The gill symbiont of the hydrothermal vent mussel Bathymodiolus thermophilus is a psychrophilic,chemoautotrophic, sulfur bacterium

Certain hydrothermal vent invertebrates, e.g. Riftia pachyptila and Calyptogena magnifica, are clearly established as harboring dense populations of chemoautotrophic sulfur bacteria in specialized tissues. By contrast, the physiological characteristics of the abundant intracellular gill symbiont of the vent mussel Bathymodiolus thermophilus have been questioned. The low activities of enzymes diagnostic for CO₂ fixation (Calvin cycle) and for sulfur-driven energy generation, as measured by other investigators, have been attributed to bacterial contamination of the gill surface. Based on research at the Galápagos Rift hydrothermal vents in 1988 and subsequent laboratory experiments, the current study confirms that the B. thermophilus symbiont is a psychrophile for which thiosulfate and sulfide stimulate CO₂ fixation. It strongly indicates that the symbiont is a chemoautotroph by establishing the following: (1) Sulfide and thiosulfate can stimulate CO₂ fixation by partially purified symbionts by up to 43-fold and 120-fold, respectively; (2) the ribulose-1,5-bisphosphate carboxylase/oxygenase activity of the symbiont is sufficient to account for its sulfide- or thiosulfate-stimulated CO₂ incorporation; (3) the symbiont's molar growth yield on thiosulfate, as judged by CO₂ incorporation, is indistinguishable from that of free-living chemoautotrophs. Due to the high protein-degrading activity of B. thermophilus gill lysate, it is also suggested that host lysis of symbionts plays a more important role in the nutrition of the vent mussel than in R. pachyptila or C. magnifica, for which no comparable protein-degrading activity was found.     

A new bathymodioline mussel symbiosis at the Juan de Fuca hydrothermal vents

Until recently, the only major hydrothermal vent biogeographic province not known to include bathymodioline mussels was the spreading centers of the northeast Pacific, but deep-sea dives using DSV Alvin on the Endeavor segment of the Juan de Fuca Ridge (47°56N 129°06W; ∼2,200 m depth) in August 1999 yielded the only recorded bathymodioline mytilids from these northeastern Pacific vents. One specimen in good condition was evaluated for its relatedness to other deep-sea bathymodioline mussels and for the occurrence of chemoautotrophic and/or methanotrophic symbionts in the gills. Phylogenetic analyses of the host cytochrome oxidase I gene show this mussel shares evolutionary alliances with hydrothermal vent and cold seep mussels from the genus Bathymodiolus, and is distinct from other known species of deep-sea bathymodiolines, suggesting this mussel is a newly discovered species. Ultrastructural analyses of gill tissue revealed the presence of coccoid bacteria that lacked the intracellular membranes observed in methanotrophic symbionts. The bacteria may be extracellular but poor condition of the fixed tissue complicated conclusions regarding symbiont location. A single gamma-proteobacterial 16S rRNA sequence was amplified from gill tissue and directly sequenced from gill tissue. This sequence clusters with other mussel chemoautotrophic symbiont 16S rRNA sequences, which suggests a chemoautotrophic, rather than methanotrophic, symbiosis in this mussel. Stable carbon (δ¹³C = −26.6%) and nitrogen (δ¹⁵N = +5.19%) isotope ratios were also consistent with those reported for other chemoautotroph-mussel symbioses. Despite the apparent rarity of these mussels at the Juan de Fuca vent sites, this finding extends the range of the bathymodioline mussels to all hydrothermal vent biogeographic provinces studied to date.     

Methanotrophic symbiont location and fate of carbon incorporated from methane in a hydrocarbon seep mussel

Seep Mytilid Ia (SMIa), an undescribed mussel found at hydrocarbon seeps in the Gulf of Mexico, harbors intracellular methanotrophic symbionts. Two techniques were used to address the hypothesis that host digestion of symbionts is a significant mechanism of carbon transfer from symbiont to host in the SMIa association: lysosomal enzyme cytochemistry and ¹⁴C tissue autoradiography. Acid phosphatase activity was consistently localized in the Golgi apparatus and associated vesicles of gill cells, but was detected around bacteria in only three of approximately 50 bacteriocytes examined. These results indicate that the cellular equipment necessary for lysosomal digestion of symbionts is present in host bacteriocytes, but that acid phosphatase activity in symbiont vacuoles is rare at a given point in time. Tissue autoradiography was conducted with mussels collected in September 1992 to document carbon fixation by symbionts and follow the time course of transfer to host tissues. No asymbiotic host cell type showed a significant increase in relative grain density until at least 1 d after the end of incubation with ¹⁴C-methane. The ratio of label in the basal portion of bacteriocytes to total bacteriocyte label did not show a significant increase until 10 d after the end of the incubation period, indicating a slow increase of labeled carbon in the putative residual bodies, containing the remnants of lysosomal digestion. These results are consistent with the hypothesis that host digestion of symbionts is one route of nutrient acquisition in SMIa. Intracellular methanotrophic bacteria were found outside of the gill in SMIa juveniles, in mantle and foot epithelial tissues previously believed to be symbiont-free. These extra-gill symbionts and their host cells are morphologically similar to their gill counterparts and, like the gill symbionts, actively fix carbon from methane. Received: 29 March 1997 / Accepted: 12 May 1997     

The methane mussel: roles of symbiont and host in the metabolic utilization of methane

Methane mussels (Bathymodiolus sp., undescribed; personal communication by R. Turner to CRF) were collected in September 1989 and April 1990 from offshore Louisiana in the Gulf of Mexico. These mussels contain endosymbiotic methane-oxidizing bacteria and are capable of utilizing environmental methane as a source of energy and carbon. Oxygen consumption, methane consumption, and carbon dioxide production were measured in mussels with intact symbionts, functionally aposymbiotic mussels, and separated symbiont preparations under controlled oxygen and methane conditions, in order to study the roles of the symbionts and the hosts in methane utilization. The association was found to be very efficient in fixing methane carbon (only 30% of CH₄ consumed is released as CO₂), and to be capable of maximal rates of net carbon uptake of nearly 5 mol g^-1 h^-1. Rates of oxygen and methane consumption were dependent upon oxygen and methane concentrations. Maximal consumption rates were measured at 250 to 300 M O₂ and 200 to 300 M CH₄, under which conditions, oxygen consumption by the gill tissues (containing symbionts) had increased more than 50-fold over rates measured in the absence of methane. A model is proposed for the functioning of the intact association in situ, which shows the symbiosis to be capable of achieving growth rates (net carbon assimilation) in the range of 0.003 to 0.50% per day depending upon oxygen and methane concentrations. Under the conditions measured in the seep environment (200 M O₂, 60 M CH₄), a mussel consuming methane at rates found to be typical (4 to 5 mol g^-1 h^-1) should have a net carbon assimilation rate of about 0.1% per day. We suggest that the effectiveness of this symbiosis arises through integration of the morphological and physiological characteristics inherent to each of the symbiotic partners, rather than from extensive specialization exhibited by other deep-sea chemotrophic associations.     

Phenotypic variations in the gills of the symbiont-containing bivalve Lucinoma aequizonata

The marine bivalve Lucinoma aequizonata (Lucinidae) maintains a population of sulfide-oxidizing chemoautotrophic bacteria in its gill tissue. These are housed in large numbers intracellularly in specialized host cells, termed bacteriocytes. In a natural population of L. aequizonata, striking variations of the gill colors occur, ranging from yellow to grey, brown and black. The aim of the present study was to investigate how this phenomenon relates to the physiology and numbers of the symbiont population. Our results show that in aquarium-maintained animals, black gills contained fewer numbers of bacteria as well as lower concentrations of sulfur and total protein. Nitrate respiration was stimulated by sulfide (but not by thiosulfate) 33-fold in homogenates of black gills and threefold in yellow gill homogenates. The total rates of sulfide-stimulated nitrate respiration were the same. Oxygen respiration could be measured in animals with yellow gills but not in animals with black gills. The cumulative data suggest that black-gilled clams maintained in the aquarium represent a starvation state. When collected from their natural habitat black gills contain the same number of bacteria as yellow gills. Also, no significant difference in glycogen concentrations of the host tissues was observed. Therefore, starvation is unlikely the cause of black gill color in a natural population. Alternative sources of nutrition to sulfur-based metabolism are discussed. Denaturing gradient gel electrophoresis (DGGE) performed on the different gill tissues, as well as on isolated symbionts, resulted in a single gill symbiont amplification product, the sequence of which is identical to published data. These findings provide molecular evidence that one dominant phylotype is present in the morphologically different gill tissues. Nevertheless, the presence of other phylotypes cannot formally be excluded. The implications of this study are that the gill of L. aequizonata is a highly dynamic organ which lends itself to more detailed studies regarding the molecular and cellular processes underlying nutrient transfer, regulation of bacterial numbers and host–symbiont communication. Received: 1 September 1999 / Accepted: 1 February 2000     

Chemoautotrophic symbionts and translocation of fixed carbon from bacteria to host tissues in the littoral bivalve Loripes lucinalis (Lucinidae)

Specimens of Loripes lucinalis (Lucinidae) living in reducing sediments were collected near a sewage outfall at low tide on the Moulin Blanc beach, Brest, France, from January to March 1987. Electron microscope studies revealed numerous Gram-negative-type bacteria in the gill cells. Ribulosebiphosphate carboxylase, a diagnostic enzyme of the Calvin-Benson cycle of CO₂-fixation was measured only in the gill extracts. Various tissues of L. lucinalis were examined for activity of APS reductase, (EC 1.8.99.2), ATP sulphurylase (EC 2.7.7.4) and rhodanese (EC 2.8.1.1), enzymes involved in sulphide oxidation. APS reductase was only found in symbiont-containing tissues, i.e., gills. These enzymatic studies characterise the symbionts as chemoautotrophic sulphide-oxidizing bacteria. Histoautoradiography demonstrated that part of the carbon dioxide fixed by symbiotic bacteria in the gills is translocated to symbiont-free tissues of the bivalve. The ultrastructure of the gill is detailed and a nomenclature based on established and new terminology is proposed to describe the various cellular types comprising the gill filament.     

Allometry of gill weights,gill surface areas,and foot biomass δ<Superscript>13</Superscript>C values of the chemoautotroph–bivalve symbiosis<Emphasis Type="Italic"> Solemya velum</Emphasis>

The protobranch bivalve Solemya velum Say, 1822 has large gills, which harbor chemolithoautotrophic bacteria that supply the majority of the clams organic carbon. A substantial portion of the CO₂, O₂, H₂S, and other nutrients necessary for symbiont autotrophy and host heterotrophy are acquired from the environment through the gills, whose large size may be necessary to facilitate the acquisition of sufficient O₂ from S. velums habitat to meet the combined demands of the host and symbionts. Large gills may also result in an oversupply of CO₂, which may in turn be responsible for the isotopically depleted ¹³C values observed in S. velum biomass (–31 to –34). Alternatively, gill hypertrophy may simply be an adaptation to house a large population of symbionts adjacent to their environmental source of dissolved gases and other nutrients. To better understand gill function in this symbiosis, gill weights, gill surface areas, and foot ¹³C values were measured as a function of total weights. S. velum gill weights were found to be a substantial portion of total clam weight, averaging 38% of wet weight, compared to nonsymbiotic protobranch bivalves Yoldia limatula Say, 1831 (5%) and Nucula proxima Say, 1822 (11%). Gill weights are a smaller percentage of total weight in larger individuals; the allometric equation for gill weight (G) as a function of total weight (M) is G=0.26M^0.85. Dry weights scale similarly. Gill surface areas are immense; the average gill surface area measured was 107 cm² g^–1 total soft tissue wet weight, the highest value for any marine invertebrate. Gill surface area (SA) also scales with size (SA=69.8M^0.85). When gill surface areas were calculated with respect to gill wet weights, they did not scale with size. The ¹³C values do not scale with size either, consistent with high rates of CO₂ supply at all sizes. Extraordinarily high rates of CO₂ supply relative to demand are supported by a model for CO₂ delivery based on Ficks law and the allometric relationship between surface areas and total weight, consistent with a role for large gill surface areas in the generation of isotopically depleted tissue ¹³C values.Communicated by J. P. Grassle, New Brunswick     

Physiological and immunological evidence for two distinct C1-utilizing pathways in Bathymodiolus puteoserpentis (Bivalvia: Mytilidae), a dual endosymbiotic mussel from the Mid-Atlantic Ridge

The existence of endosymbiotic sulfur-oxidizing chemoautotrophic and methanotrophic bacteria associating with marine mytilid mussels has previously been inferred by 16S rDNA analysis in Bathymodiolus puteoserpentis Von Cosel et al. 1994, a hydrothermal vent mussel from a site on the Mid-Atlantic Ridge. In mussels collected in June 1993, we found evidence of enzymes diagnostic of two distinct C₁ assimilation pathways in this symbiosis. Assays for the utilization of radiolabelled methane and for immunodetection of methanol dehydrogenase were positive, indicating that oxidation and incorporation of this substrate are occurring in this symbiosis. Sulfide or thiosulfate had no detectable stimulatory effect on CO₂ incorporation, and assays for the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), an enzyme diagnostic for the Calvin–Benson cycle, were negative. RubisCO was detected in all samples examined by immunoblot analysis, indicating this enzyme is expressed in the B. puteoserpentis symbiosis. Stable isotope data showed that carbon isotope values were in agreement with previously reported values, and nitrogen isotope values were among the most depleted ever reported for bivalve symbioses. The carbon isotope values do not preclude the utilization of vent-derived methane. These data could be explained by the presence of two metabolically distinct bacterial symbionts or a Type X methanotrophic symbiont. Received: 3 October 1997 / Accepted: 23 July 1998     

Role of filter-feeding in the nutritional biology of a deep-sea mussel with methanotrophic symbionts

The ability of an undescribed deep-sea hydrocarbon-seep mussel which contains endosymbiotic methanotrophic bacteria to clear, ingest, and assimilate radiolabeled bacteria (Vibrio pelagicus andEscherichia coli) and algae (Dunaliella tertiolecta) was compared with that of the bay musselMytilus edulis. The seep mussel, collected in August 1987 from the Louisana Slope in the Gulf of Mexico, was slower to clear bacteria and algae thanM. edulis. The ingestion and assimilation of filtered bacteria and algae was established from the presence of radiolabel in mussel tissues and feces. The seep mussel was somewhat less efficient in assimilating radiolabeled components from bacteria and algae thanM. edulis. The dietary carbon maintenance-requirement of the seep mussel could potentially be met at environmental concentrations of greater than 10⁶ bacteria ml^–1. At lower concentrations of particulate organic matter, filter-feeding could be an important source of nitrogen and essential nutrients not supplied by the endosymbionts.     

Cytoenzymatic investigation of intracellular digestion in the symbiont-bearing hydrothermal bivalve <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis>

Invertebrates harbouring endosymbiotic chemoautotrophic bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. Bathymodiolids are dominants of the biomass at geochemically distinct vent sites of the Mid Atlantic Ridge (MAR) and thus are good candidates to study biological processes in response to site-specific conditions. To satisfy their nutritional requirements, these organisms depend to varying extent on two types of chemoautotrophic symbionts and on filterfeeding. The quantitative relationships of the nutritional modes are poorly understood. Using enzyme cytochemistry, electron microscopy and X-ray microanalysis, the structural and functional aspects of the cellular equipment necessary for lysosomal digestion was studied. We provide evidence for the following: (1) the basis of intracellular digestion of symbionts in Bathymodiolus azoricus from two geochemically distinct vent sites was not mainly in the large lysosomal bodies as previously thought (based on the membranous content resembling bacteria); (2) senescent bacteria are autolysed, possibly by bacterial acid phosphatase, that is more likely a cell cycling of the symbionts rather than an active lysosomal digestion by the host; (3) the consistent absence of hydrolases may indicate the improper use of the name “lysosome” for large vesicles at the base of the gill bacteriocytes (4) nutrient transfer in B. azoricus, therefore, may more likely be accomplished through leaking of metabolites from the symbiont to the host, not excluding lysosomal resorption of dead bacteria as an auxiliary strategy for organic molecule transfer; (5) evidence is provided for microvillar transfer of substances from the seawater that may indicate filter-feeding, in non-symbiotic ciliated gill cells of mussels from Lucky Strike; (6) two types of lysosomal vesicles can be distinguished in digestive cells based on their enzymatic content and their elemental composition.     

Presence of hydrocarbon-degrading bacteria in the gills of mussel Mytilus galloprovincialis in a contaminated environment: a mesoscale simulation study

A number of previous studies have shown that the relationships of symbiosis existing between mussels and microorganisms are directly dependent on the environmental conditions. However, little is known about existing relationships between mussels and bacteria in hydrocarbon-impacted marine environments. The aim of this preliminary study is to investigate the presence of oil-degrading bacteria in the mussel Mytilus galloprovincialis during growth in polluted ecosystems. All the experiments were carried out in a mesocosm system designed to simulate chronic pollution and to enable direct exposure of mussels to chemicals. Quantitative (4′,6-diamidino-2-phenylindole, colony-forming units, Most Probable Number) analyses and screening (presence/absence) of metabolic functional genes were performed to analyse bacterial populations inside the gills of mussels exposed and not exposed to hydrocarbons. The data obtained show that the presence of hydrocarbons affected the abundance of bacteria inside the gills of specimens and determines selection for specific (hydrocarbon-degrading) bacteria (i.e. Alcanivorax sp. and Marinobacter sp.). However, is not yet clear whether the presence of such genera of bacteria inside the mussel is due to symbiosis or as a result of filtration.     

Association of bacteria with larvae of the gutless protobranch bivalve Solemya reidi (Cryptodonta: Solemyidae)

Rod-shaped bacteria were consistently observed by transmission electron microscopy in the locomotory test of larvae and in the perivisceral cavity of post-larvae of Solemya reidi, a gutless protobranch bivalve known to possess intracellular chemoautotrophic bacterial symbionts in the adult gill. Bacteria develop within granular vesicles in the larval test, where they either remain to be ingested at metamorphosis, or are released into the space separating the test and embryo, to be subsequently ingested through the larval mouth. In either case, bacteria lie within the perivisceral cavity following metamorphosis. Bacteria were not seen either in or on gametes or in gills of juveniles. It is hypothesized that these bacteria represent a transmission stage of the gill symbionts present in adult S. reidi and are not evident in gametes or gills of juveniles due to cryptic packaging within granular vesicles. Perpetuation of this symbiosis would therefore be assured through vertical transmission, as is typical of other marine invertebrate-bacteria endosymbioses.Harbor Branch Oceanographic Institution Contribution No. 602     

Detrimental effects of the isopod, <Emphasis Type="Italic">Edotia doellojuradoi,</Emphasis> on gill morphology and host condition of the mussel, <Emphasis Type="Italic">Mytilus edulis platensis</Emphasis>

There are many reported associations between mussels and other invertebrates, such as pea crabs, polychaetes, turbellarians and copepods, which live in their mantle cavities. The boundary between commensalism and parasitism is often indistinguishable because of insufficient knowledge or because the interaction is variable. Preliminary evidence led to a closer examination of the relationship between the mussel, Mytilus edulis platensis, and an isopod, Edotia doellojuradoi, previously described as commensalism. Monthly intertidal samples of mussels were taken from September 2004 to August 2005 at Caleta Cordova Norte (45°43′S, 67°22′W) in southern Argentina and assessed for the prevalence and abundance levels of isopods. Mussels with and without isopods were measured, examined for evidence of gill damage and their condition (soft tissue dry weight) was determined. The overall isopod prevalence in mussels was 57.9% and infestation varied with mussel length, with maximum occurence at 30.2 mm (medium-sized mussels). Experimental evidence indicated that the position of the isopod inside the mussel depended on the feeding activity of the mussel. Female isopods were observed grasping the ventral food groove of the gill demibranchs and feeding on the mucous food strand produced by the mussel. Juveniles and males were observed clustered together on the dorsal side of the single female in each occupied mussel, suggesting extended maternal care. Gill damage was observed in 58.2% of mussels at the Argentine site and was significantly associated with isopod occurrence. Categorical regression analysis showed that the most important factor associated with the degree of gill lesions was the number of male and juvenile isopods per mussel, followed by the length of female isopods and the developmental stage of juveniles. Conversely, the degree of gill damage decreased with increasing mussel length. Overall, E. doellojuradoi had a significant effect on mussel condition throughout the year, with low flesh weight in mussels with isopods, except during the austral summer and early autumn. In contrast to previous studies, which concluded that the isopod was a commensal, the present study clearly demonstrates that E. doellojuradoi is a parasite of M. edulis platensis. Other symbiotic interactions formerly classified as commensal might not be innocuous on further investigation, especially if samples are taken at multiple sites and at different times of the year.     

Assessment of the effect of environmental pollution in a marina on caged mussels using chemical and genotoxic analysis

An integrated approach using the contamination levels and DNA damage in mussels (Mytilus galloprovincialis) was applied in order to assess the chemical contamination in a marina (Eastern coastline of Aegean Sea). Mussels, which were harvested from a reference site (Foca), were transplanted into a marina situated along the coast of Izmir Bay. The transplanted mussels were collected at the 14th, 30th and 60th day of the experimental period. Polycyclic aromatic hydrocarbon (PAH) levels (27–51?ng?g^?1?wet?weight) detected in the mussels were similar to the levels detected in other coastal areas of the Mediterranean Sea. The marina’s sediment was found to be contaminated with PAHs (∑PAH?=?25?µg?g^?1) of pyrolytic origin and may become a source of pollution and a threat to the marine environment. In order to assess the DNA damage, the haemolymph and gill cells of the mussels were used for the comet analysis and considered as an indicator of exposure to genotoxic chemicals including 16 PAH compounds and metals. The highest levels of DNA damage expressed as %Tail-DNA (%T-DNA) were observed at the end of the experiment (21.5% T-DNA). The correlation analyses conducted between 2-, 3-, 4-ring PAHs in mussels and %T-DNA in haemolymph and gill cells showed a significant positive correlation. This investigation confirmed that transplanted mussel can be a useful tool to determine PAH contamination in marinas.     

Elemental sulfur in the gills of three species of clams containing chemoautotrophic symbiotic bacteria: a possible inorganic energy storage compound

Sulfur content and fine structure were studied for tissues of three species of clams, Lucinoma annulata, Calyptogena elongata and Lucina floridana, which inhabit sulfide-rich environments and whose gills harbor symbiotic sulfur bacteria. Lucinoma annulata and C. elongata were dredged from the Santa Barbara basin, California, USA, at a depth of 480 to 490 m, and Lucina floridana were dug from below the roots of seagrasses in Saint Joseph Bay, Florida, at a depth of 0.25 to 2m. Foot tissue of Lucinoma annulata, without symbionts, had a total sulfur content of 1.4±0.1 (SD) mg 100 mg^-1 dry weight of tissue (%DW). The symbiont-containing gill tissue of different individuals of L. annulata varied in color from dark red to pale yellow, and the total sulfur content was 2.5±0.4% DW in red gills and was 5.6±3.3 % DW in the yellowest gills. Maintenance of L. annulata in the laboratory for 21 d in the absence of sulfide resulted in the loss from the gill of yellow deposits which were elemental sulfur in the form of liquid-crystalline sulfur globules rather than solid orthorhombic sulfur crystals. The foot tissue did not contain elemental sulfur. When examined by freeze-etch microscopy, sulfur globules were found only within bacteria and not in the animal host cytoplasm. Sulfur globules were confined to the periplasmic space of the bacteria. C. elongata and Lucina floridana resembled Lucinoma annulata in the physical form and distribution of elemental sulfur. The absence of elemental sulfur in the animal cytoplasm suggests that its formation from sulfide is not a detoxification scheme to protect animal tissue from sulfide toxicity. The sulfur deposits probably represent inorganic energy reserves that permit the symbiotic bacteria to function even during the temporary absence of external sulfide.     

Morphological adaptations to chronic hypoxia in deep-sea decapod crustaceans from hydrothermal vents and cold seeps

Animals inhabiting hydrothermal vents and cold seeps face conditions that are challenging for survival. In particular, these two habitats are characterized by chronic hypoxia, sometimes reaching complete anoxia. The characteristics of the scaphognathite and gills were studied in four species of shrimp and three species of crabs from hydrothermal vents and cold seeps, in order to highlight potential adaptations that could enhance oxygen acquisition in comparison with shallow-water relatives. All the vent and seep species studied here exhibit significantly larger scaphognathites, likely allowing more water to flow over their gills per stroke of this appendage. This is probably more energetically efficient that prolonged hyperventilation. In contrast to annelids, vent and seep decapods usually do not possess enlarged gills, a phenomenon likely due to the physical limitations imposed by the size of the gill chamber. In the vent shrimp Rimicaris exoculata and the vent crab Bythograea thermydron, however, there is a significantly higher specific gill surface area linked to a higher number of lamellae per gram of gill. Again in contrast to annelids, the diffusion distance through the gills is not strikingly different between the vent shrimp Alvinocaris komaii and the shallow-water species Palaemon spp. This may indicate that the epithelium and cuticle of the decapod gills are already optimized for oxygen uptake and that reducing the thickness of these compartments is not physically possible without affecting the physical integrity of the gills.     

Physiological role of branchial carbonic anhydrase in the shore crabCarcinus maenas

Excretion of total CO₂ and uptake of sodium and chloride ions across the branchial epithelium of the posterior gills of the shore crabCarcinus maenas, collected from Kiel Bay (Baltic Sea) in 1989, were measured using isolated perfused gill preparations. Total CO₂ effluxes depended on the HCO ₃ ^- concentration of the internal perfusate in a saturable mode and were inhibited by internally and externally applied acetazolamide at 10^–4 M. Potential differences between hemolymph space and medium did not change significantly during experimental treatments. Neither a bicarbonate gradient (6 mM) directed from the internal perfusate to external bath solution nor symmetrically applied 10^–4 M acetazolamide significantly influenced the influxes of Na⁺ and Cl^–. Results confirmed the role of carbonic anhydrase in CO₂ excretion but called into question the assumed functioning of the enzyme in branchial ion transport processes.     

Response of mussel Brachidontes variabilis to chlorination

Brachidontes variabilis is a common fouling mussel species in cooling water systems of tropical coastal power stations. However, there are hardly any data available on the response of B. variabilis to chlorine, a commonly used antifouling biocide. Therefore, lethal and sublethal responses of this mussel to chlorine are of considerable interest to the industry. The response of mussels in terms of mortality pattern (LT₅₀ and LT₁₀₀) and physiological activities (oxygen consumption, filtration rate, foot activity and byssus thread production) in different size groups (with shell lengths of 7–24 mm) of B. variabilis was studied in the laboratory under different chlorine concentrations (0.25, 0.50, 0.75 and 1.00 mg l^?1 for sublethal responses and 1, 2, 3 and 5 mg l^?1 for mortality). The results showed that the exposure time for 100% mortality of mussels decreased significantly with increasing chlorine concentration. However, mussel size was not a determinant of its chlorine tolerance: all size groups tested (with shell lengths of 7–24 mm) took comparable exposure times to reach 100% mortality at a given chlorine concentration (1–5 mg l^?1). All size groups of B. variabilis showed a progressive reduction in physiological activities such as oxygen consumption, filtration rate, foot activity and byssus thread production, when chlorine residuals were increased from 0 to 1 mg l^?1. The data generated in the present work are compared with similar data available for other tropical fouling mussel species to see how far relative chlorine toxicity could have influenced the relative distribution of the mussels inside the seawater intake tunnel of a power station at Kalpakkam in India. It is shown that in this insufficiently chlorinated system, the relative distribution of Brachidontes striatulus, B. variabilis and Modiolus philippinarum reflects the relative tolerance of the species to chlorine.     

Interactions of 233Pa with tissues of Mytilus galloprovincialis and Carcinus mediterraneus

Uptake of ²³³Pa from sea water by mussels Mytilus galloprovincialis L. and shore crabs Carcinus mediterraneus Csrn. was studied. Results indicate a high contamination factor in digestive tract, gills and skeleton of both investigated species and an increment of the contamination factor in mussel byssus. In spite of this, reproductive system, muscle and hemolymph were only negligibly contaminated. The presence of complexone EDTA in sea water has no effect on uptake of ²³³Pa in the animal tissues tested.     

Ultrastructural,biochemical, and immunological characterization of two populations of the mytilid mussel <Emphasis Type="Italic">Bathymodiolus</Emphasis><Emphasis Type="Italic">azoricus</Emphasis> from the Mid-Atlantic Ridge: evidence for a dual symbiosis

A recently described species of mytilid mussel, Bathymodiolus azoricus Von Cosel et al., 1999, was observed to be the dominant organism at the hydrothermal vents off the Azores, at both the Lucky Strike and Menez Gwen sites. Evidence suggests this species of Bathymodiolus represents yet another example of the intriguing dual symbiosis known in three other species of deep-sea mytilid mussels. Transmission electron micrographs (TEM) show the majority of gill bacteriocytes in mussels sampled from both populations to contain two distinct symbiont morphotypes. One morphotype is characterized by large size (mean diameter, 1.25 µm), coccoid shape, and stacked intracytoplasmic membranes that are consistent with the morphology of type I methanotrophs. The second morphotype is smaller (mean diameter, 0.35 µm) and was observed in coccoid or rod shapes. Immunoblots revealed the presence of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and methanol dehydrogenase (MeDH) in both populations of mussels. Activities of these enzymes, as well as sulfate adenylyl transferase (ATP sulfurylase) and adenylyl sulfate reductase (APS reductase), were detected in gill extracts. The activities measured for the two populations were highly variable, though the population sampled from Lucky Strike showed higher RubisCO activity. Stable carbon isotope values (Lucky Strike, '¹³C=-32.6ǂ.3‰; Menez Gwen, '¹³C=-22.8ǂ.4‰) are in the range of previously reported stable carbon isotope measurements for mytilid mussels hosting a dual symbiosis. Collectively, these results provide evidence for the activity of both sulfur-oxidizing and methane-oxidizing metabolic pathways in B. azoricus. Furthermore, evidence for a greater dependence on methanotrophy in the Menez Gwen mussel population is offered by analysis of cell counts from TEMs. Higher methanotroph numbers, and putatively activity, in this population of mussels are further supported by published geochemical data indicating higher methane concentrations in the vent fluids at Menez Gwen. This finding suggests that environmental conditions may regulate a balance between the physiological activities of different symbiont populations associated with these mussels. The existence of a dual symbiosis could thus confer greater environmental tolerance and increased niche space to the mytilid host in the stochastic hydrothermal vent habitat.