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     

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     

Carbonic anhydrase in deep-sea chemoautotrophic symbioses

Carbonic anhydrase (CA) facilitates the rapid interconversion of carbon dioxide and bicarbonate. It is ubiquitous among organisms, and participates in numerous cellular processes. In several photoautotrophic marine organisms, CA facilitates uptake of inorganic carbon by catalyzing the reversible dehydration of bicarbonate to carbon dioxide, which diffuses more rapidly across cell membranes. In algal/invertebrate symbioses, this mechanism is correlated with significantly elevated CA activity in tissues where zooxanthellae are housed. Here, we determine whether elevated CA activity is also characteristic of tissues involved in inorganic carbon uptake by chemosynthetic invertebrates. Measurements of CA activity in several chemosynthetic clam and vestimentiferan species indicate that CA facilitates inorganic carbon uptake, with high activities present in clam gill tissue, and vestimentiferan plume and trophosome tissues. However levels of CA activity among species varied widely, from very low in mytilids to extraordinarily high in some vesicomyids and vestimentiferans. High CA activity is generally correlated with other adaptations for control of internal chemical environments where symbionts are housed, such as the presence of serum-borne sulfide-binding moieties. These findings suggest that a CA-based mechanism of carbon uptake may be one of a suite of adaptations to provide symbionts with essential metabolites.     

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.     

Bacterial symbionts as an additional cytological marker for identification of sponges without a skeleton

Symbiotic bacteria from six Oscarella species (adults and embryos) collected in the Mediterranean Sea (O. lobularis, O. tuberculata, O. imperialis, O. microlobata, O. viridis) and the Sea of Japan (O. malakhovi) were investigated by scanning electron microscopy and transmission electron microscopy. In most cases, symbionts are rather numerous. Each sponge species has a definite set of bacterial morphological types. All bacteria are extracellular. Symbionts occupy the mesohyl of adult sponges or intercellular space in embryos and are often in contact with mesohylar filaments or cells. Bacteria of some morphotypes have characteristic blebs. Most symbionts are gram-negative, and two types of bacteria have traits of Archaea and one type of bacteria is similar to Planctomycetes. Data on morphology of bacterial symbionts can be a good additional character for identification of Oscarella species, which have no skeleton.     

The bacterial symbiont from the hydrothermal vent tubewormRiftia pachyptila is a sulfide specialist

We have investigated the metabolic adaptations of the chemolithotrophic bacterial symbionts ofRiftia pachyptila. Specimens of the tubeworm were collected by submersible from depths of 2600 m at 13°N on the East Pacific Rise in 1987, and 2450 m at the Galápagos Rift in 1988. Isolated bacteria utilize sulfide, but not thiosulfate or sulfite, as their sole reduced-sulfur energy source. The bacteria rapidly oxidize a wide range of sulfide concentrations (5µM to 2 mM), with maximal respiration rates at concentrations >1 mM, and unlike many sulfur-oxidizing bacteria, show no inhibition in oxygen consumption at sulfide concentrations up to 2 mM. Incubations of freshly homogenized trophosome tissue or isolated bacteria with sodium [³⁵S] sulfide and subsequent analysis of sulfur products by high-performance liquid chromatography and flow-through scintillation counting showed that sulfide disappeared almost completely within 1 min. Both soluble and insoluble products of sulfide oxidation were produced. The soluble fraction contained sulfate and polysulfides, with no thiosulfate produced. However, the majority of the radioactivity was in the water-insoluble fraction, mostly as elemental sulfur. Whole-worm experiments under pressure showed a rapid removal of³⁵S-sulfide from the incubation water, with sulfide, sulfate, and polysulfides appearing in the blood within 4 h. There was no utilization of thiosulfate by the whole worms, freshly homogenized trophosome tissue, or isolated bacteria.     

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).     

Evolutionary relationships among deep-sea mytilids (Bivalvia: Mytilidae) from hydrothermal vents and cold-water methane/sulfide seeps

A protein electrophoretic survey of mytilids inhabiting deep-sea hydrothermal vents and cold-water methane/sulfide seeps revealed electromorph patterns diagnostic of 10 distinct species. From hydrothermal vents located at sites on the Galápagos Rift, the Mid-Atlantic Ridge, and the Mariana Back Arc Basin, we detected four species of mytilids. Six additional species were detected from three cold-water seep sides in the Gulf of Mexico. The patchy distribution and temporal stability of seeps may provide a greater opportunity for mytilid diversification and persistence than vent sites Nei's genetic distances (D) between species were relatively large (range: 0.528 to ) both within and among habitat types. This pronounced degree of genetic differentiation suggests a relatively ancient common ancestor for the group. Phylogenetic trees were generated using distance Wagner and parsimony analyses of allozyme and morphological characters. The tree topologies obtained from both methods support: (1) the hypothesis that a seep ancestor gave rise to the deep-sea hydrothermal vent mytilids, (2) a historical progression from shallow-water to deep-water habitats, and (3) a co-evolutionary progression from external to internal localization of bacterial symbionts. Whether the seep mytilid taxa constitute paraphyletic or polyphyletic groups remains unresolved. Our phylogenetic hypotheses also provide a benchmark for the phylogeny of mytilid bacterial symbionts.     

Metabolic diversity in epibiotic microflora associated with the Pompeii wormsAlvinella pompejana andA. caudata (Polychaetae: Annelida) from deep-sea hydrothermal vents

Specimens of alvinellid polychaetes (Alvinella pompejana Desbruyères and Laubier, 1980 andA. caudata Desbruyères and Laubier, 1986) and their tubes were sampled from deep-sea hydrothermal vents at 13°N from the manned submersible Nautile during the Hydronaut cruise (October to November 1987) on the East Pacific Rise. Samples were subjected to bacterial analysis aboard the mother ship Nadir to detect bacteria involved in the nitrogen and sulphur cycles, in non-specific heterotrophic processes, and displaying resistance to selected heavy metals. Cultures were incubated at different temperatures under atmospheric and in situ (250 atm) pressures. Bacterial growth was observed in enrichment cultures for most metabolic types screened. Heavy-metal-resistant bacteria were also detected in many samples. No filamentous bacterial form was observed in the cultures. The results demonstrate a high metabolic diversity in episymbiotic flora, indicating that the worm (A. pompejana orA. caudata), its tube and its epiflora represent a complex micro-ecosystem.     

Microbial associations in sponges. II. Numerical analysis of sponge and water bacterial populations

Three taxonomically distant sponges Pericharax heteroraphis, Jaspis stellifera and Neofibularia irata contain phenotypically similar bacterial symbionts which differ from bacteria in the ambient water. These symbionts are predominant in the sponges and were detected after computer analysis of 526 heterotrophic bacterial strains tested for 76 characters. These facultative anaerobic symbionts metabolize a wide range of compounds and may be important in removing waste products while the sponges are not circulating water. The bacteria produce sticky-mucoid colonies and thus would contribute to sponge structural rigidity. The fourth sponge Ircinia wistarii contains a mixed aerobic population similar to that in the ambient water. The majority of the bacteria are located around the inhalant canals, facilitating the uptake of dissolved organic matter and oxygen from the incoming water.     

Comparison of bacterial and algal utilization of orthophosphate in an estuarine environment

Bacterial utilization of orthophosphate in an estuarine environment has been differentiated from algal utilization by using flow-filters of 5.0, 1.2 and 0.45 m poresize. Examination by light microscopy showed that most of the bacterial population passed through a 5.0-m filter, whereas most algae were retained. In all experiments, bacterial and algal cell numbers and biomass were estimated. P-uptake by algae and bacteria was closely correlated with cell biomass. P-uptake by algae was high only in the summer months, whereas P-uptake by bacteria was high throughout the year. Neither algal nor bacterial P-uptake, however, was correlated with temperature or dissolved orthophosphate, total organic phosphate or total phosphate concentrations. Cell biomass of algae at a given time had a high correlation with dissolved organic phosphate levels in 2 weeks prior to sampling (r=0.830) and a low correlation in the 2 weeks following sampling (r=0.0005). Algal cell numbers had a high correlation with bacterial cell numbers (r=0.950). The biomass of algae and bacteria also had a high correlation (r=0.902). The rate of P-uptake from the water by algae and bacteria varied with season and with the species composition of the natural population.     

Microbial associations in sponges. III. Ultrastructure of the in situ associations in coral reef sponges

Symbiotic cyanobacteria are associated with marine sponges in three ways: the majority are free-living in the mesohyl; large aggregates occur in cyanocytes (specialized, vacuolated archeocytes); and few are present in digestive vacuoles. The cyanobacteria in Jaspis stellifera and Neofibularia irata are morphologically similar to those described in Mediterranean sponges, whereas those in Pericharax heteroraphis are different. The freeliving bacterial populations are morphologically similar, although the number of bacteria varies between the species. The fourth sponge Ircinia wistarii contains a mixed bacterial population unlike those in the other sponges. Sponge digestion of microbial associates is rare and not considered to contribute significant nutrients.     

Cortical and endosomal structure of the marine sponge Stelletta grubii

The fine structure of the marine astrophorid sponge Stelletta grubii is described for the first time. The following new data are presented: spongin is present, choanocyte chambers are diplodal, intercellular symbiotic bacteria are numerous and unequally distributed in the cortex and endosome, and collagenous fibril bundles are associated with lophocyte activity and are not elastic fibers. The cortex contains numerous fibril bundles, fewer symbionts, very few cells, and transitional zones with higher archeocyte density near the surface and endosome. Limited phagocytosis of the bacterial symbionts is observed. This species appears to be dioecious and oviparous. These observations suggest that the enigmatic species Chondrosia reniformis is closely related to S. grubii and that it should be placed within or near the astrophorids. The rhizoids of the red alga Phyllophora palmettoides penetrate the sponge tissue without eliciting the development of a structurally specialized contact zone in the sponge matrix or of a limiting epithelium.     

Strobilation,budding and initiation of scyphistoma morphogenesis in the rhizostome Cassiopea andromeda (Cnidaria: Scyphozoa)

Scyphistomae of Cassiopea andromeda Forskål, 1775 containing symbiontic zooxanthellae did not develop medusae at a constant temperature of 20°C, but monodisc strobilation was initiated after transfer of the polyps to 24°C. After release of the ephyrae and regeneration of the hypostome and tentacular region, the recovered polyps either produced vegetative buds or entered a new strobilation phase. Formation of motile, planula-like buds was not found to be indicative of unfavourable environmental conditions. Intensity of budding was positively correlated with available food and with increase of temperature. Budding was negatively correlated with the number of polyps maintained per dish and with the conditioning of the sea water. Under optimal feeding and temperature conditions, polyps could simultaneously produce chains of buds at 2 to 4 budding regions. Settlement and development of buds into scyphistomae was suppressed in pasteurized sea water and in pasteurized sea water containing antibiotics, but polyps developed from buds in the presence of algal material taken from the aquarium, debris or egg shells of Artemia salina, or on glass slides which had been incubated in used A. salina culture medium. Several species of marine bacteria were detected after staining these slides. One, a Gramnegative coccoid rod, which was identified as a nonpathogenic Vibrio species, was isolated, cultivated as a pure strain, and was proved to induce the development of C. andromeda buds into polyps. Millipore filter-plates coated with Vibrio sp. cells grown in suspension culture were ineffectual, but diluted filtrate initiated polyp morphogenesis. The inducing factor is obviously not a constituent of the bacterial cell surface, but is a product of growing Vibrio sp. cells released into the medium. This product was found to be relatively heat-stable and dialyzable. As to the basic mechanism involved in the induction of polyp formation, it is suggested that the inducing factor (s) acts bimodally by inducing pedal disc development and by eliminating a head inhibitor originating from the basal end of the bud. The life history, and various aspects of medusa-formation and of vegetative reproduction in scyphozoans are reviewed and discussed with particular reference to rhizostome species. Special attention has been paid to some reports of larval metamorphosis controlled by marine bacteria.     

Nemertean,copepod, and amphipod symbionts of the dimorphic ascidian Pyura stolonifera near Melbourne,Australia: specificities to host morphs,and factors affecting prevalences

On the central coast of Victoria. Australia, the dimorphic ascidian Pyura stolonifera (Heller, 1878) harbors three endosymbionts: the nemertean Gononemertes australiensis Gibson, 1974, the copepod Doropygus pulex (Thorell, 1859), and the amphipod Paraleucothoe novaehollandiae (Haswell, 1880). The specificities of these symbionts to two host colour morphs were studied during 1989 to 1991 as part of a multidisciplinary investigation aimed at determining whether the two morphs are genetically distinct. Distributional surveys revealed that nemerteans and copepods occur only in yellow and brown ascidians, respectively, and that amphipods live in both forms. These specificities held true not only when the two morphs were in allopatry, but also in sympatry. These observations, especially the sympatric data, suggest that the two host morphs might be genetically distinct. For example, the two morphs might have different genetically encoded internal milieus that favour the survival of nemerteans in yellow ascidians, and copepods in brown hosts. In transplant experiments, which involved moving ascidian morphs within and between habitats, the wrong symbionts never colonised the wrong hosts. These results, although consistent with the hypothesis of genetic maintenance of specificity, were deemed inconclusive because of the difficulty of establishing reliable controls (i.e. vacant hosts). The relationships between symbiont prevalences and several factors (season, year, site within host, host individual, host habitat, host size/age, host breeding condition, and co-occurrence of other symbiont species) were also analysed. Both simple (e.g. greater prevalences for large hosts) and complex (e.g. prevalence x season x gonad state of host) interactions were detected for all three symbiont species. These are among the very few quantitative analyses of factors affecting prevalences of ascidicolous nemerteans and amphipods. The present report identifies one of very few definite nemertean-ascidian symbioses. Since no differences in gross condition were ever noticed between occupied and vacant hosts, it is suggested that all three symbionts are commensals rather than parasites or mutuals.     

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     

Origins and microenvironments of bacteria mediating fecal pellet decomposition in the sea

The fecal pellets of zooplankton are thought to be major carriers of organic matter from surface to deeper waters of the oceans. As the pellets descend, they release soluble components, partially due to breakdown by associated microorganisms. Previous laboratory work of other investigators has suggested that the surface of a fecal pellet rapidly acquires bacteria, which increase in abundance until they and their protozoan consumers disrupt the pellet membrane, spilling contents into the water. In contrast, our field collections of fecal pellets from free-floating particle interceptor traps (from the Vertex project off Central California in 1980 and off Mexico in 1981), suggest that microbial decomposition probably is initiated in the sea from inside the fecal pellets. Transmission and scanning electron microscopy indicate that bacterial populations are most abundant in the interior of fecal pellets obtained from the sea, but that the same pellets will acquire the surface bacterial lawn typically observed in laboratory studies if maintained aboard ship. If the fecal pellets are decomposed from the inside, then the principal agents are enteric bacteria or ingested, digestion-resistant bacteria, or both. Such bacteria may differ metabolically from those that colonize the fecal pellet surfaces. Further-more, the abundance of healthy-appearing bacteria inside the pellets suggests that their metabolic activities may produce microhabitats of reduced oxygen tension that could differ considerably from that of the pellet exteriors. Decomposition in these semi-enclosed microenvironments may proceed in a manner not yet predicted by models that attribute decomposition to well-aerated, surface-dwelling bacterial populations on fecal pellets in the sea.     

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.     

Extracellular enzymatic activity and secondary production in free-living and marine-snow-associated bacteria

Abundance, production (measured as thymidine incorporation) and extracellular enzymatic activity in free-living and marine-snow-associated bacteria were measured in the northern Adriatic Sea. Although bacterial density and production were similar in both free-living and marine-snow-associated bacteria, hydrolytic activity (- and -glucosidase and l-aminopeptidase) was significantly higher in marine-snow-associated bacteria, in terms of both absolute and per-cell rates. As concentrations of dissolved total and monomeric carbohydrates and free amino acids in marine snow were very close to those in the ambient water, we suggest that the observed differences between free-living and marine-snow-associated baycteria do not simply reflect catabolic repression of enzyme expression in one of the bacterial components. Whether substrate induction is responsible for the observed higher hydrolase activity in marine-snow bacteria and/or whether there are distinct bacterial species obligatorily associated with marine snow remains unknown.