Microbial diversity associated with the hydrothermal shrimp Rimicaris exoculata gut and occurrence of a resident microbial community

Rimicaris exoculata dominates the megafauna of several Mid-Atlantic Ridge hydrothermal sites. Its gut is full of sulphides and iron-oxide particles and harbours microbial communities. Although a trophic symbiosis has been suggested, their role remains unclear. In vivo starvation experiments in pressurized vessels were performed on shrimps from Rainbow and Trans-Atlantic Geotraverse sites in order to expel the transient gut contents. Microbial communities associated with the gut of starved and reference shrimps were compared using 16S rRNA gene libraries and microscopic observations (light, transmission and scanning electron microscopy and FISH analyses). We show that the gut microbiota of shrimps from both sites included mainly Deferribacteres, Mollicutes, Epsilon - and Gammaproteobacteria . For the first time, we have observed filamentous bacteria, inserted between microvilli of gut epithelial cells. They remained after starvation periods in empty guts, suggesting the occurrence of a resident microbial community. The bacterial community composition was the same regardless of the site, except for Gammaproteobacteria retrieved only in Rainbow specimens. We observed a shift in the composition of the microbiota of long-starved specimens, from the dominance of Deferribacteres to the dominance of Gammaproteobacteria . These results reinforce the hypothesis of a symbiotic relationship between R. exoculata and its gut epibionts.     

Pathways of carbon and energy metabolism of the epibiotic community associated with the deep-sea hydrothermal vent shrimp Rimicaris exoculata

Background

The shrimp Rimicaris exoculata dominates the faunal biomass at many deep-sea hydrothermal vent sites at the Mid-Atlantic Ridge. In its enlarged gill chamber it harbors a specialized epibiotic bacterial community for which a nutritional role has been proposed.

Methodology/Principal Findings

We analyzed specimens from the Snake Pit hydrothermal vent field on the Mid-Atlantic Ridge by complementing a 16S rRNA gene survey with the analysis of genes involved in carbon, sulfur and hydrogen metabolism. In addition to Epsilon- and Gammaproteobacteria, the epibiotic community unexpectedly also consists of Deltaproteobacteria of a single phylotype, closely related to the genus Desulfocapsa. The association of these phylogenetic groups with the shrimp was confirmed by fluorescence in situ hybridization. Based on functional gene analyses, we hypothesize that the Gamma- and Epsilonproteobacteria are capable of autotrophic growth by oxidizing reduced sulfur compounds, and that the Deltaproteobacteria are also involved in sulfur metabolism. In addition, the detection of proteobacterial hydrogenases indicates the potential for hydrogen oxidation in these communities. Interestingly, the frequency of these phylotypes in 16S rRNA gene clone libraries from the mouthparts differ from that of the inner lining of the gill chamber, indicating potential functional compartmentalization.

Conclusions

Our data show the specific association of autotrophic bacteria with Rimicaris exoculata from the Snake Pit hydrothermal vent field, and suggest that autotrophic carbon fixation is contributing to the productivity of the epibiotic community with the reductive tricarboxylic acid cycle as one important carbon fixation pathway. This has not been considered in previous studies of carbon fixation and stable carbon isotope composition of the shrimp and its epibionts. Furthermore, the co-occurrence of sulfur-oxidizing and sulfur-reducing epibionts raises the possibility that both may be involved in the syntrophic exchange of sulfur compounds, which could increase the overall efficiency of this epibiotic community.     

Recent population expansion and connectivity in the hydrothermal shrimp Rimicaris exoculata along the Mid‐Atlantic Ridge

Aim Deep‐sea hydrothermal vents are unstable habitats that are both spatially and temporally fragmented. In vent species, a ‘short‐term insurance’ hypothesis would lead us to expect mostly self‐recruitment, limiting the loss of larvae in the deep ocean or water column and increasing genetic differentiation over the time elapsed since colonization. Alternatively, a ‘long‐term insurance’ hypothesis would support the prediction of selection for large‐scale dispersal, to ensure long‐term persistence in these ephemeral habitats. The main goal of this study was to infer the spatial and temporal distribution of genetic diversity of the shrimp Rimicaris exoculata, which forms high‐density local populations on hydrothermal vents along the Mid‐Atlantic ridge. Location Deep‐sea hydrothermal vents along the Mid‐Atlantic Ridge. Methods We used sequences of mitochondrial cytochrome c oxidase subunit I (COI, 710 bp) to assess the spatio‐temporal distribution of genetic diversity across five hydrothermal fields from 36° N to 4° S. Results In contrast to previous results from pioneer studies, very high haplotype diversity was observed in vents across the entire region (i.e. 0.69–0.82), indicating current large effective population size and low drift. The star‐like shape of the network of haplotypes, the lack of spatial genetic structure and the significance of tests reflecting demographic effects, together with the fitting of a population expansion model, all support a recent population expansion. Main conclusions Our results suggest a very recent common history of R. exoculata populations/demes along the Mid‐Atlantic Ridge, derived after a common bottleneck or founder event and followed by a concomitant demographic expansion. This study therefore suggests a large effective population size and/or high dispersal capacity, as well as a possible recent (re)colonization of Mid‐Atlantic hydrothermal vents by R. exoculata.     

Acquisition of epibiotic bacteria along the life cycle of the hydrothermal shrimp Rimicaris exoculata

The caridean shrimp Rimicaris exoculata dominates the fauna at several Mid-Atlantic Ridge hydrothermal vent sites. This shrimp has an enlarged gill chamber, harboring a dense ectosymbiotic community of chemoautotrophic bacteria associated with mineral oxide deposits. Until now, their acquisition is not fully understood. At three hydrothermal vent sites, we analyzed the epibionts diversity at different moult stages and also in the first stages of the shrimp life (eggs, hatched eggs (with larvae) and juveniles). Hatched eggs associated with young larvae were collected for the first time directly from gravid females at the Logachev vent site during the Serpentine cruise. An approach using 16S rRNA clone libraries, scanning and transmission electron microscopy, and fluorescent in situ hybridization was used. Molecular results and microscope observations indicated a switch in the composition of the bacterial community between early R. exoculata life cycle stage (egg libraries dominated by the Gammaproteobacteria) and later stages (juvenile/adult libraries dominated by the Epsilonproteobacteria). We hypothesized that the epibiotic phylotype composition could vary according to the life stage of the shrimp. Our results confirmed the occurrence of a symbiosis with Gammaproteobacteria and Epsilonproteobacteria, but more complex than previously assumed. We revealed the presence of active type-I methanotrophic bacteria colonizing the cephalothorax of shrimps from the Rainbow site. They were also present on the eggs from the Logachev site. This could be the first ‘epibiotic'' association between methanotrophic bacteria and hydrothermal vent crustacean. We discuss possible transmission pathways for epibionts linked to the shrimp life cycle.     

Branchial chamber tissues in two caridean shrimps: the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata

The structure of the epithelia of the branchial chamber organs (gills, branchiostegites, epipodites) and the localization of the Na(+),K(+)-ATPase were investigated in two caridean shrimps, the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata. The general organization of the phyllobranchiate gills, branchiostegites and epipodites is similar in P. adspersus and in R. exoculata. The gill filaments are formed by a single axial epithelium made of H-shaped cells with thin lateral expansions and a basal lamina limiting hemolymph lacunae. In P. adspersus, numerous ionocytes are present in the epipodites and in the inner-side of the branchiostegites; immunofluorescence reveals their high content in Na(+),K(+)-ATPase. In R. exoculata, typical ionocytes displaying a strong Na(+),K(+)-ATPase specific fluorescence are observed in the epipodites only. While the epipodites and the branchiostegites appear as the main site of osmoregulation in P. adspersus, only the epipodites might be involved in ion exchanges in R. exoculata. In both species, the gill filaments are mainly devoted to respiration.     

ϵ-Proteobacterial diversity from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge

The prokaryotic phylogenetic diversity was determined for a sample associated with an in situ growth chamber deployed for 5 days on a Mid-Atlantic Ridge hydrothermal vent (23 degrees 22'N, 44 degrees 57'W). The DNA was extracted from the sample and the 16S rDNA amplified by PCR. No Archaea were detected in the sample. Eighty-seven clones containing bacterial 16S rDNA inserts were selected. Based on restriction fragment length polymorphism analysis, 47 clones were unique, however, based on comparative sequence analysis some of these were very similar, and thus only 22 clones were selected for full sequence and phylogenetic analysis. The phylotypes were dominated by epsilon-Proteobacteria (66%). The remainder formed a novel lineage within the Proteobacteria (33%). One clone formed a distinct deeply branching lineage, and was a distant relative of the Aquificales. This report further expands the growing evidence that epsilon-Proteobacteria are important members in biogeochemical cycling at deep-sea hydrothermal ecosystems, participating as epibionts and free living bacteria.     

深海热液区化能自养菌Caminibacter profundus氢酶及其对H2的响应特点

化能自养菌中的氢酶在深海热液区生态系统的物质和能量转化中具有重要作用。以Caminibacter profundus为研究对象,通过设计PCR引物,克隆编码膜结合的类型I NiFe-氢酶大亚基基因序列hynL并进行生物信息学分析;研究hynL相对表达、甲基紫晶(MV)还原氢酶活性以及菌株生长对H2浓度变化的响应特点。结果表明,从C.profundus克隆获得864 bp的hynL基因片段,其编码的氨基端序列与Lebetimonas acidiphila的相似性为99%,与热液区化能自养的Epsilonproteobacteria D类群的类型I NiFe氢酶大亚基属同一分支。hynL相对表达量和MV还原的氢酶活性分别于12 h和24 h达到最高,此时菌体处于指数生长期;hynL相对表达量和MV还原的氢酶活性和菌株生长的最适H2浓度均为60%。提示C.profundus通过调控hynL的表达,响应环境中H2浓度的变化,以影响菌株能量代谢的催化过程和生长繁殖。     

Biotic and abiotic controls on iron oxyhydroxide formation in the gill chamber of the hydrothermal vent shrimp Rimicaris exoculata

A unique feature of the shrimp, Rimicaris exoculata , from the Rainbow hydrothermal vent field is the abundance of iron oxyhydroxides in its branchial chamber. These minerals accumulate throughout the molting cycle and are intimately associated with the shrimps' epibiotic microflora. In this study, an enhancement of the iron oxidation rate through shrimp swarms in the vicinity of vents is highlighted. This process is sustained by the high molting frequency of the shrimp, and potentially has large biogeochemical and ecological consequences for the associated hydrothermal ecosystem. The calculated rate for abiotic (homogeneous and heterogeneous) iron oxidation suggests that autocatalytic oxidation is the predominant reaction pathway leading to the accumulation of iron oxyhydroxides throughout the molting cycle. The occurrence of iron-oxidizing bacteria is not excluded, but their growth is most probably restricted to the first molting stage when competition with the abiotic iron oxidation is low. The influence of epibiont activity on local oxygen conditions and on the surface properties of the formed mineral, combined with the position of the shrimp in the hydrothermal mixing gradient, is expected to drive the relative contribution of abiogenic and biogenic iron oxidation.     

Dual symbiosis of the vent shrimp Rimicaris exoculata with filamentous gamma- and epsilonproteobacteria at four Mid-Atlantic Ridge hydrothermal vent fields

The shrimp Rimicaris exoculata from hydrothermal vents on the Mid-Atlantic Ridge (MAR) harbours bacterial epibionts on specialized appendages and the inner surfaces of its gill chamber. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH), we examined the R. exoculata epibiosis from four vents sites along the known distribution range of the shrimp on the MAR. Our results show that R. exoculata lives in symbiosis with two types of filamentous epibionts. One belongs to the Epsilonproteobacteria, and was previously identified as the dominant symbiont of R. exoculata. The second is a novel gammaproteobacterial symbiont that belongs to a clade consisting exclusively of sequences from epibiotic bacteria of hydrothermal vent animals, with the filamentous sulfur oxidizer Leucothrix mucor as the closest free-living relative. Both the epsilon- and the gammaproteobacterial symbionts dominated the R. exoculata epibiosis at all four MAR vent sites despite striking differences between vent fluid chemistry and distances between sites of up to 8500 km, indicating that the symbiosis is highly stable and specific. Phylogenetic analyses of two mitochondrial host genes showed little to no differences between hosts from the four vent sites. In contrast, there was significant spatial structuring of both the gamma- and the epsilonproteobacterial symbiont populations based on their 16S rRNA gene sequences that was correlated with geographic distance along the MAR. We hypothesize that biogeography and host-symbiont selectivity play a role in structuring the epibiosis of R. exoculata.     

Structural studies of the main exopolysaccharide produced by the deep-sea bacterium Alteromonas infernus

The structure of the extracellular polysaccharide produced by the mesophilic species, Alteromonas infernus, found in deep-sea hydrothermal vents and grown under laboratory conditions, has been investigated using partial depolymerization, methylation analysis, mass spectrometry and NMR spectroscopy. The repeating units of this polysaccharide is a nonasaccharide with the following structure: [carbohydrate: see text].     

Ascorbic acid catabolism by gut microflora: studies in germ-free and conventional guinea pigs

The role of gut microflora in ascorbic acid catabolism was investigated in both conventional and germ-free guinea pigs. In vitro studies demonstrated extensive degradation of the vitamin by fresh feces, cecal, and colonic contents of conventional guinea pigs. Direct injection of [1-¹⁴C] ascorbic acid into the cecum of conventional guinea pigs in vivo yielded a 70% recovery of the label as respiratory ¹⁴CO₂ within 6 hr compared with only 5% recovery following injection into the virtually sterile peritoneum in a comparable group of conventional guinea pigs. Thus, ascorbic acid not absorbed prior to reaching the lower gastrointestinal tract stands to be extensively decarboxylated by microflora in the cecum. In a companion study of germ-free guinea pigs, 10% of an administered dose of [1-¹⁴C] ascorbic acid was expired as ¹⁴CO₂ within 36 hr post-injection following intraperitoneal injection compared with 16% recovery in a matched group of conventional animals injected at the same site. Results of this series of studies suggest that hepatic decarboxylation and gut microflora, in tandem, contribute to ascorbic acid decarboxylation in this species.     

Diversity in mussel beds at deep-sea hydrothermal vents and cold seeps

Remarkably little is known about fundamental distinctions (or similarities) between the faunas of deep‐sea hydrothermal vents and seeps. Low species richness at vents has been attributed to the transient nature of vent habitats and to toxic effects of hydrogen sulphide and heavy metals in vent effluents. Seeps are arguably more stable and more chemically benign than vents. They have also been regarded as more diverse, but until now there has not been a rigorous test of this hypothesis. We evaluated diversity indices for invertebrates associated with mussel beds at six vents and two seeps and found that invertebrate diversity was significantly higher at seeps than vents, although some vent mussel beds supported nearly the same diversity as seep mussel beds. Lower diversity at vents may be a consequence of a greater physiological barrier to invasion at vents than at seeps. Diversity was lowest where spacing between vents was greatest, suggesting that risks of extinction as a result of dispersal‐related processes may contribute to the pattern of diversity observed at vents.     

Host associated mixed probiotic bacteria induced digestive enzymes in the gut of tiger shrimp Penaeus monodon

The shrimp Penaeus monodon was used for the isolation of digestive enzyme producing host-associated probiotic bacteria. Gut was isolated from a healthy animal completely and morphologically different bacterial isolates were screened for the production of hydrolytic enzymes, such as, protease, amylase, lipase and cellulases. Based on their ability to produce enzymes, the potent probiotic bacteria were identified as Bacillus subtilis and B. licheniformis and these two were used for the preparation of probiotic diet for experimental trials. Probiotic diet was prepared by mixing the shrimp feed with 2 g probiotic/100 g artificial diet (F1), 4 g/100 g (F2), 6 g/100 g (F3), 8 g/100 g (F4) and 10 g/100 g (F5). Juvenile shrimp was fed with probiotic and control diet for a period of 7 weeks at 5 and 8% body weight for the first 3 and 7 weeks, respectively. After seven weeks, whole gut was dissected out and protease activity was estimated as 145 ± 12.3 U/g in control animal and increased as 710 ± 15.2 U/ g in F5 feed groups. Amylase activity was 139 ± 10.4 U/g in control and increased as 209 ± 13. 3 U/g in F5 group. Cellulase activities were 171 ± 9.3 in F5 groups and the control group showed only 102 ± 12.4 U/g. Lipase activity was 78 ± 3 U/g in F1 groups and it increased as 85 ± 5 U/g in F3 groups. These findings indicate the potential of host-associated bacteria to enhance the production of enzymes in the gut of juvenile P. monodon.     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed. An erratum to this article can be found at     

昆虫肠道微生物的多样性、功能及应用

昆虫肠道微生物种类繁多、数量巨大,在与宿主长期的协同进化过程中,不仅形成极为多样的种群结构,也进化出多样的生物学功能,对宿主的营养、生理、发育、防御、抗逆等方面都产生显著影响。近年来,越来越多的昆虫肠道微生物的多样性和生物学特性被揭示,具有农业、能源和环保价值的众多微生物种类和活性基因得到了开发,展现出巨大的应用潜力。本文将从昆虫肠道微生物的多样性、生物学功能、应用三个方面对近年来的研究进展进行总结,并进行展望。     

<Emphasis Type="Italic">Pyrolycus manusanus</Emphasis>, a new genus and species of deep-sea eelpout from a hydrothermal vent field in the Manus Basin,Papua New Guinea (Zoarcidae,Lycodinae)

A new genus and species of deep-sea zoarcid fish, Pyrolycus manusanus, is described on the basis of eight specimens collected from a hydrothermal vent field in the Manus Basin, Papua New Guinea, at depths of 1623–1971 m. This genus is distinguished from other scaleless eelpout genera in the subfamily Lycodinae by the following combination of characters: six suborbital bones; six to seven suborbital pores; flesh gelatinous; gill slit large, extending ventrally beyond pectoral fin base; three postorbital pores; single occipital pore; two to three pelvic fin rays; palatopterygoid series weak; vomer and palatine teeth present; pseudobranch, scales, lateral line absent; pyloric caeca present; 16–17 pectoral fin rays; 22–23 + 56–59 = 78–81 vertebrae. Received: August 5, 1999 / Revised: July 5, 2001 / Accepted: August 26, 2001     

New insigths on the metabolic diversity among the epibiotic microbial communitiy of the hydrothermal shrimp Rimicaris exoculata

The Rimicaris exoculata dominates the megafauna of some of the Mid Atlantic ridge hydrothermal vent sites. This species harbors a rich community of bacterial epibionts inside its gill chamber. Literature data indicate that a single 16S rRNA phylotype dominates this epibiotic community, and is assumed to be a sulfide-oxidizing bacteria. However attempts of cultivation were not successful and did not allow to confirm it. The aim of our study was to test the hypothesis of sulfide oxidation in the gill chamber, by a multidisciplinary approach, using in vivo experiments at in situ pressure in the presence of sulfide, microscopic observations and a molecular survey. Morphology of microorganisms, before and after treatment, was analyzed to test the effect of sulfide depletion and re-exposure. Our observations, as well as molecular data indicate a wider diversity than previously described for this shrimp's epibiotic community. We observed occurrence of bacterial intracellular sulfur- and iron-enriched granules and some methanotrophic-like bacteria cells for the first time. Genes that are characteristic of methane-oxidizing (pmoA) and sulfide-oxidizing (APS) bacteria were identified. These results suggest that three metabolic types (iron, sulfide and methane oxidation) may co-occur within the epibiont community associated with Rimicaris exoculata. As this shrimp colonizes chemically contrasted environments, the relative abundance of each metabolic type could vary according to the local availability of reduced compounds.