The importance of methane and thiosulfate in the metabolism of the bacterial symbionts of two deep-sea mussels

Undescribed hydrocarbon-seep mussels were collected from the Louisiana Slope, Gulf of Mexico, during March 1986, and the ultrastructure of their gills was examined and compared to Bathymodiolus thermophilus, a mussel collected from the deep-sea hydrothermal vents on the Galápagos Rift in March 1985. These closely related mytilids both contain abundant symbiotic bacteria in their gills. However, the bacteria from the two species are distinctly different in both morphology and biochemistry, and are housed differently within the gills of the two mussels. The symbionts from the seep mussel are larger than the symbionts from B. thermophilus and, unlike the latter, contain stacked intracytoplasmic membranes. In the seep mussel three or fewer symbionts appear to be contained in each host-cell vacuole, while in B. thermophilus there are often more than twenty bacteria visible in a single section through a vacuole. The methanotrophic nature of the seep-mussel symbionts was confirmed in ¹⁴C-methane uptake experiments by the appearance of label in both CO₂ and acid-stable, non-volatile, organic compounds after a 3 h incubation of isolated gill tissue. Furthermore, methane consumption was correlated with methanol dehydrogenase activity in isolated gill tissue. Activity of ribulose-1,5-biphosphate (RuBP) carboxylase and ¹⁴CO₂ assimilation studies indicate the presence of either a second type of symbiont or contaminating bacteria on the gills of freshly captured seep mussels. A reevaluation of the nutrition of the symbionts in B. thermophilus indicates that while the major symbiont is not a methanotroph, its status as a sulfur-oxidizing chemoautotroph, as has been suggested previously, is far from proven.     

Carbon dioxide use by chemoautotrophic endosymbionts of hydrothermal vent vestimentiferans: affinities for carbon dioxide, absence of carboxysomes, and δ13C values

The hydrothermal vent vestimentiferans Riftia pachyptila Jones, 1981 and Ridgeia piscesae Jones, 1985 live in habitats with different abundances of external CO₂. R. pachyptila is found in areas with a high input of hydrothermal fluid, and therefore with a high [CO₂]. R. piscesae is found in a range of habitats with low to high levels of hydrothermal fluid input, with a correspondingly broad range of CO₂ concentrations. We examined the strategies for dissolved inorganic carbon (DIC) use by the symbionts from these two species. R. pachyptila were collected from the East Pacific Rise (9°50′N; 104°20′W) in March 1996, and R. piscesae were collected from the Juan de Fuca Ridge (47°57′N; 129°07′W) during September of 1996 and 1997. The differences in the hosts' habitats were reflected by the internal pools of DIC in these organisms. The concentrations of DIC in coelomic fluid from R. piscesae were 3.1 to 10.5 mM, lower than those previously reported for R. pachyptila, which often exceed 30 mM. When symbionts from both hosts were incubated at in situ pressures, their carbon fixation rates increased with the extracellular concentration of CO₂, and not HCO₃ ⁻, and symbionts from R. piscesae had a higher affinity for CO₂ than those from R. pachyptila (K _1/2 of 7.6 μM versus 49 μM). Transmission electron micrographs showed that symbionts from R. piscesae lack carboxysomes, irrespective of the coelomic fluid [DIC] of their host. This suggests that the higher affinity for CO₂ of R. piscesae symbionts may be their sole means of compensating for lower DIC concentrations. The δ¹³C values of tissues from R. piscesae with higher [DIC] in the coelomic fluid were more positive, opposite to the trend previously described for other autotrophs. Factors which may contribute to this trend are discussed. Received: 24 September 1998 / Accepted: 12 May 1999     

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.     

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     

Response of hydrothermal vent vestimentiferan <Emphasis Type="Italic">Riftia pachyptila</Emphasis> to differences in habitat chemistry

Vestimentiferan tubeworms, which rely on intracellular sulfide-oxidizing autotrophic bacteria for organic carbon, flourish at deep-sea hydrothermal vents despite the erratic nature of their habitat. To assess the degree to which differences in habitat chemistry (sulfide, pH/CO₂) might impact host and symbiont metabolic activity, Riftia pachyptila tubeworms were collected from habitats with low (H₂S < 0.0001 mM) and high (up to 0.7 mM) sulfide concentrations. The elemental sulfur content of the symbiont-containing trophosome organ was lower in specimens collected from the low-sulfide site. Symbiont abundance, RubisCO activity, and trophosome carbon fixation rates were not significantly different for individuals collected from low- versus high-sulfide habitats. Carbonic anhydrase activities were higher in the anterior gas exchange organs of R. pachyptila from the low-sulfide habitat. Despite large differences in habitat chemistry, symbiont abundance and autotrophic potential were consistent, while the host appears to tailor carbonic anhydrase activity to environmental CO₂ availability.     

An autoradiographic examination of carbon fixation, transfer and utilization in the Riftia pachyptila symbiosis

Riftia pachyptila, the giant vestimentiferan tubeworm from the East Pacific Rise, harbors abundant chemolithoautotrophic, sulfide-oxidizing bacteria in an internal organ, the trophosome. Several facts, such as the lack of a digestive system in the host, stable carbon isotope values and net carbon dioxide uptake all suggest that the tubeworms obtain the bulk of their nutrition from their symbionts. Using tissue autoradiography, we investigated the mode of nutritional transfer between symbionts and host, and the site of early incorporation of symbiont fixed-carbon in the host. Fast labeling in the trophosome clearly demonstrates that the symbionts are the primary site of carbon fixation. Appearance of label in some symbiont-free host tissues in as little as 15 min indicates that the symbionts release a significant amount of organic carbon immediately after fixation. The organic carbon is largely incorporated into specific, metabolically active host tissues such as fast-growing body regions in the trunk and plume, and into tube-secreting glands. In addition to immediate release of fixed carbon by the symbionts, there is evidence of a second possible nutritional mode, digestion of the symbionts, which is consistent with previous suggestions based on trophosome ultrastructure. Results suggest that symbiont-containing host cells migrate in a predictable pattern within trophosome lobules and that symbiont division occurs predominately in the center of a lobule, followed eventually by autolysis/digestion at the periphery of the lobule. Received: 1 July 1999 / Accepted: 30 December 1999     

Metabolic and blood characteristics of the hydrothermal vent tube-worm Riftia pachyptila

Specimens of the hydrothermal vent pogonophoran Riftia pachyptila Jones were collected by submersible at a depth of 2 600 m at the 21°N hydrothermal vent site on the East Pacific Rise (20°50N, 109°06W) in April and May of 1982. The worms were maintained in pressurized aquaria for up to 45 d for metabolic studies. Consumption of O₂ was regulated down to low P_{O 2} (oxygen partial pressure) values; O₂ consumption rates were 0.63 and 1.12 mol g^-1 wet wt h^-1 at 2.5° and 8°C, respectively; such rates were comparable to those previously measured for other pogonophorans. Intact specimens of R. pachyptila (including bacterial symbionts) did not consume significant amounts of CH₄ from the environment. The respiratory quotients, in the absence of added sulfide, indicated that metabolism was mainly heterotrophic. High rates of uptake of dissolved amino acids were recorded for one specimen. The total [CO₂] in the vascular blood and the Hb-containing coelomic fluid were high. Under anaerobic conditions, there were equilibrium distributions of pH, total [CO₂] and sulfide concentrations between the vascular blood and the coelomic fluid, apparently because these metabolites were readily exchanged between the two compartments. The vascular blood bound neither CH₄ nor H₂. However, sulfide was reversibly bound by both the vascular blood and coelomic fluid; because this binding depended strongly on pH (with a maximum at about 7.5), HS^- was probably the molecular species bound. Under anaerobic, but not aerobic conditions, the trophosome bound substantial amount of sulfide; thus, the high concentrations of sulfide in the trophosome may have resulted mainly from sulfide bound to sulfide oxidases under anaerobic conditions. The coelomic fluid had a relatively low buffering capacity (2.2 mmol CO₂pH^-1).     

Digestive enzymes in marine invertebrates from hydrothermal vents and other reducing environments

The present study demonstrates the potential hydrolytic activities in the symbiont-containing tissues of the vent invertebrates Riftia pachyptila, Bathymodiolus thermophilus (collected in 1991 at the East Pacific Rise) and the shallow-water bivalve Lucinoma aequizonata (collected in 1991 from the Santa Barbara Basin). Activities of phosphatases, esterases, -glucuronidase and leucineaminopeptidase were comparable to those of digestive tract tissues of other marine invertebrates. A lack in most glycosidases as well as in trypsin and chymotrypsin was observed. Activities of lysozyme and chitobiase were rather high. In all vent invertebrates with symbionts and in L. aequizonata, the symbiont-containing tissues and the symbiont-free tissues had similar levels of enzymatic activities, indicating that polymeric nutrients could be hydrolysed after release from the symbionts and cellular uptake. The high activities of -fucosidase in all vent invertebrates as well as in the shallow-water bivalve L. aequizonata could point to the existence of a yet undescribed substrate available to hydrolysation. The ectosymbionts-carrying polychaete Alvinella pompejana (collected in 1991 at the East Pacific Rise, EPR) shows high lysozyme activities in its gut, consistent with the proposed food source of bacteria. For the vent crab Bythogrea thermydron (also collected in 1991 at the EPR) hydrolytic activities were highest in the gut, dominated by esterase and peptidase activities which support their proposed carnivorous food source. A snail and a limpet collected from R. pachyptila tubes showed high levels of chitobiase suggesting a food source of grazed bacteria or ingested R. pachyptila tube.     

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     

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.     

Elemental composition of the hydrothermal vent clam Calyptogena magnifica from the East Pacific Rise

Specimens of the hydrothermal vent clam Calyptogena magnifica were collected at a depth of 2 600 m from 21° N on the East Pacific Rise in spring, 1982. The elemental composition was determined for the total soft tissues, individual organs, and the shell. The soft parts contained high concentrations of a number of trace metals. Iron, copper, and zinc, at 760, 148 and 2 152 g g^-1 dry weight, respectively, were present at the highest concentrations. Silver and antimony exhibited the greatest enrichments when their concentrations in C. magnifica were compared with those measured in a shallow-water marine mussel, Mytilus edulis. In contrast to the soft parts, the shell was not a site of metal accumulation or deposition. Within the soft parts; gills, kidney, pericardium, and mantle were involved in the concentration of various metals; the degree of concentration varied depending on the particular metal under consideration. C. magnifica possesses metal body burdens which are ordinarily considered to be very high and potentially toxic in other species.     

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.     

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     

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     

Bacterial symbiosis in Northeast Pacific Vestimentifera: a TEM study

A number of new vestimentiferan species occur at northeast Pacific hydrothermal vent sites. The trophosome and bacterial symbionts of three species, collected from the Juan de Fuca and Explorer Ridges between 1984 and 1986, were studied by transmission electron microscopy (TEM). As in Riftia pachyptila, trophosome tissue is organised into lobules each having an axial blood vessel, and intracellular bacterial symbionts are contained in membrane vacuoles. The bacteria have many cytoplasmic inclusions including tubular membrane systems, glycogen-like particles and poly--hydroxybutyrate (PHB) or sulfur bodies. Glycogen production may be quantitatively important to both the symbionts and the host. Glycogen-like granules appear to first accumulate in the bacterial cells and then be released into the bacteriocyte cytoplasm as bacteria are degraded. Although various stages of bacterial growth and degradation are observed, data are insufficient to verify any across-lobule progression of these processes. Morphological comparison of the symbionts reveals that similar symbionts are found in different vestimentiferan species and that one to two bacterial types exist within single individuals. Two possible models of trophosome function and nutrient exchange are discussed.Deceased     

Effects of sulfide exposure history and hemolymph thiosulfate on oxygen-consumption rates and regulation in the hydrothermal vent crab Bythograea thermydron

The hydrothermal vent crab Bythograea thermydron is exposed to high environmental concentrations of sulfide and low levels of oxygen for extended periods of time. It has previously been shown that hydrogen sulfide is oxidized to the relatively non-toxic thiosulfate (S₂O ₃ ^2– ), which accumulates in the hemolymph. Hemolymph thiosulfate levels in freshly captured crabs vary significantly among crabs from different hydrothermal vent sites as well as between crabs from different microhabitats within the same site. Hemolymph thiosulfate concentrations were not significantly different between crabs captured at the same site 6 mo apart. Hemolymph thiosulfate concentrations ranged from 66 mol 1^–1 in a crab captured at a site with relatively low sulfide venting, to 3206 mol 1^–1 in an individual that was netted from an active smoker vent with much higher sulfide exposure. The differences in hemolymph thiosulfate between sites and the stability of hemolymph thiosulfate in crabs captured at the same site at different times suggest that sulfide exposure is significantly different between sites and that this exposure may not vary significantly over the course of a few months. B. thermydron experimentally exposed to sulfide had high levels of thiosulfate in their hemolymph and increased abilities to regulate oxygen consumption in conditions of low oxygen. This enhancement of regulatory abilities suggests that the previously demonstrated increased hemocyaninoxygen (Hc–O₂) affinity due to elevated thiosulfate may be adaptive in vivo. Average oxygen-consumption rates were much higher in crabs experimentally exposed to sulfide than in unexposed crabs. Crabs injected with isosmotic thiosulfate did not have increased oxygen-consumption rates as did the sulfide-exposed individuals, but did show a similar reduction in P _c (the critical partial pressure of oxygen at which crabs can no longer regulate oxygen consumption). This suggests that it is the sulfide exposure and/or detoxification rather than the elimination of thiosulfate that causes the increase in metabolic rate. Thiosulfate diffuses into dead crabs and into live crabs exposed to 15 mmol S₂O ₃ ^2- l^–1, indicating substantial permeability, and yet live crabs are able to eliminate thiosulfate when incubated in sea water containing 1.5 mmol S₂O ₃ ^2- l^–1, suggesting a process that has an active component.     

Persistence,morphology, and nutritional state of a gastropod hosted bacterial symbiosis in different levels of hydrothermal vent flux

The limpet, Lepetodrilus fucensis McLean, is found in prominent stacks around hydrothermal vents on the Juan de Fuca Ridge. L. fucensis hosts a filamentous episymbiont on its gill lamellae that may be ingested directly by the gill epithelium. To assess the persistence of this symbiosis I used microscopy to examine the gills of L. fucensis from sites representing its geographic range and different habitats. The symbiosis is present on all the specimens examined in this study, including both sexes and a range of juvenile and adult sizes. Next, I aimed to determine if patterns in bacterial abundance, host condition, and gill morphology support the hypotheses that the bacteria are chemoautotrophic and provide limpets with a food resource. To do so, I compared specimens from high and low flux locations at multiple vents. My results support the above hypotheses: (1) gill bacteria are significantly less abundant in low flux where the concentrations of reduced chemicals (for chemoautotrophy) are negligible, (2) low flux specimens have remarkably poor tissue condition, and (3) the lamellae of high flux limpets have greater surface area: the blood space and bacteria-hosting epithelium are deeper and have more folds than low flux lamellae, modifications that support higher symbiont abundances. I next asked if the morphology of the lamellae could change. To test this, I moved high flux limpets away from a vent and after 1 year the lamellar depth and shape of the transplanted specimens resembled low flux gills. Last, I was interested in whether bacterial digestion by the gill epithelium is a significant feeding mechanism. As bacteria-like cells are rarely apparent in lysosomes of the gill epithelium, I predicted that lysosome number would be unrelated to bacterial abundance. My data support this prediction, suggesting that digestion of bacteria by the gill epithelium probably contributes only minimally to the limpet’s nutrition. Overall, the persistence and morphology of the L. fucensis gill symbiosis relates to the intensity of vent flux and indicates that specimens from a variety of habitats may be necessary to characterize the morphological variability of gill-hosted symbioses in other molluscs.     

Gene flow among vestimentiferan tube worm (Riftia pachyptila) populations from hydrothermal vents of the eastern Pacific

Allozyme data are presented for six discrete populations of the giant hydrothermal vent tube worm Riftia pachyptila Jones, 1981 collected throughout the species' known range along mid-ocean spreading ridges of the eastern Pacific Ocean. Contrary to an earlier report, levels of genetic variation are relatively high in this species. Estimates of gene flow based on F-statistics revealed that dispersal throughout the surveyed region is sufficiently high to counter random processes that would lead to losses of genetic diversity and significant population differentiation. R. pachyptila, like other species of tube worms, displays considerable morphologic variation among populations, but this diversity is not reflected in allozyme variation. Vestimentifera, in general, appear to show extensive phenotypic plasticity. In the light of the available genetic data, caution is warranted when making inferences about the taxonomic status of collections based on morphological variation alone. A general decrease in estimated rates of gene flow between geographically more distant populations supports the hypothesis that dispersal in this species follows a stepping-stone model, with exchange between neighboring populations in great excess of long-distance dispersal. High levels of gene flow have been recorded in a variety of vent fauna and may be a prerequisite for success of species found in the ephemeral habitats associated with regions of sea-floor hydrothermal activity.     

Contrasting effects of sulfide and thiosulfate on symbiotic CO2-assimilation of Phallodrilus leukodermatus (Annelida)

Experiments were conducted in order to specify the reductants responsible for the carbon dioxide fixation of the symbiotic sulfur bacteria in the gutless marine oligochaete Phallodrilus leukodermatus (Annelida) from shallow calcareous sediments in Bermuda. Carbon dioxide-uptake rates were suppressed by S⁼ and stimulated by S₂O₃ ⁼. Individuals which hosted bacteria containing reserve energy substances maintained a high short-term CO₂-uptake activity, while bacteria in worm homogenates and in worms treated with an antibiotic (Baypen) did not show any significant metabolic activity. Absolute uptake rates in P. leukodermatus were usually considerably higher than those reported for other animals harbouring prokaryotic sulfuroxidizing symbionts. Utilization of thiosulfate rather than sulfide is compatible with the preferred occurrence of the worms around the redox discontinuity layer and has been confirmed in other thiobiotic animals. Sulfur stored in the symbiotic bacteria appears to be oxidized to sulfate and be excreted when the worms are held under energy-limited conditions. The data emphasize the complexity of the possible metabolic pathways involved in the oxidation of reduced-sulfur compounds by bacterial symbionts in marine invertebrates.     

Ultrastructure of the gill of the hydrothermal vent bivalveCalyptogena magnifica,with a discussion of its nutrition

Calyptogena magnifica Boss and Turner, 1980, a new Vesicomyidae found during the Galápagos expedition in hydrothermal vents of the East Pacific Rise, was collected in the same Rise at 21°N during the Oasis expedition (March 1982), and samples of the gill were fixed for ultrastructural observations. The large size and structure of the gill indicate that this is the organ mainly involved in the nutritional processes ofC. magnifica. Despite the classic structural appearance of the external cilia of its gill, and an obvious production of mucus,C. magnifica is not a filter-feeder, as it does not use filtering processes to provide its major source of nutrition. Negligible particulate transfer is evidenced by reduction of the ciliary groove, of the labial palps and of the digestive tube, as well as by the absence of mucous strings. Histological and ultrastructural observations endorse the hypothesis that endocellular chemoautotrophic bacteria play an important role in the nutrition of the clam. Except for a superficial zone of ciliated cells, most of the gill tissue is comprised of cells which appear to be bacteriocytes, and which are perfectly integrated into the gill tissue and contain abundant and normally reproducing bacteria. The differences observed in the structure of the bacteriocytes suggest a cyclic process of their colonization by bacteria, their possible resorption, and their replacement by new bacteria-infected cells. Energetic substrates (sulfides and organic molecules) are probably directly absorbed by the bacteriocytes through the microvilli of the epithelial cells. Abundant fingerprint-like mitochondria in ciliate cells attest to a particularly high metabolic activity, perhaps related to active biosynthesis.