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Isolated bacteriocyte cell suspensions from the hydrothermal-vent tubeworm Riftia pachyptila, a potent tool for cellular physiology in a chemoautotrophic symbiosis
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The hydrothermal-vent tubeworm Riftia pachyptila relies entirely on its intracellular chemoautotrophic symbionts to sustain its metabolism. The host must therefore provide them with inorganic metabolites, including carbon. This study describes a tool for studying cell processes occurring in a bacteria-containing cell by the dissociation of trophosome cell types. The physiological assays performed on cell preparations focused on carbon dioxide conversion and transport processes. Trophosome tissue was mechanically dissociated, resulting in cell suspensions enriched in small (7-20 m) bacteriocytes, which were viable for several hours. In addition, medium-term cell cultures were also attempted. As a start to the understanding of the CO2 metabolism of these cells, we were interested in evidence of carbonic anhydrase (CA) isoforms, ATPases and chloride exchangers. Variations in intracellular and extracellular pH, and in intracellular concentrations of sodium, potassium and chloride, were followed after addition of selective inhibitors. The data presented here suggest the occurrence of potential cytosolic and membrane-associated carbonic anhydrase isoforms in the bacteriocytes, proton-driven sodium-ATPases and a well represented anion transporter exchanging intracellular chloride against extracellular anions.
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... The cellular physiology of bacteriocytes is thus central to the functioning of the symbiosis in Vestimentifera. Although bacteriocyte isolation has proved feasible in R. pachyptila (De Cian et al. 2003b), few studies have exploited this approach, and most of these have been done on trophosome tissue incubations. The rest of current knowledge on bacteriocyte-symbiont relationships is based on indirect evidence, mostly from biochemical and molecular studies. ...
... immunolocalisation of cytosolic CA and in situ hybridisation with CA-cDNA probe in Riftia pachyptila trophosome lobules demonstrated that CA is expressed and found in bacteriocytes and peritoneal cells (De Cian et al. 2003a). A physiological investigation using various inhibitors on isolated bacteriocyte suspensions from the trophosome tissue of R. pachyptila revealed a complex interaction of CA with two important ion-transporting enzymes: the vacuolar-type V-h + ATPase and the Na + k + -ATPase (De Cian et al. 2003b). These enzymes seem to be involved in the transepithelial transport processes for electrolytes and Co 2 , and the authors suggested that in the trophosome, the proton efflux might generate a local acidification of the outer layer of the membrane, facilitating the influx of Co 2 . ...
Vestimentiferan tubeworms, once erected at a phylum level, are now known to comprise a part of the specialised deep-sea polychaete family siboglinidae. Their widespread and abundant occurrence at hydrothermal vents and hydrocarbon seeps has fostered numerous studies of their evolution and biogeography, ecology and physiology. harbouring autotrophic, sulphide-oxidising, intracellular bacterial symbionts, they form large populations of 'primary' producers with contrast-ing characteristics, from fast-growing, short-living species at vents, to slow-growing, long-living spe-cies at seeps. These different life strategies and the ways they modify the biogeochemistry of their respective environments have consequences on the macro-and meiofaunal assemblages that develop within vestimentiferan bushes. New findings indicate that postlarval recruits get infected through the skin by free-living bacteria for which growth is rapidly and specifically limited by the host to mesoderm cells around the gut that further transform into the characteristic trophosome. The result-ing internal location of symbionts prompts specific adaptations of the hosts to fulfil their metabolic requirements, including unusual sulphide and carbon dioxide assimilation and transport mecha-nisms. symbiont genome sequencing has improved our knowledge of potential bacterial metabolism and should rapidly open the way for new research approaches to resolve the intricate physiological relationships between a eukaryotic host and its chemoautotrophic bacterial symbionts.
... CA facilitates CO 2 diffusion into the plume by converting it to HCO 3 Ϫ (63, 64) and likely back-converts the HCO 3 Ϫ to CO 2 for fixation by the symbionts in the trophosome. Our analysis suggests that three of the Riftia CAs are membrane bound (Text S1, section 7) and could thus facilitate CO 2 diffusion into bacteriocytes by converting HCO 3 Ϫ to CO 2 on the bacteriocyte cell surface (65,66). Transport of HCO 3 Ϫ to the bacteriocytes could be mediated by sodium bicarbonate exchangers, which we identified in trophosome and plume samples (Table S1a). ...
All animals are associated with microorganisms; hence, host-microbe interactions are of fundamental importance for life on earth. However, we know little about the molecular basis of these interactions. Therefore, we studied the deep-sea Riftia pachyptila symbiosis, a model association in which the tubeworm host is associated with only one phylotype of endosymbiotic bacteria and completely depends on this sulfur-oxidizing symbiont for nutrition. Using a metaproteomics approach, we identified both metabolic interaction processes, such as substrate transfer between the two partners, and interactions that serve to maintain the symbiotic balance, e.g., host efforts to control the symbiont population or symbiont strategies to modulate these host efforts. We suggest that these interactions are essential principles of mutualistic animal-microbe associations.
The mutualistic interactions between Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone (short Endoriftia) have been extensively researched. However, the closed Endoriftia genome is still lacking. Here, by employing single‐molecule real‐time sequencing we present the closed chromosomal sequence of Endoriftia. In contrast to theoretical predictions of enlarged and mobile genetic element‐rich genomes related to facultative endosymbionts, the closed Endoriftia genome is streamlined with fewer than expected coding sequence regions, insertion‐, prophage‐sequences and transposase‐coding sequences. Automated and manually curated functional analyses indicated that Endoriftia is more versatile regarding sulphur metabolism than previously reported. We identified the presence of two identical rRNA operons and two long CRISPR regions in the closed genome. Additionally, pangenome analyses revealed the presence of three types of secretion systems (II, IV and VI) in the different Endoriftia populations indicating lineage‐specific adaptations. The in‐depth mobilome characterisation identified the presence of shared genomic islands in the different Endoriftia drafts and in the closed genome, suggesting that the acquisition of foreign DNA predates the geographical dispersal of the different endosymbiont populations. Finally, we found no evidence of epigenetic regulation in Endoriftia, as revealed by gene screenings and absence of methylated modified base motifs in the genome. As a matter of fact, the restriction‐modification system seems to be dysfunctional in Endoriftia, pointing to a higher importance of molecular memory‐based immunity against phage via spacer incorporation into CRISPR system. The Endoriftia genome is the first closed tubeworm endosymbiont to date and will be valuable for future gene oriented and evolutionary comparative studies.
The deep-sea tubeworm Riftia pachyptila lacks a digestive system, but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which enabled comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. We demonstrate that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion, and substrate transfer to the symbiont by abundant host proteins. Our analysis further suggests that Riftia maintains its symbiont by protecting the bacteria from oxidative damage, while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis.
Animal Biology studies have always benefited from the achievement of experiments on live animals, which obviously provide data on dynamic aspects of physiology. When it comes to deep-sea fauna, in vivo experiments are impaired, and in some cases impossible, due to the severe and often lethal stress experienced by animals throughout the sampling process. However, freshly collected deep fauna may be maintained alive and in good condition, by using specific aquaria which restore environmental conditions prevailing at depth in situ. Here we describe the pressure device named "Incubateur Pressurisé pour l'Observation et la Culture d'Animaux Marins Profonds", or IPOCAMP, and provide an overview of the studies which were achieved using this pressure aquarium, with a particular focus on thermal biology of deep-sea vent fauna.
The symbiotic tubeworm Riftia pachyptila needs to fuel its chemoautotrophic symbiotic bacteria with inorganic carbon. CO2 is transported from the surrounding water to the bacteriocytes located in the trophosome, through the branchial plume and the body fluids. Previous studies have demonstrated the implication of carbonic anhydrase (CA) and proton pumps (ATPases) at various steps of CO2 transport. The present study describes the expression pattern of cytosolic CA using an RNA probe and its histochemical and immunocytochemical localization in the trophosome and branchial plume of Riftia. Immunolocalization of V-H⁺ATPase and Na⁺K⁺-ATPase were also performed and related to CA localization. In the branchial plume, CA is expressed and localized in the most apical region of the branchial epithelium, close to the surrounding water. V-H⁺ATPase is mostly colocalized with CA and both enzymes probably allow CO2 entry against the concentration gradient while regulating intracellular pH. Na⁺K⁺-ATPase is mostly restricted to the basal part of epithelial cells and probably participates in CO2 transport to the body fluids. In the trophosome lobules, cytosolic CA is expressed and found in bacteriocytes and peritoneal cells. Hypotheses on the role of CA in bicarbonate and CO2 interconversion to fuel the symbiotic bacteria are discussed.
Many deep-sea organisms need hydrostatic pressure for their survival. Thus, when retrieved from their natural environment for biological studies that cannot be conducted at the bottom of ocean, they must be returned to high pressure using appropriate pressure vessels. Our studies of organisms living at deep-sea hydrothermal vents lead us to develop such pressure vessels. Here, we present a system of small pressure reactors that were specifically conceived for the study of embryonic development, but may be also used for the study of small organisms, larvae, cells or bacteria. The system consists of two reactors fed by a common pressure line which can be manipulated separately using a set of valves. It is possible to carry out different operations (renewal of water, sampling) without depressurizing the reactors, a feature which proves crucial when carrying out long-term development experiments (over several days). The system has special chambers equipped with sapphire windows that allow the observation of embryos under pressure using a standard optical microscope. Using this system, we studied for the first time the embryonic development of Alvinella pompejana, one of the most intriguing species colonizing the hydrothermal vents.
The deep-sea vent worm Alvinella pompejana is a biological enigma, regarding the adaptative features that allow it to prosper in the harsh environment of hydrothermal chimney walls (East Pacific Rise). Moreover, attempts to maintain this invertebrate alive following recovery (2600 m depth) have so far failed, thus forbidding in vivo laboratory studies. For the first time, it is shown that most of the specimens reach the surface alive, as witnessed by video monitoring of these animals after native pressure conditions have been restored. Furthermore, some individuals may show active behaviour at least 20 h after recovery, under controlled temperature and oxygen level conditions. These promising results open up a new field of investigation, and call for a major breakthrough towards our understanding of A. pompejana's biology, in the near future.
Preparations of trophosome tissue from Riftia pachyptila containing viable endosymbiotic bacteria were incubated with several substrates under a variety of conditions to characterize the symbionts physiologically. Of all the potential substrates tested, only sulfide stimulated carbon fixation by the trophosome preparations; neither hydrogen, ammonia, nor thiosulfate were effective. Trophosome preparations did not oxidize 14 C-methane to either 14 C-organic compounds or 14 CO2, nor did they reduce acetylene under the conditions tested. Carbon fixation by the endosymbionts appears barotolerant. The symbionts require both sulfide and oxygen to fix carbon through autotrophic pathways, but are inhibited by free oxygen and by sulfide concentrations in the 300 μM range. Maximal rates of carbon fixation were documented in incubations in dilute Riftia blood, which protects the symbionts from the inhibitory effects of free sulfide and oxygen while providing them with an abundant pool of both substrates, bound by the vestimentiferan hemoglobins.
Deep-sea hydrothermal vents are home to a variety of invertebrate species, many of which host chemosynthetic bacteria in unusual symbiotic arrangements. Riftia pachyptila Jones, 1981, found to be symbiotic with intracellular chemoautotrophic bacteria, is probably the most successful invertebrate host living at the vents along the East Pacific Rise. Unlike most metazoans, these animals must take up and transport CO2 and HS- to their symbionts, as well as maintain the proper extracellular pH, in the face of many internal processes that act to disrupt acid-base balance. We present here our findings on the physiology of R. pachyptila; specifically the uptake of CO2 and HS- from the environment and H+ elimination via high concentrations of H+ -ATPases (see also Goffredi et al. 1997a,b).
Ultrastructural and microanalytic study of the Vestimentiferan trophosome shows that the important sulphur quantities present in the organ are not stored as globules as it was previously hypothesized. We assume that the elemental sulphur could exist under S8 octaedral structures, whose size is not observable at the microscopic scale inside the bacteria hyaloplasm. Metallic bioaccumulations are not considerable in bacteria and in bacteriocytes and are due to a physiological purifying metabolism. Metallic salts, abundant in the hydrothermal fluid, are probably poorly soluble and then less toxic than it appeared previously.
The sequencing of 16S and 23S ribosomal RNA (rRNA) molecules is currently the gold standard for the classification of new microbial isolates. Comparative analyses of these sequences are for the first time in the history of microbiology facilitating the reconstruction of universal phylogenetic trees [38]. Among many other important findings the work of Carl Woese and his colleagues demonstrated that only certain (by far not all) phenotypic/physiological groups of micro-organisms are monophyletic (e.g., methanogenes, cyanobacteria, spirochetes). About 10 years ago it has been proposed to use an rRNA approach for studies in microbial ecology [21]. The microbial diversity should be analyzed in a cultivation-independent way by direct rRNA sequence retrieval, whereas nucleic acid probes complementary to rRNA or rRNA genes should be the tools to monitor population dynamics in the environmental samples. By their own nature rRNA-targeted probes track genotypes which are not necessarily linked to one phenotype. Microbial ecologists who want to apply this approach to investigate correlations between community structures and functions should be aware of this fact and design or apply rRNA-targeted probes accordingly.
Based on comparative analyses of 16S and 23S ribosomal RNA sequences we have located sites specific for the alpha-, beta-, and gamma-subclasses of Proteobacteria. Short oligodeoxynucleotides complementary to these signature regions were evaluated as potential nucleic acid probes for the differentiation of the major subclasses of Proteobacteria. Hybridization conditions were optimized by the addition of formamide to the hybridization buffer and high stringency post-hybridization washing. Single-mismatch discrimination of probes was further improved by blocking nontarget probe binding sites with competitor oligonucleotides. Nonisotopic dot-blot hybridization to reference strains demonstrated the expected probe specificities, whole cell hybridization with fluorescent probe derivatives allowed the classification of individual microbial cells. The probes will be useful for determinative studies and for the in situ monitoring of population distribution and dynamics in microbial communities.
Riftia pachyptila, the giant hydrothermal vent tube worm, lives in a thermally and chemically complex environment It apparently derives the bulk of its nutrition from its chemoautotrophic symbionts, which are found at very high densities in a large internal organ, the trophosome. Although somewhat buffered from the environmental variation, the internal symbionts likely are exposed to significantly different temperatures in different host individuals, in different positions in the same host individual, and at different times in the same host individual Temperature optima for autotrophic carbon fixation varied from 25° to 35°C in trophosome preparations from eight individuals. Because of a variety of factors, both the mode of supply of inorganic carbon to the symbionts in the trophosome and the adequacy of that supply (with respect to saturation of demand) are not understood. On the basis of experiments varying the concentrations of bicarbonate and dissolved carbon dioxide, an apparent K, for dissolved carbon dioxide of between 0.021 and 0.035 mM is demonstrated, and dissolved carbon dioxide is determined to be the form of inorganic carbon taken up and used by the symbionts.
Hydrothermal activity at mid-ocean ridge spreading centers has an
influence on all aspects of oceanography. The heat input from these
systems affects the mid-depth circulation of the oceans, although the
magnitude of this effect has not been rigorously quantified [Stommel,
1982]. The rising hydrothermal plumes also entrain deeper, and often
saltier water, carrying it up in the water column thereby also affecting
the thermohaline circulation of the oceans [Lupton et al., 1985]. The
topography of the ridges may also influence deep circulation patterns.
It is the inputs of heat and reduced chemical species from the
hydrothermal systems that provide the energy for communities of
chemosynthetic organisms, unknown prior to the discovery of venting.
Hydrothermal activity also affects the chemistry of seawater by the
direct addition and removal of various chemical species, and the more
indirect scavenging of mid depth chemical constituents onto the Fe- and
Mn-particles fanned in hydrothermal plumes. The composition of the
oceanic crust is also changed by the addition and removal of chemical
constituents as a result of hydrothermal alteration. Recycling of the
oceanic crust into the Earth's mantle and the eventual transfer of some
gases input by hydrothermal activity from the ocean to the atmosphere,
extend the Influence of hydrothermal activity to beyond the oceans
themselves. While the magnitude and importance of these linkages are
still poorly quantified, we were unaware these linkages even existed
prior to the discovery of seafloor hydrothermal systems.