Relationship between the occurrence of filamentous bacteria on Bathymodiolus azoricus shell and the physiological and toxicological status of the vent mussel

The edifice walls of the Eiffel Tower hydrothermal vent site (Mid-Atlantic Ridge, Lucky Strike vent field) are populated with dense communities of dual symbioses harboring vent mussel Bathymodiolus azoricus, some of which are covered by white filamentous mats belonging to sulfur-oxidizing bacteria. Mussels were collected in both the presence and absence of the filamentous bacteria. A sample of the filamentous bacteria was collected and water measurements of temperature, CH₄ and H₂S were recorded at the collection area. The whole soft tissues were analyzed for total lipid, carbohydrate and total protein. Metallothioneins and metals (Cu, Fe and Zn) levels were determined in the major organs. The results showed no significant physiological and toxicological evidence that emphasizes the influence of associated sulfur-oxidizing filamentous bacteria on B. azoricus mussel shells. However, B. azoricus mussel seems to be well adapted to the assorted physico-chemical characteristics from the surrounding environment since it is able to manage the constant fluctuation of physico-chemical compounds.     

Multiple I-Type Lysozymes in the Hydrothermal Vent Mussel Bathymodiolus azoricus and Their Role in Symbiotic Plasticity

The aim of this study was first to identify lysozymes paralogs in the deep sea mussel Bathymodiolus azoricus then to measure their relative expression or activity in different tissue or conditions. B. azoricus is a bivalve that lives close to hydrothermal chimney in the Mid-Atlantic Ridge (MAR). They harbour in specialized gill cells two types of endosymbiont (gram—bacteria): sulphide oxidizing bacteria (SOX) and methanotrophic bacteria (MOX). This association is thought to be ruled by specific mechanism or actors of regulation to deal with the presence of symbiont but these mechanisms are still poorly understood. Here, we focused on the implication of lysozyme, a bactericidal enzyme, in this endosymbiosis. The relative expression of Ba-lysozymes paralogs and the global anti-microbial activity, were measured in natural population (Lucky Strike -1700m, Mid-Atlantic Ridge), and in in situ experimental conditions. B. azoricus individuals were moved away from the hydrothermal fluid to induce a loss of symbiont. Then after 6 days some mussels were brought back to the mussel bed to induce a re-acquisition of symbiotic bacteria. Results show the presence of 6 paralogs in B. azoricus. In absence of symbionts, 3 paralogs are up-regulated while others are not differentially expressed. Moreover the global activity of lysozyme is increasing with the loss of symbiont. All together these results suggest that lysozyme may play a crucial role in symbiont regulation.     

A New Screening Method for C-P Compound Producing Organisms by the Use of Phosphoenolpyruvate Phosphomutase

Phosphoenolpyruvate phosphomutase (PEPPM) catalyzes the C-P bond forming reaction by intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate, and shows stronger activity in catalyzing the reverse reaction than the forward reaction. By exploiting the activity of PEPPM to catalyze the reverse reaction, 81 strains were screened as possible C-P compound producing microorganisms from 230 soil samples. Two of these strains, B-1 and L-b, were found to produce C-P compounds by ³¹P-NMR spectral analysis of their broth filtrates. The activity of PEPPM was detected in the cell extracts of these two strains. The strain B-1 was identified as Pseudomonas gladioli, and hydroxyethylphosphonic acid (HEP) was isolated from the broth filtrate of this strain as a C-P compound.     

Observations on parasitism in deep-sea hydrothermal vent and seep limpets

Parasite burdens of shallow-water molluscs have been well documented, but little is known about parasite burdens of molluscs from deep-sea chemosynthetic environments (e.g. hydrothermal vents and seeps). Chemosynthetic habitats are characterized by high concentrations of reduced sulfur and, in the case of vents, high heavy metal concentrations. These compounds are noxious and even stress-inducing in some environments, but are part of the natural chemical milieu of vents and seeps. To examine parasite types and infection intensities in limpets from vents and seeps we documented parasite burdens in 4 limpet species from 4 hydrothermal vent fields (3 on the East Pacific Rise, 1 on the Mid-Atlantic Ridge) and 1 seep site (Florida Escarpment). Approximately 50 % of all limpets examined were infected with 1 or more types of parasites. Limpet parasites were predominantly rickettsia-like inclusions in the digestive and gill epithelia. Limpets collected from the vent field on the Mid-Atlantic Ridge were free of parasites. We detected no histopathological effects that we could attribute to parasites.     

Microbial Iron Mats at the Mid-Atlantic Ridge and Evidence that Zetaproteobacteria May Be Restricted to Iron-Oxidizing Marine Systems

Chemolithoautotrophic iron-oxidizing bacteria play an essential role in the global iron cycle. Thus far, the majority of marine iron-oxidizing bacteria have been identified as Zetaproteobacteria, a novel class within the phylum Proteobacteria. Marine iron-oxidizing microbial communities have been found associated with volcanically active seamounts, crustal spreading centers, and coastal waters. However, little is known about the presence and diversity of iron-oxidizing communities at hydrothermal systems along the slow crustal spreading center of the Mid-Atlantic Ridge. From October to November 2012, samples were collected from rust-colored mats at three well-known hydrothermal vent systems on the Mid-Atlantic Ridge (Rainbow, Trans-Atlantic Geotraverse, and Snake Pit) using the ROV Jason II. The goal of these efforts was to determine if iron-oxidizing Zetaproteobacteria were present at sites proximal to black smoker vent fields. Small, diffuse flow venting areas with high iron(II) concentrations and rust-colored microbial mats were observed at all three sites proximal to black smoker chimneys. A novel, syringe-based precision sampler was used to collect discrete microbial iron mat samples at the three sites. The presence of Zetaproteobacteria was confirmed using a combination of 16S rRNA pyrosequencing and single-cell sorting, while light micros-copy revealed a variety of iron-oxyhydroxide structures, indicating that active iron-oxidizing communities exist along the Mid-Atlantic Ridge. Sequencing analysis suggests that these iron mats contain cosmopolitan representatives of Zetaproteobacteria, but also exhibit diversity that may be uncommon at other iron-rich marine sites studied to date. A meta-analysis of publically available data encompassing a variety of aquatic habitats indicates that Zetaproteobacteria are rare if an iron source is not readily available. This work adds to the growing understanding of Zetaproteobacteria ecology and suggests that this organism is likely locally restricted to iron-rich marine environments but may exhibit wide-scale geographic distribution, further underscoring the importance of Zetaproteobacteria in global iron cycling.     

Numerical taxonomic study of thermophilic Bacillus isolated from three geographically separated deep-sea hydrothermal vents

Abstract: A numerical taxonomic study has been carried out with 80 strains, newly isolated, from three geographically separated deep-sea hydrothermal vents (Mid-Atlantic Ridge, Guaymas Basin and Lau Basin) and eleven thermophilic reference strains representing 11 Bacillus species. The deep-sea isolates were all halotolerant spore-forming rods and grew aerobically above 65°C. Results from unweighted average linkage cluster analysis of a similarity matrix derived from the simple matching coefficient, showed formation of nine major phena, which were defined at the 83% similarity level or above. Seven phena were composed exclusively of strains isolated from the same site (4 from Mid-Atlantic Ridge, 1 from Guaymas Basin and 2 from Lau Basin). The majority of the Lau Basin isolates clustered with 6 of the reference strains in one phenon, while isolates from Mid-Atlantic Ridge and Guaymas Basin were found separated from this phenon at the 69% similarity level. The other reference strains showed less than 69% similarity with the deep-sea isolates.     

Cytonuclear disequilibrium in a hybrid zone involving deep-sea hydrothermal vent mussels of the genus Bathymodiolus

A hybrid zone involving the deep-sea mussels, Bathymodiolus azoricus and B. puteoserpentis, was recently discovered at Broken Spur hydrothermal vent field (29 degrees 10' N, 43 degrees 10' W) along an intermediate segment of the Mid-Atlantic Ridge axis. Examination of nuclear (allozymes) and cytoplasmic (mitochondrial DNA) gene markers in a new sample from Broken Spur revealed significant cytonuclear disequilibrium caused by an excess of the parental types (coupling phase) and a deficiency of recombinants (repulsion phase). An assignment test of individual multilocus genotypes also revealed an excess of parental genotypes in the admixed population. These results support the hypothesis that the Broken Spur mussel population comprises a nonequilibrium mixture of parental immigrants and hybrid individuals.     

A dual symbiosis shared by two mussel species, Bathymodiolus azoricus and Bathymodiolus puteoserpentis (Bivalvia: Mytilidae), from hydrothermal vents along the northern Mid-Atlantic Ridge

Bathymodiolus azoricus and Bathymodiolus puteoserpentis are symbiont-bearing mussels that dominate hydrothermal vent sites along the northern Mid-Atlantic Ridge (MAR). Both species live in symbiosis with two physiologically and phylogenetically distinct Gammaproteobacteria: a sulfur-oxidizing chemoautotroph and a methane-oxidizer. A detailed analysis of mussels collected from four MAR vent sites (Menez Gwen, Lucky Strike, Rainbow, and Logatchev) using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH) showed that the two mussel species share highly similar to identical symbiont phylotypes. FISH observations of symbiont distribution and relative abundances showed no obvious differences between the two host species. In contrast, distinct differences in relative symbiont abundances were observed between mussels from different sites, indicating that vent chemistry may influence the relative abundance of thiotrophs and methanotrophs in these dual symbioses.     

Deep-sea mussels from a hybrid zone on the Mid-Atlantic Ridge host genetically indistinguishable symbionts

The composition and diversity of animal microbiomes is shaped by a variety of factors, many of them interacting, such as host traits, the environment, and biogeography. Hybrid zones, in which the ranges of two host species meet and hybrids are found, provide natural experiments for determining the drivers of microbiome communities, but have not been well studied in marine environments. Here, we analysed the composition of the symbiont community in two deep-sea, Bathymodiolus mussel species along their known distribution range at hydrothermal vents on the Mid-Atlantic Ridge, with a focus on the hybrid zone where they interbreed. In-depth metagenomic analyses of the sulphur-oxidising symbionts of 30 mussels from the hybrid zone, at a resolution of single nucleotide polymorphism analyses of ~2500 orthologous genes, revealed that parental and hybrid mussels (F2–F4 generation) have genetically indistinguishable symbionts. While host genetics does not appear to affect symbiont composition in these mussels, redundancy analyses showed that geographic location of the mussels on the Mid-Atlantic Ridge explained most of the symbiont genetic variability compared to the other factors. We hypothesise that geographic structuring of the free-living symbiont population plays a major role in driving the composition of the microbiome in these deep-sea mussels.Subject terms: Metagenomics, Population genetics, Symbiosis, Microbiome     

Genetic and morphometric characterization of mussels (Bivalvia: Mytilidae) from mid-atlantic hydrothermal vents.

Mussels were collected from deep-sea hydrothermal vents along the Mid-Atlantic Ridge. Specimens from the Snake Pit site were previously identified genetically and anatomically as Bathymodiolus puteoserpentis, but the relationships of mussels from other sites (Logatchev and Lucky Strike) were unclear. Molecular genetic and morphological techniques were used to assess differences among these mussel populations. The results indicate that the range for B. puteoserpentis extends from Snake Pit to Logatchev, and that an unnamed second species, B. n. sp., occurs at Lucky Strike. Analysis of mitochondrial NADH dehydrogenase subunit 4 (ND4) revealed 13% sequence divergence between the two species. Nei's genetic distance (D) based on 14 allozyme loci was 0.112. A multivariate morphometric analysis yielded a canonical discriminant function that correctly identified individuals from these sites to species 95% of the time.     

Biomarkers Reveal Diverse Microbial Communities in Black Smoker Sulfides from Turtle Pits (Mid-Atlantic Ridge,Recent) and Yaman Kasy (Russia,Silurian)

The steep biogeochemical gradients near deep sea hydrothermal vents provide various niches for microbial life. Here we present biosignatures of such organisms enclosed in a modern and an ancient hydrothermal sulfide deposit (Turtle Pits, Mid-Atlantic Ridge, Recent; Yaman Kasy, Russia, Silurian). In the modern sulfide we found high amounts of specific bacterial and archaeal biomarkers with δ¹³C values between ?8 and ?37‰ VPDB. Our data indicate the presence of thermophilic members of the autotrophic Aquificales using the reductive tricarboxylic acid (rTCA) cycle as well as of methanogenic and chemolithoheterotrophic Archaea. In the ancient sample, most potential biomarkers of thermophiles were obscured by compounds derived from allochthonous organic matter (OM), except for an acyclic C₄₀ biphytane and its C₃₉ breakdown product. Both samples contained high amounts of unresolved complex mixtures (UCM) of hydrocarbons. Apparently, OM in the sulfides had to withstand high thermal stress, indicated by highly mature hopanes, steranes, and cheilanthanes with up to 41 carbon atoms.     

Evidence for Methylotrophic Symbionts in a Hydrothermal Vent Mussel (Bivalvia: Mytilidae) from the Mid-Atlantic Ridge

Symbioses between chemolithoautotrophic bacteria and the major macrofaunal species found at hydrothermal vents have been reported for numerous sites in the Pacific Ocean. We present microscopical and enzymatic evidence that methylotrophic bacteria occur as intracellular symbionts in a new species of mytilid mussel discovered at the Mid-Atlantic Ridge hydrothermal vents. Two distinct ultrastructural types of gram-negative procaryotic symbionts were observed within gill epithelial cells by transmission electron microscopy: small coccoid or rod-shaped cells and larger coccoid cells with stacked intracytoplasmic membranes typical of methane-utilizing bacteria. Methanol dehydrogenase, an enzyme diagnostic of methylotrophs, was detected in the mytilid gills, while tests for ribulose-1,5-bisphosphate carboxylase, the enzyme diagnostic of autotrophy via the Calvin cycle, were negative. Stable carbon isotope values (δ¹³C) of mytilid tissue (−32.7 and −32.5% for gill and foot tissues, respectively) fall within the range of values reported for Pacific vent symbioses but do not preclude the use of vent-derived methane reported to be isotopically heavy relative to biogenically produced methane.     

Off-axis symbiosis found: Characterization and biogeography of bacterial symbionts of Bathymodiolus mussels from Lost City hydrothermal vents

Organisms at hydrothermal vents inhabit discontinuous chemical 'islands' along mid-ocean ridges, a scenario that may promote genetic divergence among populations. The 2003 discovery of mussels at the Lost City Hydrothermal Field provided a means of evaluating factors that govern the biogeography of symbiotic bacteria in the deep sea. The unusual chemical composition of vent fluids, the remote location, and paucity of characteristic vent macrofauna at the site, raised the question of whether microbial symbioses existed at the extraordinary Lost City. If so, how did symbiotic bacteria therein relate to those hosted by invertebrates at the closest known hydrothermal vents along the Mid-Atlantic Ridge (MAR)? To answer these questions, we performed microscopic and molecular analyses on the bacteria found within the gill tissue of Bathymodiolus mussels (Mytilidae, Bathymodiolinae) that were discovered at the Lost City. Here we show that Lost City mussels harbour chemoautotrophic and methanotrophic endosymbionts simultaneously. Furthermore, populations of the chemoautotrophic symbionts from the Lost City and two sites along the MAR are genetically distinct from each other, which suggests spatial isolation of bacteria in the deep sea. These findings provide new insights into the processes that drive diversification of bacteria and evolution of symbioses at hydrothermal vents.     

Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes from deep-sea microorganisms

The phylogenetic diversity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, E.C. 4.1.1.39) large-subunit genes of deep-sea microorganisms was analyzed. Bulk genomic DNA was isolated from seven samples, including samples from the Mid-Atlantic Ridge and various deep-sea habitats around Japan. The kinds of samples were hydrothermal vent water and chimney fragment; reducing sediments from a bathyal seep, a hadal seep, and a presumed seep; and symbiont-bearing tissues of the vent mussel, Bathymodiolus sp., and the seep vestimentiferan tubeworm, Lamellibrachia sp. The RuBisCO genes that encode both form I and form II large subunits (cbbL and cbbM) were amplified by PCR from the seven deep-sea sample DNA populations, cloned, and sequenced. From each sample, 50 cbbL clones and 50 cbbM clones, if amplified, were recovered and sequenced to group them into operational taxonomic units (OTUs). A total of 29 OTUs were recorded from the 300 total cbbL clones, and a total of 24 OTUs were recorded from the 250 total cbbM clones. All the current OTUs have the characteristic RuBisCO amino acid motif sequences that exist in other RuBisCOs. The recorded OTUs were related to different RuBisCO groups of proteobacteria, cyanobacteria, and eukarya. The diversity of the RuBisCO genes may be correlated with certain characteristics of the microbial habitats. The RuBisCO sequences from the symbiont-bearing tissues showed a phylogenetic relationship with those from the ambient bacteria. Also, the RuBisCO sequences of known species of thiobacilli and those from widely distributed marine habitats were closely related to each other. This suggests that the Thiobacillus-related RuBisCO may be distributed globally and contribute to the primary production in the deep sea.     

Evidence of in situ uptake and incorporation of bicarbonate and amino acids by a hydrothermal vent mussel

The hydrothermal vent mussel Bathymodiolus sp. is demonstrated to incorporate inorganic CO₂ from sea water. After ≈24 h incubation with H¹⁴CO⁻₂ the major part of the radioactivity is incorporated into macromolecules mostly in proteins but also in a notable lipidic fraction. 77 to 98% of this radioactivity is found in the gill and autoradiographs show that CO₂ fixation is only observed in cells containing high concentrations of bacteria. The results endorse the hypothesis that the associated bacteria might provide a nutritional source for the mussel.The mussel is also able to absorb and incorporate dissolved amino acids. Heterotrophic processes involving dissolved organic matter may interfere with the autotrophic pathways. Beside its capability of feeding on particulate material, the mussel may be thus able to live on reduced carbon and nitrogen compounds synthesized by its associated bacteria as well as on dissolved organic compounds present in sea water. The effective participation of the different processes is probably related to the ecological conditions experienced by the mussel in vent areas.     

A new species of sea anemone (Cnidaria: Anthozoa: Actiniaria) from Manus Basin hydrothermal vents, South-western Pacific

During the BIOACCESS Japanese cruises (1996 & 1998), active hydrothermalism and associated vent fauna were studied on the South-eastern Rift of Manus Basin (South-western Pacific). In the PACMANUS vent field, a conspicuous vent fauna was sampled, including an actinostolid sea anemone (Actiniaria) belonging to an undescribed genus and species. Pacmanactis hashimotoi gen. et spec. nov. is here described, and represents the 9th sea anemone reported from hydrothermal vents.     

Occurrence and Diversity of Yeasts in the Mid-Atlantic Ridge Hydrothermal Fields Near the Azores Archipelago

The yeast community associated with deep-sea hydrothermal systems of the Mid-Atlantic Rift was surveyed for the first time. This study relied on a culture-based approach using two different growth media: a conventional culture medium for yeasts supplemented with sea salts (MYPss) and the same medium additionally supplemented with sulfur (MYPssS). For the evaluation of species diversity, a molecular approach involving minisatellite-primed polymerase chain reaction (MSP-PCR) strain typing and sequence analysis of the D1/D2 domains of the 26S rDNA was followed. In the seven water samples that were studied, the number of colony-forming units per liter (cfu/L) ranged from 0 to 5940. The nonpigmented yeasts were much more abundant than the pink-pigmented ones. This disproportion was not observed in studies of other marine systems and may be due to the unique conditions of hydrothermal vents, characterized by a rich animal and microbial diversity and therefore by the availability of organic compounds utilizable by yeasts. Higher counts of nonpigmented yeast were obtained using MYPss, whereas for pink yeasts, higher counts were obtained using MYPssS. Moreover, among pink yeasts, some of the MSP-PCR classes obtained were composed of isolates obtained only on MYPssS, which might be an indication that these isolates are adapted to the ecosystems of the hydrothermal vents. Twelve phylotypes belonged to the Ascomycota and seven phylotypes belonged to the Basidiomycota. The nonpigmented yeasts were identified as Candida atlantica, C. atmosphaerica, C. lodderae, C. parapsilosis, Exophiala dermatitidis, Pichia guilliermondii, and Trichosporon dermatis, whereas the pigmented yeasts were identified as Rhodosporidium diobovatum, R. sphaerocarpum, R. toruloides, and Rhodotorula mucilaginosa. Some of the yeasts that were found belong to phylogenetic groups that include species reported from other marine environments, and eight phylotypes represent undescribed species. The new phylotypes found at Mid-Atlantic Ridge hydrothermal fields represent 33% of the total number of yeast taxa that were found.     

Do the Anemonefish Amphiprion ocellaris (Pisces: Pomacentridae) Imprint Themselves to Their Host Sea Anemone Heteractis magnifica (Anthozoa: Actinidae)?

Juvenile anemonefishes detect their host sea anemone by olfactory stimuli; in order to investigate whether this behaviour is innate or acquired, the anemonefish species Amphiprion ocellaris was bred in two different ways: 1. With no host sea anemone present at all (–A); and 2. With the specific host sea anemone Heteractis magnifica present in the hatching aquarium, so that these eggs were laid and hatched close to the sea anemone, as in nature (+A). The two different types of juvenile A. ocellaris were presented to the odours of the host sea anemone H. magnifica in two sets of short-term experiments with the host (a) visually hidden in a net cage, and (b) visible but physically separated from the anemonefishes. In both cases, a water flow was established between fishes and host. The +A-fishes found their host by olfactory and not by visual stimuli. In both series, the –A-fishes showed a significantly lower affinity behaviour towards the odour compounds from the host sea anemone than the +A-fishes did. A third type of experiment was a direct confrontation between fishes and host; here, the –A-fishes were indifferent towards the host sea anemone for almost 48 h, while the +A-fishes acclimated to the host sea anemone within the first 5 min of the direct confrontation. The results of this study suggest that Amphiprion ocellaris imprints itself olfactorily to its species-specific host sea anemone Heteractis magnifica, and, furthermore, may be genetically disposed towards olfactory recognition of the host sea anemone.