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     

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.     

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.     

The color of the trophosome: elemental sulfur distribution in the endosymbionts of<Emphasis Type="Italic"> Riftia pachyptila</Emphasis> (Vestimentifera; Siboglinidae)

Riftia pachyptila Jones, 1981, lives in association with a chemoautotrophic, sulfide-oxidizing -Proteobacterium that occurs in a specialized organ, the trophosome. Ultrastructurally different bacterial subpopulations occur in different regions of the trophosome lobules (central rods, median small cocci, peripheral large cocci) and contain vesicles, which have been proposed to be sites of elemental sulfur storage. Differently colored trophosomes have been suggested to reflect different amounts of elemental sulfur in the tissue. In this study, the presence of elemental sulfur (S⁰) was confirmed in the vesicles of the symbionts of R. pachyptila by electron energy loss spectrography (EELS). The proportion of (two-dimensional) area occupied by sulfur vesicles in the cells was found to be strongly correlated with trophosome color, both in individuals with uniformly colored trophosomes and individuals that exhibited a gradual color change along the length of their trophosomes. Elemental sulfur content was highly variable between individuals from a single collection, suggesting a high degree of microhabitat heterogeneity within vestimentiferan aggregations.Communicated by O. Kinne, Oldendorf/Luhe     

Microsensor studies of photosynthesis and respiration in larger symbiotic foraminifera. I The physico-chemical microenvironment of Marginopora vertebralis, Amphistegina lobifera and Amphisorus hemprichii

 The physico-chemical microenvironment of larger benthic foraminifera was studied with microsensors for O₂, CO₂, pH, Ca²⁺ and scalar irradiance. Under saturating light conditions, the photosynthetic activity of the endosymbiotic algae increased the O₂ up to 183% air saturation and a pH of up to 8.6 was measured at the foraminiferal shell surface. The photosynthetic CO₂ fixation decreased the CO₂ at the shell down to 4.7 μM. In the dark, the respiration of host and symbionts decreased the O₂ level to 91% air saturation and the CO₂ concentration reached up to 12 μM. pH was lowered relative to the ambient seawater pH of 8.2. The endosymbionts responded immediately to changing light conditions, resulting in dynamic changes of O₂, CO₂ and pH at the foraminiferal shell surface during experimentally imposed light–dark cycles. The dynamic concentration changes demonstrated for the first time a fast exchange of metabolic gases through the perforate, hyaline shell of Amphistegina lobifera. A diffusive boundary layer (DBL) limited the solute exchange between the foraminifera and the surrounding water. The DBL reached a thickness of 400–700 μm in stagnant water and was reduced to 100–300 μm under flow conditions. Gross photosynthesis rates were significantly higher under flow conditions (4.7 nmol O₂ cm⁻³ s⁻¹) than in stagnant water (1.6 nmol O₂ cm ⁻³ s⁻¹), whereas net photosynthesis rates were unaffected by flow conditions. The Ca²⁺ microprofiles demonstrated a spatial variation in sites of calcium uptake over the foraminiferal shells. Ca²⁺ gradients at the shell surface showed total Ca²⁺ uptake rates of 0.6 to 4.2 nmol cm⁻² h⁻¹ in A. lobifera and 1.7 to 3.6 nmol cm⁻² h⁻¹ in Marginopora vertebralis. The scattering and reflection of the foraminiferal calcite shell increased the scalar irradiance at the surface up to 205% of the incident irradiance. Transmittance measurements across the calcite shell suggest that the symbionts are shielded from higher light levels, receiving approximately 30% of the incident light for photosynthesis. Received: 6 July 1999 / Accepted: 28 April 2000     

Multiple trophic resources for a chemoautotrophic community at a cold water brine seep at the base of the Florida Escarpment

The biological community that surrounds the hypersaline cold water brine seeps at the base of the Florida Escarpment is dominated by two macrofaunal species: an undescribed bivalve of the family Mytilidac and a vestimentiferan worm, Escarpia laminata. These animals are apparently supported by the chemoautotrophic fixation of carbon via bacterial endosymbionts. Water column and sediment data indicate that high levels of both sulfide and methane are present in surface sediments around the animals but absent from overlying waters. Stable isotopic analyses of pore water indicate that there are two sources of sulfide: the first is geothermal sulfide carried in groundwater leaching from the base of the escarpment, and the second is microbial sulfide produced in situ. The vestimentiferan E. laminata, and the mytilid bivalve (seep mussel) live contiguously but rely on different substrates for chemoautotrophy. Enzyme assays, patterns of elemental sulfur storage and stable isotopic analyses indicate that E. laminata relies on sulfide oxidation and the seep mussel on methane oxidation for growth.     

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.     

Hepatopancreatic endosymbionts in coastal isopods (Crustacea: Isopoda), and their contribution to digestion

Three isopod species (Crustacea: Isopoda), commonly found in the intertidal and supratidal zones of the North American Pacific coast, were studied with respect to symbiotic microbiota in their midgut glands (hepatopancreas). Ligia pallasii (Oniscidea: Ligiidae) contained high numbers of microbial symbionts in its hepatopancreatic caeca. Numbers of endosymbionts were strongly reduced by ingestion of antibiotics. By contrast, the hepatopancreas of Idotea wosnesenskii (Valvifera: Idoteidae) and Gnorimosphaeroma oregonense (Sphaeromatidea: Sphaeromatidae) did not contain any microbiota. Results of feeding experiments suggest that microbial endosymbionts contribute to digestive processes in L. pallasii, the most terrestrial of the three isopods that we studied. The acquisition of digestion-enhancing endosymbionts may have been an important evolutionary step allowing isopods to colonize terrestrial habitats where relatively indigestible leaf litter is the primary food source. By contrast, the ability to digest phenolic compounds was most developed in one of the more marine species, suggesting that this trait may have evolved independently in isopod species that consume a phenolic-rich diet, whether in marine habitats or on land. Received: 28 August 2000 / Accepted: 8 December 2000     

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     

Three party symbiosis: acoelomorph worms,corals and unicellular algal symbionts in Eilat (Red Sea)

Epizoic worms were found to occur on certain coral colonies from reefs off the coast of Eilat (Red Sea). We identified 14 coral species infested by acoelomorph worms at a depth range of 2–50 m. The host corals were all zooxanthellate and included both massive and branching stony corals and a soft coral. Worms from all hosts were identified as belonging to the genus Waminoa and contained two distinct algal symbionts differing in size. The smaller one was identified as Symbiodinium sp. and the larger one is presumed to belong to the genus Amphidinium. Worm-infested colonies of the soft coral, Stereonephthya cundabiluensis, lacked a mucus layer and exhibited distinct cell microvilli, a phenotype not present in colonies lacking Waminoa sp. In most cases, both cnidarian and Acoelomorph hosts displayed high specificity for genetically distinctive Symbiodinium spp. These observations show that the epizoic worms do not acquire their symbionts from the “host” coral.     

Activities of sulfur-oxidizing bacteria at the 21°N East Pacific Rise vent site

The incorporation of inorganic and organic carbon into cell material, as well as the activities of carboxylating enzymes (ribulose bisphosphate carboxylase and phosphoenol pyruvate carboxylase), were measured in waters emitted from warm vents at the 21°N ocean spreading site (depth 2 600 m) of the East Pacific Rise. Both obligately and facultatively chemoautotrophic bacteria were present and comprised a significant but variable portion of the total microbial population as indicated by comparisons of microscopic cell counts with liquid enrichments and colony counts on media containing reduced sulfur compounds. The proportion of chemoautotrophic, sulfur-oxidizing bacteria maximally reached 79% of the total microbial population based on ribulose bisphosphate carboxylase activity. Variability of chemoautotrophic activity occurred between vents at different locations, but was also observed in one individual vent. Maximum rates of CO₂ incorporation in warm vent water were similar to levels measured previously in the O₂/H₂S interface of the Black Sea. In shipboard experiments, these rates were virtually unaffected by in-situ pressures (ca 260 atm). Rate measurements at various temperatures, as well as the observed mesophilic characteristics of all isolates obtained, suggest that the microbial, chemoautotrophic activity decreases rapidly as freshly emitted vent water is diluted with cold, ambient, deep-sea water.Contribution No. 6071 of the Woods Hole Oceanographic InstitutionContribution No. 1708 of the Center for Environmental and Estuarine Studies of the University of Maryland     

Growth of the scyllarid lobsters Ibacus peronii and I. chacei

The growth rates of the morphologically similar scyllarid lobsters Ibacus peronii (Leach, 1815) and I. chacei (Brown and Holthuis, 1998) are described using data from a tag/recapture study and from tagged lobsters kept in captivity. Within particular size classes, we found no differences in moult increments between male and female I. peronii nor between male and female I. chacei. Small individuals of both species always had larger moult increments than larger individuals. For I. peronii, females moulted more frequently than males, and smaller size classes moulted more frequently than larger size classes. Female I. peronii therefore grew more quickly than males and reached their estimated size at sexual maturity (51 mm carapace length) after ∼2 yr. Moulting of I. peronii was seasonal, with most lobsters (96.3%) moulting between October and January. We found no differences in growth rates of I. peronii at two locations along the east coast of Australia: Coffs Harbour in New South Wales (30°18′S; 153°08′E), and Lakes Entrance in Victoria (37°53′S; 148°00′E). For I. chacei, we found no differences in the frequency of moulting between males and females and, because we also found no differences in the moult increments between males and females, the growth rates of both sexes were the same. Received: 14 August 1999 / Accepted: 20 January 2000     

Impact of the polychaete Capitella sp. I on microbial activity in an organic-rich marine sediment contaminated with the polycyclic aromatic hydrocarbon fluoranthene

Polychaetes belonging to the genus Capitella are often present in high numbers in organic-rich sediments polluted with, e.g., oil components, and Capitella spp. may have a great impact on the biogeochemistry of these sediments. We examined the influence of Capitella sp. I on microbial activity in an organic-rich marine sediment contaminated with the polycyclic aromatic hydrocarbon, fluoranthene. Capitella sp. I were added to microcosms (10 000 ind m⁻²) and the impact of a pulse-sedimentation of fluoranthene-contaminated sediment (3 mm layer) was studied for a period of 12 d after sedimentation. The sediment oxygen uptake and total sediment metabolism (TCO₂ production) increased in cores with worms (71 to 131%), whereas the anaerobic activity, measured as sulfate reduction rate 12 d after sedimentation, was lower compared to cores without worms. The effect of fluoranthene on sulfate reduction was most pronounced in the presence of worms, with a 34% reduction versus 16% in cores without worms. The reduced sulfur pools in cores with worms were smaller than in cores without worms, suggesting that the reduced anaerobic activity was caused by increased oxidation of the sediment, which may favor O₂ and other electron-acceptors (e.g. NO₃ ⁻, Fe³⁺, Mn⁴⁺) in organic matter decomposition. The sediment oxygen uptake and TCO₂ production did not show significant changes due to fluoranthene treatment, indicating that these parameters were either less sensitive to fluoranthene stress or recovered more rapidly (i.e. within 48 h) than sulfate reduction rates. Bioturbation by Capitella sp. I altered the depth profile of fluoranthene such that fluoranthene was found in deeper sediment layers (down to 2 cm) where diffusional loss and microbial breakdown probably are reduced relative to surface layers. In cores without worms, fluoranthene was found down to 1 cm, with 75% remaining in the upper 5 mm. Received: 5 December 1996 / Accepted: 11 February 1997     

Microsensor studies of photosynthesis and respiration in the symbiotic foraminifer Orbulina universa

Oxygen and pH microelectrodes were used to investigate the microenvironment of the planktonic foraminifer Orbulina universa and its dinoflagellate endosymbionts. A diffusive boundary layer surrounds the foraminiferal shell and limits the O₂ and proton transport from the shell to the ambient seawater and vice versa. Due to symbiont photosynthesis, high O₂ concentrations of up to 206% air saturation and a pH of up to 8.8, i.e. 0.5 pH units above ambient seawater, were measured at the shell surface of the foraminifer at saturating irradiances. The respiration of the host–symbiont system in darkness decreased the O₂ concentration at the shell surface to <70% of the oxygen content in the surrounding air-saturated water. The pH at the shell surface dropped to 7.9 in darkness. We measured a mean gross photosynthetic rate of 8.5 ± 4.0 nmol O₂ h⁻¹ foraminifer⁻¹. The net photosynthesis averaged 5.3 ± 2.7 nmol O₂ h⁻¹. In the light, the calculated respiration rates reached 3.9 ± 1.9 nmol O₂h⁻¹, whereas the dark respiration rates were significantly lower (1.7 ± 0.7 nmol O₂ h⁻¹). Experimental light–dark cycles demonstrated a very dynamic response of the symbionts to changing light conditions. Gross photosynthesis versus scalar irradiance curves (P vs E _o curves) showed light saturation irradiances (E _k) of 75 and 137 μmol photons m⁻² s⁻¹ in two O. universa specimens, respectively. No inhibition of photosynthesis was observed at irradiance levels up to 700 μmol photons m⁻² s⁻¹. The light compensation point of the symbiotic association was 50 μmol photons m⁻² s⁻¹. Radial profile measurements of scalar irradiance (E _o) inside the foraminifera showed a slight increase at the shell surface up to 105% of the incident irradiance (E _d). Received: 26 January 1998 / Accepted: 11 April 1998     

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     

Size-dependent photosynthetic performance in the giant clam <Emphasis Type="Italic">Tridacna maxima</Emphasis>, a mixotrophic marine bivalve

Giant clams form a symbiosis with photosynthetic algae of the genus Symbiodinium that reside in clam mantle tissue. The allometry of symbiont photosynthetic performance was investigated as a mechanism for the increasing percentage of giant clam carbon respiratory requirements provided by symbionts as clam size increases. Chlorophyll fluorescence measurements of symbionts of the giant clam Tridacna maxima were measured during experiments conducted in September of 2009 using specimens 0.5–200 g tissue wet weight (3–25 cm long), collected from waters around southern Taiwan (N 21°36′, E 120°47′) from July to August of 2009. Light-dependent decreases in effective quantum yield (∆F/F _m′) calculated as the noontime maximum excitation pressure over PSII (Q _m), relative electron transport rates (rETR), and dark-adapted maximum quantum yield (F _v/F _m) all varied as a quadratic function of clam size. Both Q _m and rETR increased as clam size increased up to ~10–50 g then decreased as clam size increased. F _v/F _m decreased as clam size increased up to ~5–50 g then increased as clam size increased. Chlorophyll fluorescence measurements of rETR were positively correlated with gross primary production measured during chamber incubations. Overall, symbionts of mid-sized clams ~5–50 g exhibited the highest light-dependent decreases in effective photosynthetic efficiencies, the highest relative electron transport rates, and the lowest maximum photosynthetic efficiencies, and symbiont photosynthetic performance is allometric with respect to host clam size.     

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.     

Specificity of two temperate dinoflagellate–anthozoan associations from the north-western Pacific Ocean

Whilst many studies of symbiotic dinoflagellate diversity have focused on tropical reef environments, only a few have explored the degree and pattern of divergence of these endosymbionts at high latitudes. In this study, the genetic diversity and specificity of symbiotic dinoflagellates associated with two common anthozoan hosts in the north-western Pacific Ocean was studied in four different seasons during a period of 1 year. Partial nucleotide sequences of 28S and complete ITS1 ribosomal DNA regions were used to identify, genetically, the endosymbionts extracted from the scleractinian Alveopora japonica and the actinarian Heteractis sp. A. japonica harbours symbionts belonging to Symbiodinium of clade F, while Heteractis sp. associates with Symbiodinium of clade C. Moreover, no seasonal changes in the endosymbiont community were detected in these two associations during this study. This is the first evidence that these two temperate cnidarian–microalgae symbioses are stable. Furthermore, we tested the apparent specificity of the Heteractis sp.– Symbiodinium sp. clade C association, by performing alga-infection experiments with aposymbiotic hosts, and monitoring the uptake and persistence of homologous and heterologous symbionts. The findings confirm the association patterns detected in the field and show that Heteractis sp. only establishes a successful association with Symbiodinium cells of clade C, at least among the heterologous symbionts occurring in the study area. Our results are consistent with the idea that selective pressures in highly fluctuating temperate environments might have granted symbiosis-specificity an adaptive value.Communicated by T. Ikeda, Hakodate