Bathymodiolus Thermophilus Research Articles

Comparative Ultrastructure of Digestive Diverticulae in Bathymodiolin Mussels: Discovery of an Unknown Spherical Inclusion (SIX) in Digestive Cells of a Seep Mussel

Mussels in the genus Bathymodiolus host endosymbiotic bacteria in their gills, from which the mussel derives much of its nutrition. Bathymodiolin mussels also have functional digestive systems and, as in shallow-water mytilid mussels, cells of the digestive diverticulae are of two types: basophilic secretory cells and columnar digestive cells. Cellular contents of secretory and digestive cells of Bathymodiolus thermophilus and Bathymodiolus brevior from deep-sea hydrothermal vents are comparable to cellular contents of these cell types observed in shallow-water mytilids. In the seep mussel Bathymodiolus heckerae, cellular contents of columnar cells were anomalous, being dominated by an unknown cellular inclusion herein called spherical inclusion unknown or SIX. SIX was observed in all digestive cells and some basophilic cells of B. heckerae examined with TEM. It is a large (2–10-μm diameter) and abundant (7 ± 1.5 inclusions per epithelial cell section) inclusion, with a double external membrane and stacked internal lamellae. No microbial DNA was detected in digestive tubules of B. heckerae using molecular probes, preferential DNA amplification techniques, or DAPI staining, suggesting that SIX is not a unicellular parasite or symbiont. The ubiquity and abundance of SIX within cells of the digestive diverticula suggest that it has an important cellular function (positive or negative), yet to be determined.

Evidence for a slightly deleterious effect of intron polymorphisms at the EF1α gene in the deep-sea hydrothermal vent bivalve Bathymodiolus

A multilocus analysis was initiated in order to infer the general effect of demography and the indirect effect of positive selection on some chromosome segments in Bathymodiolus. Mussels of the genus Bathymodiolus inhabit the very hostile, fragmented and variable environment of deep-sea hydrothermal vents which is thought to cause recurrent population bottlenecks via extinction/colonisation processes and adaptation to new environmental conditions. In the course of this work we discovered that the assumption of neutrality of non-coding polymorphisms usually made in genome scan experiments was likely to be violated at one of the loci we analysed. The direct effect of slight purifying selection on non-coding polymorphisms shares many resemblances with the indirect effect of positive selection through genetic hitchhiking. Combining polymorphism with divergence data for several closely related species allowed us to obtain different expectations for the direct effect of negative selection and the indirect effect of positive selection. We observed a strong excess of rare non-coding polymorphisms at the second intron of the EF1α gene in the two species Bathymodiolus azoricus and Bathymodiolus thermophilus, while two other loci, the mitochondrial COI gene and an intron of the Lysozyme gene, did not exhibit such a deviation. In addition, the divergence rate of the EF1α intron was estimated to be unexpectedly low when calibrated using the closure of the Panama Isthmus that interrupted gene flow between the two species. The polymorphism to divergence ratio was similar to the one observed for the other two loci, in accordance to the hypothesis of purifying selection. We conclude that slight purifying selection is likely to act on polymorphic intronic mutations of the EF1α second intron and discuss the possible relationship with the specific biology of Bathymodiolus mussels.

Hypotaurine and thiotaurine as indicators of sulfide exposure in bivalves and vestimentiferans from hydrothermal vents and cold seeps

AbstractVesicomyid clams, vestimentiferans, and some bathymodiolin mussels from hydrothermal vents and cold seeps possess thiotrophic endosymbionts, high levels of hypotaurine and, in tissues with symbionts, thiotaurine. The latter, a product of hypotaurine and sulfide, may store and/or transport sulfide non‐toxically, and the ratio to hypotaurine plus thiotaurine (Th/[H + Th]) may reflect an animal's sulfide exposure. To test this, we analyzed seep and vent animals with in situ sulfide measurements. Calyptogena kilmeri clams occur at high‐sulfide seeps in Monterey Canyon, while C. (Vesicomya) pacifica clams occur at seeps with lower levels but take up and metabolize sulfide more effectively. From one seep where they co‐occur, both had gill thiotaurine contents at 22–25 mmol kg−1 wet mass, and while C. (V.) pacifica had a higher blood sulfide level, it had a lower Th/[H + Th] (0.39) than C. kilmeri (0.63). However, these same species from different seeps with lower sulfide exposures had lower ratios. Bathymodiolus thermophilus [East Pacific Rise (EPR 9°50′ N)] from high‐(84 μm) and a low‐(7 μm) sulfide vents had gill ratios of 0.40 and 0.12, respectively. Trophosomes of Riftia pachyptila (EPR 9°50′ N) from medium‐(33 μm) and low‐(4 μm) sulfide vents had ratios of 0.23 and 0.20, respectively (not significantly different). Ridgeia piscesae vestimentiferans (Juan de Fuca Ridge) have very different phenotypes at high‐ and low‐sulfide sites, and their trophosomes had the greatest differences: 0.81 and 0.04 ratios from high‐ and low‐sulfide sites, respectively. Thus Th/[H + Th] may indicate sulfide exposure levels within species, but not in interspecies comparisons, possibly due to phylogenetic and metabolic differences. Total H + Th was constant within each species (except in R. piscesae); the sum may indicate the maximum potential sulfide load that a species faces.

Interspecific Comparison of the Mechanical Properties of Mussel Byssus

Byssally tethered mussels are found in a variety of habitats, including rocky intertidal, salt marsh, subtidal, and hydrothermal vents. One key to the survival of mussels in these communities is a secure attachment, achieved by the production of byssal threads. Although many studies have detailed the unique biomechanical properties of byssal threads, only a few prevalent species have been examined. This study assesses the variation in the mechanical properties of byssus in a broad range of mussel species from diverse environments, including intertidal and subtidal Mytilus edulis, Modiolus modiolus, Geukensia demissa, Bathymodiolus thermophilus, and Dreissena polymorpha. A tensometer was used to measure quasi-static and dynamic mechanical properties of individual threads, and several aspects of morphology were quantified. The results indicate that thread mechanical properties vary among mussel species, and several novel properties were observed. For example, of the species examined, D. polymorpha threads were the strongest, stiffest, least resilient, and fastest to recover after partial deformation. Threads of M. modiolus were characterized by the presence of two distinct yield regions prior to tensile failure. This comparative study not only provides insight into the ecological limitations and evolution of mussels, but also suggests new models for the design of novel biomimetic polymers.

Morphological variability of Bathybdella sawyeri (Hirudinida: Piscicolidae) from hydrothermal vents on the Galápagos Rift and the South East Pacific Rise

The deep-sea fish leech Bathybdella sawyeri Burreson has recently been collected from two sites on the South East Pacific Rise at approximately14° and 17°S, 2623–2586 m depth. Leeches were obtained from washings of vent mussels Bathymodiolus thermophilus (Mytilidae) and clams Calyptogena magnifica (Vesicomyidae) collected with a grab by a submersible, or were found attached to the cheliped of the crab Cyanagraea praedator (Bythograeidae) collected in baited traps at 14°S. Most individual leeches had morphology typical of B. sawyeri as originally described, with suckers of approximately equal size, but all specimens collected from the crab C. praedator had unusually large oral suckers that were distinctly larger than the caudal sucker. However, internal morphology of the specimens from C. praedator was identical to B. sawyeri on the basis of serial sections; thus all specimens were identified as B. sawyeri. The only previous report of B. sawyeri is from the Galápagos Rift, so these collections extend the range to the South East Pacific Rise.

Hydrothermal vent meiobenthos associated with mytilid mussel aggregations from the Mid-Atlantic Ridge and the East Pacific Rise

Deep-sea hydrothermal vents occur along the mid-ocean ridges and back-arc basins around the globe. There are very few community analyses of vent meiobenthos. The central objectives of this study were to identify and quantify for the first time the entire metazoan meiobenthic community associated with mussel aggregations of Bathymodiolus thermophilus Kenk and Wilson, 1985 from the EPR, 11°N and of Bathymodiolus puteoserpentis Cosel et al., 1994 from the Mid-Atlantic Ridge (MAR), 23°N. Using a quantitative sampling method, abundance, biomass, sex ratio, species richness, diversity, evenness, and trophic structure were studied based on three samples from each site. Meiobenthic abundance in each sample was unexpectedly low, but similar between sites. The community was composed of nematodes, copepods, ostracods, and mites, with a total of 24 species at EPR vents, and 15 species at MAR vents. While most copepod species were vent endemics within the family Dirivultidae, nematodes and harpacticoid copepods belonged to generalist genera, which occur at a variety of habitats and are not restricted to hydrothermal vents or the deep sea. The meiobenthos of hydrothermal-vent mussel beds constitutes a unique community unlike those of other sulfidic habitats, including the thiobios of shallow-water sediments and the meiobenthos of deep-sea, cold-seep sediments. The trophic structure was dominated by primary consumers, mainly deposit feeders, followed by parasites. Predatory meiofaunal species were absent.

Metal influence on metallothionein synthesis in the hydrothermal vent mussel Bathymodiolus thermophilus

The present study reports on the metallothionein expression in the hydrothermal vent mussel Bathymodiolus thermophilus. Metallothioneins (MT) are proteins involved in intracellular metal regulation and conserved throughout the animal kingdom. The hydrothermal vent environment presents peculiarities (high levels of sulfides and metals, low pH, anoxia) that may have driven associated species to develop original evolutionary ways to face these extreme living conditions. Mussels were exposed to different metal solutions at the atmospheric pressure. The MT mRNA levels and MT contents were measured in gills and mantles of each exposed mussel. The intracellular metal distribution was estimated in fractions obtained after the centrifugation of tissue homogenates. A few of the tested metals (Ag, Cu, Cd, Hg and Zn) were able to significantly induce MT mRNA levels. Silver was the only one that produced a significant increase of the MT protein level in both mantle and gills. The gills always presented higher MT protein levels than the mantle did, while their MT mRNA levels were similar. Our data show that MT mRNA and MT protein levels do not follow a clear relationship in the gills and mantle of B. thermophilus and we assume that a posttranscriptional control occurs in these mussels.

Small Metallothionein MT-10 Genes in Coastal and Hydrothermal Mussels

Metallothioneins (MTs) are important proteins in the intracellular regulation of metals. In the Mytilidae family, which includes many economically important species, 2 major forms of MTs have been reported: MT-10 (10 kDa) and MT-20 (20 kDa). Many different MT-10 proteins have been isolated from the common species Mytilus edulis, which suggests that distinct MT-10 genes may occur in a single specimen. Some MT genes, involving 3 exons and 2 large introns, have been isolated in Mytilidae. Our aim was to determine whether intron-free forms of the MT-10 genes can exist, which could allow rapid transcription in response to exposure to metals. Our study focused on 2 species living under very different environmental conditions: Mytilus edulis (a coastal mussel) and Bathymodiolus thermophilus (a hydrothermal mussel). We report here the first description of small, intron-free MT-10 genes, possessing a correct open reading frame in these 2 species.

Composition of a One-Year-Old Riftia pachyptila Community Following a Clearance Experiment: Insight to Succession Patterns at Deep-Sea Hydrothermal Vents

In the basalt-hosted hydrothermal vent habitat around 9°50 N on the East Pacific Rise, the vestimentiferan tubeworms Tevnia jerichonana and Riftia pachyptila (Polychaeta: Siboglinidae) commonly settle before the mussel Bathymodiolus thermophilus (Bivalvia: Mytilidae). We removed six aggregations of R. pachyptila and deployed mussels on the cleared sources of diffuse flow to test the effect of the B. thermophilus on the subsequent colonization by the tubeworms. None of the transplanted mussels persisted on the cleared sources of diffuse flow; however, aggregations of R. pachyptila grew in half of the clearances. We collected one of the aggregations of R. pachyptila along with the associated fauna for determination of relative abundance and biomass in this one-year-old community. This aggregation consisted of 647 specimens of R. pachyptila that hosted individuals of 24 species, including small individuals of T. jerichonana and B. thermophilus. The abundance of the associated fauna was numerically dominated by gastropods, and the biomass was dominated by the Alvinellid polychaete Paralvinella grasslei. Following an eruption, the vestimentiferan tubeworms Tevnia jerichonana and Riftia pachyptila (Polychaeta: Siboglinidae) quickly colonize the basalt-hosted hydrothermal vents around 9°50 N (East Pacific Rise) and soon grow to visually dominate these habitats (1, 2, 3). Colonization by the vent mussel Bathymodiolus thermophilus often follows shortly, and the mussels eventually overgrow the tubeworms at most sites (2, 4, 5). Both tubeworms and mussels harbor sulfur-oxidizing chemoautotrophic endosymbionts, which provide the bulk of their nutrition (6, 7). However, mussels have also maintained the ability to filter-feed (8), which apparently allows them to occupy a wider range of environmental conditions and better tolerate declining hydrothermal vent activity than the tubeworms (9). The mechanism of ecological succession in these hydrothermal vent habitats is not fully understood, but may be regulated by a combination of pre-settlement factors, such as the use of biogenic cues (3) and the response to geochemical changes (2), and post-settlement factors, such as physical overgrowth (4) and resource competition (5). We conducted a manipulative experiment to test the hypothesis that the mussel B. thermophilus prevents further colonization of a vent by tubeworms. In May 1998, six aggregations of the giant tubeworm R. pachyptila were cleared from a site named Riftia Field (9°50.705 N 104°17.593 W). The original location and source of diffuse flow for each aggregation were marked and recorded on video. After the aggregations were cleared, the temperatures of the venting fluid were measured, using the low-temperature probe of the DSV Alvin, and ranged from 20–22 °C above ambient at the six locations. Then about 600 mussels were collected from a nearby site (“East Wall”, 9°50.614 N 104° 17.509 W) and about 200 were deployed over each of three cleared sources of diffuse flow (treatments). Mussels were not deployed on the three other cleared areas (conReceived 28 April 2004; accepted 4 October 2004. * To whom correspondence should be addressed. E-mail: breea@psu.edu † Present address: Department of Fisheries and Aquatic Sciences, University of Florida, Gainesville, FL 32653. Reference: Biol. Bull. 207: 177–182. (December 2004) © 2004 Marine Biological Laboratory

Do organisms living around hydrothermal vent sites contain specific metallothioneins? The case of the genus Bathymodiolus (Bivalvia, Mytilidae)

The unusual characteristics of the hydrothermal vent environment (high pressure and metal concentrations, low pH, etc.) leads us to wonder how species living in this particular biotope have adjusted to these severe living conditions. To investigate the consequences of high metal concentrations, filter-feeding organisms are commonly used in ecotoxicological studies. Metallothioneins (MTs) are proteins conserved throughout the animal kingdom and involved in intracellular metal regulation Therefore, we tried here to find out whether the metallothioneins of hydrothermal bivalves are different from those of coastal bivalves. The characterization of DNA sequences coding MTs from some of the most common hydrothermal bivalves, belonging to the genus Bathymodiolus (Mytilidae) was performed by reverse transcription polymerase chain reactions (RT-PCRs). The complementary DNAs (cDNAs) of MT-10 and MT-20 isoforms were obtained for the Atlantic and Pacific hydrothermal mussels ( Bathymodiolus azoricus and Bathymodiolus thermophilus). The MT-10 transcripts were 222 nucleotides long and the MT-20 transcripts, 207 nucleotides. The polymorphism of the MT cDNAs in these two hydrothermal species is discussed. The comparison between metallothionein cDNA sequences of the Mytilus and the Bathymodiolus genera shows strong homologies among metallothioneins of coastal and hydrothermal mussels.

Tissue and symbiont condition of mussels (Bathymodiolus thermophilus) exposed to varying levels of hydrothermal activity

Bathymodiolus thermophilus is a mixotrophic mussel capable of deriving nourishment from sulphur-oxidizing, bacterial endosymbionts in its gills and from suspended particulates through mucociliary feeding. These mussels become nutritionally stressed when removed from sulphide-rich fluid, but the dynamics of the host/symbiont relationship during this process are still not fully understood. Bathymodiolus thermophilus mussels were collected from an active and a waning hydrothermal site on the southern East Pacific Rise (EPR) and were removed from active venting for 12 d at a site on the northern EPR. Light and transmission electron microscopy showed that gill symbionts were lost from gill bacteriocytes in transplant mussels and that mussels exposed to longer periods of sulphide deprivation suffered deterioration of gill structure and body condition. There was no evidence to support the hypothesis of a shift toward reliance on photosynthetically derived organic material in mussels at the waning vent site.

Aspects of the feeding biology of the pectinacean Bathypecten vulcani, a peri-hydrothermal vent bivalve

The feeding biology of Bathypecten vulcani, a peri-hydrothermal vent pectinacean, was investigated using histological and scanning electron microscope studies of the gills and stomach contents of specimens sampled from the 9° and 13°N sites of the East Pacific Rise. Salient characteristics were compared with those of Bathymodiolus thermophilus from the same and similar habitats. The gills of Bathypecten vulcani displayed heterogeneous organic and mineral particles on their frontal surface. The digestive tract possessed well-developed structures (whose anatomical and cytological characteristics indicated full functionality), typically found in bivalves from littoral aerobic environments, as well as in Bathymodiolus thermophilus, which is capable of suspension-feeding: oesophagus, stomach, crystalline style, digestive gland, and intestine. Observations of stomach contents revealed diverse particles from the photic zone, including debris from diatoms and coccolithophorans. In contrast to Bathymodiolus thermophilus, bacteria were rarely observed in the digestive tract, indicating a more complete reliance on surface-originating particles.

16S rDNA-based phylogeny of sulphur-oxidising bacterial endosymbionts in marine bivalves from cold-seep habitats

The phylogenetic relationship of sulphur-oxidising endosymbiotic bacteria from bivalves of the families Vesicomyidae (Calyptogena sp. C1, Calyptogena sp. C3), Solemyidae (Acharax sp.) and Thyasiridae (Conchocele sp.) from cold-seep habitats were determined by 16S rDNA nucleotide sequence analyses. The endosymbiotic bacteria form distinct groups within the gamma-Proteobacteria and are well separated from each other and from free-living sulphur- oxidising bacteria of the genera Beggiatoa, Halothiobacillus and Thiomicrospira. The endosymbiotic bacteria of Acharax sp. from cold seeps off Oregon, Indonesia and Pakistan have sequences highly similar to each other but quite distinct from other thiotrophic endosymbionts. This includes endosym- bionts from Solemya spp., to which they are distantly related. Symbiotic bacteria of Conchocele sp. from a cold seep in the Sea of Okhotsk are similar to those of Bathymodiolus thermophilus and related species, as shown by their overall sequence similarity and by signature sequences. The endosymbiotic bacteria of Calyptogena spp. from cold seeps off Oregon and Pakistan are closely related to those of other vesicomyids. Endosymbiont species found off Oregon corresponded to 2 different clusters of Calyptogena spp. symbionts in the same samples. The results corroborate the hypothesis of a monophyletic origin of the symbionts in vesicomyid clams, and support the existence of deeply branching groups in solemyid symbionts and of divergent lines and distribution for thyasirid symbionts. The results also indicate that certain symbiont species cluster according to the depth distribution of their hosts, and that in consequence host species together with their symbionts may have undergone depth-specific adaptation and evolution.

Anaerobic sulfur metabolism in thiotrophic symbioses.

Hydrogen sulfide is generally accepted to be the energy source for the establishment of sulfur-oxidizing symbiotic communities. Here, we show that sulfur-storing symbioses not only consume but also produce large amounts of hydrogen sulfide. The prerequisite for this process appears to be the absence of oxygen. Anaerobic sulfide production is widespread among different thiotrophic symbioses from vent and non-vent sites (Riftia pachyptila, Calyptogena magnifica, Bathymodiolus thermophilus, Lucinoma aequizonata and Calyptogena elongata). The extent of H2S generation correlates positively with the amount of elemental sulfur stored in the symbiont-bearing tissues of the hosts. Sulfide production starts a few hours after anoxia sets in, with H2S initially accumulating in the circulatory system before it is excreted into the surrounding environment. We propose that not sulfate but the elemental sulfur deposited in the symbionts serves as a terminal electron acceptor during anoxia and is reduced to sulfide. In anoxia-tolerant symbioses such as L. aequizonata, anaerobic sulfur respiration may be important for producing maintenance energy to help the species survive several months without oxygen. The increased levels of cysteine in the gills of L. aequizonata may be caused by a lack of reoxidation due to the absence of oxygen.

Molecular and morphological identification of settlement‐stage vent mussel larvae, Bathymodiolus azoricus (Bivalvia: Mytilidae), preserved in situ at active vent fields on the Mid‐Atlantic Ridge

This paper describes the first successful attempt to trap and identify the larvae of a deep‐sea vent organism using a combination of sediment traps and molecular analysis. During the European Union‐funded MARVEL cruise in August and September 1997, sediment traps containing a high‐salt DNA preservative were deployed around active black smoker chimneys on the newly discovered Rainbow vent field in an attempt to collect larval stages of the dominant vent bivalve Bathymodiolus azoricus. A total of 2,055 shelled mussel larvae was collected within a 2‐week period, all of which were of identical size and shell morphology that indicated that they were at the settlement, prodissoconch II stage. Scanning electron microscopy of the shell hinge indicated that they belonged to the family Mytilidae, but it required molecular analysis to confirm their species identity. Polymerase chain reaction (PCR) amplification of the ITS2 region was performed on a subset of the larvae, and the resulting PCR products were cut with diagnostic restriction endonucleases to allow comparison with a DNA database based on adult specimens. The DNA restriction patterns typifying the Rainbow larvae were identical to those of adult B. azoricus from the Menez Gwen, Lucky Strike, and Rainbow vent sites and were significantly different from those that typified Bathymodiolus thermophilus (Pacific) and Bathymodiolus puteoserpentis (Snake Pit vent field on the Mid‐Atlantic Ridge), which clearly points to the Rainbow larvae having their origin on that part of the ridge close to the Azores. These findings point to the value of sediment traps as a way to study the temporal and spatial aspects of larval settlement in deep‐sea hydrothermal vent environments.

Tubeworm succession at hydrothermal vents: use of biogenic cues to reduce habitat selection error?

Species colonizing new deep-sea hydrothermal vents along the East Pacific Rise show a distinct successional sequence: pioneer assemblages dominated by the vestimentiferan tubeworm Tevnia jerichonana being subsequently invaded by another vestimentiferan Riftia pachyptila, and eventually the mussel Bathymodiolus thermophilus. Using a manipulative approach modified from shallow-water ecological studies, we test three alternative hypotheses to explain the initial colonization by T. jerichonana and its subsequent replacement by R. pachyptila. We show that R. pachyptila and another vestimentiferan, Oasisia alvinae, colonized new surfaces only if the surfaces also were colonized by T. jerichonana. This pattern does not appear to be due to restricted habitat tolerances or inferior dispersal capabilities of R. pachyptila and O. alvinae, and we argue the alternative explanation that T. jerichonana facilitates the settlement of the other two species and is eventually outcompeted by R. pachyptila. Unlike the classic model of community succession, in which facilitating species promote their own demise by modifying the environment to make it more hospitable for competitors, we suggest that T. jerichonana may produce a chemical substance that induces settlement of these competitors. This process of selecting habitat based on biogenic cues may be especially adaptive and widespread among later-successional species that occupy a physically variable and unpredictable environment. In these cases, the presence of weedy species implies some integrated period of environmental suitability, whereas an instantaneous assessment of physical habitat conditions, such as water temperature for vent tubeworms, provides a poorer predictor of long-term habitat suitability.

Calcium carbonate dissolution rates in deep-sea bivalve shells on the East Pacific Rise at 21°N: results of an 8-year in-situ experiment

Analysis of shell fragments of two common deep-sea hydrothermal vent bivalves, the vesicomyid clam Calyptogena magnifica and the mytilid Bathymodiolus thermophilus, deployed more than 100 m from any active hydrothermal vent location at 20°50′N, 109°06′W on the East Pacific Rise indicates significant variation of calcium carbonate dissolution in in-situ exposures of more than 8 years. Shell fragments embedded in epoxy blocks, mounted on a buoyed and anchored polypropylene line, and deployed by DSV Alvin (depth = 2615 m) were continuously exposed to ambient seawater (∼2°C) conditions immediately above the seafloor. The mean dissolution rate for the aragonitic shell of C. magnifica amounted to 27.7 μm/yr. Dissolution rates varied significantly among different shell layers of the clam, with the middle fine to irregular complex crossed lamellar shell layer exhibiting the lowest rates (mean = 22.2 μm/yr) and the irregular prismatic shell layer the highest rates (mean = 36.9 μm/yr). In the mytilid B. thermophilus, the dissolution rate of the aragonitic shell layer averaged 41.6 μm/yr, while that of the calcitic shell layer was immeasurable. The rates of calcium carbonate dissolution reported here for a ridge-crest site remote from any active hydrothermal vent are much lower than those previously documented for active vent sites at 21°N, the Guaymas Basin (Southern Trough), and Galapagos Rift (Rose Garden). Assuming a constant rate of dissolution, we estimate that empty adult shell valves of C. magnifica at the experimental site would dissolve completely in a period of ∼300 years, which has important implications for determining the longevity of hydrothermal vent activity along the rise axis.

Characterization and functional properties of the extracellular coelomic hemoglobins from the deep-sea, hydrothermal vent scalewormBranchipolynoe symmytilida

Polychaete species belonging to the genus Branchipolynoe are commensal with mussels from deep-sea hydrothermal vents and cold-seeps. Possessing hemoglobins (Hbs), the species B. symmytilida, which is found in the mussel Bathymodiolus thermophilus on the East Pacific Rise, is exceptional in a family normally devoid of respiratory pigments. In a previous paper we described two major coelomic extracellular hemoglobins with unique quaternary structures. Aiming to discern respiratory adaptations to the highly variable hydrothermal environment, this paper characterizes the functional properties of these Hbs and the coelomic fluid. The two major hemoglobins (C1 and C2) exhibit spectrophotometric characteristics of both intra- and extracellular hemoglobins. However, their amino acid content is very different from other known hemoglobins and is characterized by a high proportion of alanine and glycine (up to 40% cumulated in C1). C1 and C2 differ markedly by their cysteine content (0.8% and 13% respectively). The coelomic fluid exhibits a strong buffer capacity due to the high hemoglobin content (3 mM heme). In vitro, CO2 accumulation (up to 10-12 mM CO2 for PCO2 = 7.5 Torr) occurs with limited pH changes and is only partly accounted for by carbamino-Hb formation. The two hemoglobins exhibit high oxygen-affinities (P50 0.4 Torr for C1 and 0.9 Torr for C2, at 10 degrees C, pH 8) and a normal Bohr effect (phi values ranging from -0.54 and -0.37 at 10 degrees C, to -0.24 and -0.28 at 30 degrees C, for C1 and C2, respectively). Cooperativity values range from 0.8 to 1.9 for C1 and from 0.8 to 1.7 for C2. The temperature sensitivity of O2 affinity reflect deltaH values that decrease from -30 to -60 kJ x mol(-1) with increasing pH. C2 exhibits a slight specific effect of CO2 on oxygenation properties.

Pathways of inorganic nitrogen assimilation in chemoautotrophic bacteria-marine invertebrate symbioses: expression of host and symbiont glutamine synthetase

Symbioses between chemoautotrophic bacteria and marine invertebrates living at deep-sea hydrothermal vents and other sulfide-rich environments function autotrophically by oxidizing hydrogen sulfide as an energy source and fixing carbon dioxide into organic compounds. For chemoautotrophy to support growth, these symbioses must be capable of inorganic nitrogen assimilation, a process that is not well understood in these or other aquatic symbioses. Pathways of inorganic nitrogen assimilation were investigated in several of these symbioses: the vent tubeworms Riftia pachyptila and Tevnia jerichonana, the vent bivalves Calyptogena magnifica and Bathymodiolus thermophilus, and the coastal bivalve Solemya velum. Nitrate reductase activity was detected in R. pachyptila, T. jerichonana and B. thermophilus, but not in C. magnifica and S. velum. This is evidence for nitrate utilization, either assimilation or respiration, by some vent species and is consistent with the high levels of nitrate availability at vents. The ammonia assimilation enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were detected in all symbioses tested, indicating that ammonia resulting from nitrate reduction or from environmental uptake can be incorporated into amino acids. A complicating factor is that GS and GDH are potentially of both host and symbiont origin, making it unclear which partner is involved in assimilation. GS, which is considered to be the primary ammonia-assimilating enzyme of autotrophs, was investigated further. Using a combination of molecular and biochemical approaches, host and symbiont GS were distinguished in the intact association. On the basis of Southern hybridizations, immunoreactivity, subunit size and thermal stability, symbiont GS was found to be a prokaryote GS. Host GS was distinct from prokaryote GS. The activities of host and symbiont GS were separated by anion-exchange chromatography and quantified. Virtually all activity in symbiont-containing tissue was due to symbiont GS in R. pachyptila, C. magnifica and B. thermophilus. In contrast, no symbiont GS activity was detected in the gill of S. velum, the predominant activity in this species appearing to be host GS. These findings suggest that ammonia is primarily assimilated by the symbionts in vent symbioses, whereas in S. velum ammonia is first assimilated by the host. The relationship between varying patterns of GS expression and host-symbiont nutritional exchange is discussed.

Comparison between isolation protocols commonly used for the purification of mollusc metallothioneins

Two centrifugation methods (at 30 000 and 100 000 g) combined with two isolation procedures (heat denaturation and solvent precipitation) were tested to check if their use introduced significant differences in the quantification of metals (Cd, Cu, Zn) and metallothioneins in blue mussel organs (gills and digestive gland). No contaminating compounds originating from the lowest centrifugation speed were likely to interfere with quantitative measurements of metal, protein and metallothionein. As isolation treatments act on specific properties of proteins in general and metallothionein isoforms in particular they cannot be used interchangeably. Therefore, any comparisons of MT amounts in bivalve tissues with data from the literature have to take into account the isolation procedure. Similar results were obtained with three other molluscs, Crassostrea gigas, Bathymodiolus thermophilus (hydrothermal mussel), and Corbicula fluminea (estuarine bivalve).