents and establish the effect of the vents on biodiversity of both prokaryotes and eukaryotes. This paper presents an initial synthesis of the project results. Research was done both by land-based SCUBA diving and from several vessels at a number of active sites in the near-shore coastal regions of Milos and Kos, with some additional studies at Methana, Lesbos and Santorini. Vent water composition showed very large variations. This was due to the mixing, of hydrothermal reservoir fluids, vapour condensate and seawater altered by interactions of fluid-sediment-bacteria in different proportions, in the gasohydrothermal vents. The composition ranged from nearly sea water with only slightly reduced pH, to higher or lower salinity fluids with a pH as low as 3 and with large enrichments in heavy and trace metals. Phase separation was a common feature at these shallow vents. The dry gas phase was mainly C02, but with significant amounts of H2S, CH4 and H2. These fluids commonly passed through soft sediments before venting from the seafloor and induced a convection cell of pore-water entrainment from deeper sediment layers into the water column with a consequent ‘re-charge’ down-flow of seawater into the sediment around the vent outlets. Such complex conditions may well explain the high biodiversity of Bacteria, Archaea and epifaunal species surrounding the vents. As many as 44 % of the archaeal lineages detected were found to represent novel phyla. Epifaunal diversity was particularly high with over 200 species recorded at the shallower Milos vents. These vents may form a ‘steppingstone’ for warmer water species to colonise the surrounding areas when water temperatures permit.

Fatty acids of hydrothermal vent Ridgeia piscesae and inshore bivalves containing symbiotic bacteria

Ridgeia piscesae from a hydrothermal vent and lucinid and thyasirid bivalves from inshore Canadian and UK waters, known to contain sulphur-oxidizing symbiotic bacteria, had lipids rich in 16:0,16:l(n-7) and 18:l(n-7) fatty acids in both bacteria-rich trophosome or gill tissue and in tissues without symbiotic bacteria. The results are consistent with the animals deriving these fatty acids from their sulphur-oxidizing symbionts. Ridgeia piscesae, Lucinoma annulata, Parvilucina tenuisculpta, Lucinoma borealis and Myrtea spinifera also contained substantial amounts of the non-methylene-interrupted dienoic fatty acids 20:2A5,13 and 22:2A7,15. It is proposed that these fatty acids are produced by chain elongation and A5 desaturation in animal tissues of 18:l(n-7) produced by the bacterial symbionts. Thyasira flexuosa did not contain 20:2A5,13 or 22:2A7,15 but instead contained 18:l(n-ll) and 20:l(n-13) which were not present in the other species analysed. It is proposed that 18:l(n-ll) and 20:l(n-13) arise from the A9 desaturation of 20:0 and 22:0, respectively, followed by chain shortening of the mono-unsaturated fatty acid products of A9 desaturation. It is considered that 20:2A5,13 and 22:2A7,15 are formed in the animals in response to a relative excess of 16:0, 16:l(n-7) and 18:l(n-7), accompanied by a relative deficiency of (n-3) and (n-6) polyunsaturated fatty acids. The results are discussed in relation to the lipid nutrition of marine invertebrates containing bacterial symbionts.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

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 Ca(2+) 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.

Biological Communities at Marine Shallow-Water Vent and Seep Sites

The review compiles, for the first time, data on the communities at 62 shallow-water hydrothermal vent and cold seep sites. ‘Shallow sites’ are defined as sites no deeper than 200 m. The communities at these sites are also compared with communities in reducing sediments at similar depths. Below 200 m, vent and seep obligate species tend to dominate the fauna living in areas where reducing fluids are released from the seabed. At the shallow sites, vent and seep obligate species of fauna are rare, only eight having being reported from shallow vents. No definite seep obligates have been found. Shallow vents and seeps are colonized by communities that consist of a subset of the background fauna, especially those species that are less sensitive to hydrogen sulphide toxicity. Conversely the zones directly surrounding shallow vent and seeps sites with varied topography, substrate type and food supply, often have a higher species diversity than the background area. The reasons for these differences are discussed.

Stetteria hydrogenophila, gen. nov. and sp. nov., a novel mixotrophic sulfur-dependent crenarchaeote isolated from Milos, Greece

Abstract A new hyperthermophilic, strictly anaerobic crenarchaeote, Stetteria hydrogenophila DSM11227 representing a new genus within the family of Desulfurococcaceae, was isolated from the sediment of a marine hydrothermal system at Paleohori Bay in Milos, Greece. Cells are gram-negative irregular and disc-shaped cocci, 0.5–1.5 μm in diameter, which are flagellate and can form cytoplasmatic protrusions up to 2 μm in length. The strain grew optimally at 95°C at pH 6.0 and at a NaCl concentration of 3%. The organism grew mixotrophically on peptide substrates. It required elemental sulfur as an external electron acceptor, and in addition, its growth was completely dependent on the presence of molecular hydrogen. Sulfur could be replaced by thiosulfate. H2S, CO2, acetate, and ethanol were identified as products of metabolism. The G + C content of DNA was 65 mol%. Analysis of its phylogenetic position by sequence analysis of 16S rRNA placed this organism in the family of Desulfurococcaceae. The dependence of this organism on both hydrogen and sulfur during growth on peptide substrates distinguishes Stetteria from all previously described species of Crenarchaeota.

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

The deep-sea hydrothermal vent mussel Bathymodiolus azoricus has been the subject of several studies aimed at understanding the physiological adaptations that vent animals have developed in order to cope with the particular physical and chemical conditions of hydrothermal environments. In spite of reports describing successful procedures to maintain vent mussels under laboratory conditions at atmospheric pressure, few studies have described the mussels 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.

Red cell carbonic anhydrase levels in flounders, Platichthys flesus L., from salt water and fresh water.

1. 1. Carbonic anhydrase levels in flounder red cells were unchanged by adaptation to salt or fresh water. 2. 2. Two major red cell isoenzymes were found in flounder red cells after electrophoresis. These patterns were identical in all fish studied, whether from salt or fresh water environments. Thus no inherited or adaptive changes were observed. 3. 3. Both flounder red cell carbonic anhydrase isoenzymes split a range of ester substrates. Activity was abolished with acetazolamide.