Parasitism in species of Bathymodiolus (Bivalvia: Mytilidae) mussels from deep-sea seep and hydrothermal vents

Bivalve species, especially mussels, are biomass dominants in many deep-sea chemosynthetic ecosystems. As in shallow-water environments, parasites are likely to be important factors in the population dynamics of bivalve communities in chemosynthetic ecosystems, but there has been little study of parasitism in deep-sea seep or vent molluscs. In this study, parasite types, diversity, prevalence, infection density and non-infectious indicators of stress or disease as related to host age, reproductive condition, and endosymbiont density were assessed in mussels (Bathymodiolus heckerae) from 2 seep sites and mussels (B. puteoserpentis) from 2 vent sites. We identified 10 microbial or parasitic agents in histological sections. Parasite types included 3 viral-like gut inclusions, 2 rickettsia-like gill inclusions, a rickettsia-like mantle inclusion, a bacterial gill-rosette, a chlamydia-like gut inclusion, gill-dwelling ciliates, and an unidentified inclusion in gut tissues. Parasite species richness was greater in seep mussels than in vent mussels, with the seep mussels possessing 9 types of parasites compared to 2 in the vent mussels. One of the viral-like inclusions infecting the seep mussel B. heckerae was pathogenic, causing lysis of the digestive tubules. The prevalence and intensity of infection by this pathogen were greater in hosts with shell lengths less than 100 mm. Mussels from all 4 sites also exhibited intense infiltration of tissues and blood spaces by enlarged hemocytes. Hemocytic infiltration (hemocytosis) showed variable degrees of severity that were not associated with other host factors examined.     

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.     

A hybrid zone between hydrothermal vent mussels (Bivalvia: Mytilidae) from the Mid-Atlantic Ridge

This study provides the first example of a hybrid zone between animal taxa distributed along the mid-ocean ridge system. We examined the distribution and genetic structure of deep-sea hydrothermal vent mussels (Bivalvia: Mytilidae) along a 2888-km portion of the Mid-Atlantic Ridge between 37 degrees 50' N and 14 degrees 45' N latitude. Mitochondrial DNA (mtDNA), allozymes and multivariate-morphometric evidence discriminated between individuals of a northern species, Bathymodiolus azoricus, and a southern species, B. puteoserpentis, that were separated by an intermediate ridge segment almost devoid of mussels. A small sample of mussels from Broken Spur, a vent locality along this intermediate zone, revealed a mixed population with gene frequencies and morphology that were broadly intermediate to those of the northern and southern species. Multilocus clines in mtDNA and allozyme frequencies were centred over the intermediate zone. We consider intrinsic and extrinsic processes that might limit genetic exchange across this hybrid zone.     

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.     

2-D difference gel electrophoresis approach to assess protein expression profiles in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal vents

Hydrothermal vent mussels Bathymodiolus azoricus are naturally exposed to toxic chemical species originated directly from vent chimneys. The amount of toxic elements varies significantly among vent sites along the Mid-Atlantic Ridge and B. azoricus must be able to adapt to changes in hydrothermal fluid composition, temperature and pressure. The aim of this work was to study changes in the proteome in the "gill-bacteria complex" of mussels B. azoricus from three hydrothermal vent sites with distinct environmental characteristics using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). Results showed that 31 proteins had different expression profiles among vent sites and both cluster and principal component analysis confirm a clear separation of mussels between sites. This suggests the existence of specific parameters grouping individuals from the same hydrothermal site. Protein spots of the more abundant differentially expressed proteins were excised, digested with trypsin and identified by mass spectrometry. All identified proteins (actin, ubiquinone, S-adenosylhomocysteine hydrolase, cysteine peptidases, chaperonin and catalase) have been related previously with oxidative stress conditions and are known to be affected by ROS inducing stressors, including metals. Results point out to specific adaptations at the proteome level of B. azoricus depending on the level of toxicants present in their environment.     

Changes of gill and hemocyte-related bio-indicators during long term maintenance of the vent mussel Bathymodiolus azoricus held in aquaria at atmospheric pressure

The deep-sea hydrothermal vent mussel Bathymodiolus azoricus has been the subject of several studies aimed at understanding the physiological adaptations that vent animals have developed in order to cope with the particular physical and chemical conditions of hydrothermal environments. In spite of reports describing successful procedures to maintain vent mussels under laboratory conditions at atmospheric pressure, few studies have described the mussel's physiological state after a long period in aquaria. In the present study, we investigate changes in mucocytes and hemocytes in B. azoricus over the course of several months after deep-sea retrieval. The visualization of granules of mucopolysaccharide or glycoprotein was made possible through their inherent auto-fluorescent property and the Alcian blue-Periodic Acid Schiff staining method. The density and distribution of droplets of mucus-like granules was observed at the ventral end of lamellae during acclimatization period. The mucus-like granules were greatly reduced after 3 months and nearly absent after 6 months of aquarium conditions. Additionally, we examined the depletion of endosymbiont bacteria from gill tissues, which typically occurs within a few weeks in sea water under laboratory conditions. The physiological state of B. azoricus after 6 months of acclimatization was also examined by means of phagocytosis assays using hemocytes. Hemocytes from mussels held in aquaria up to 6 months were still capable of phagocytosis but to a lesser extent when compared to the number of ingested yeast particles per phagocytic hemocytes from freshly collected vent mussels. We suggest that the changes in gill mucopolysaccharides and hemocyte glycoproteins, the endosymbiont abundance in gill tissues and phagocytosis are useful health criteria to assess long term maintenance of B. azoricus in aquaria. Furthermore, the laboratory set up to which vent mussels were acclimatized is an applicable system to study physiological reactions such as hemocyte immunocompetence even in the absence of the high hydrostatic pressure found at deep-sea vent sites.     

Annual spawning of the hydrothermal vent mussel, Bathymodiolus azoricus, under controlled aquarium, conditions at atmospheric pressure

Dense macrofaunal communities of modioliform mussels are a major component of many hydrothermal vent and cold seep ecosystems. The hydrothermal vent mussel Bathymodiolus azoricus, that dominates hydrothermal vent communities near the Azores Triple Junction, can be maintained in aquaria at atmospheric pressure. Cages containing these mussels were placed over diffuse vent outlets and recovered at different times. Cages recovered in January 2003 contained mussels with ripe gonads while those recovered in July, August and November 2001 and in April 2003 did not. Mussels collected post-spawning in April 2003 spawned in the aquaria in January 2004. Young mussels recruited to the cages in April 2003. The data support a main single period of spawning and of juvenile recruitment for B. azoricus.     

The contrasted evolutionary fates of deep‐sea chemosynthetic mussels (Bivalvia,Bathymodiolinae)

Bathymodiolinae are giant mussels that were discovered at hydrothermal vents and harboring chemosynthetic symbionts. Due to their close phylogenetic relationship with seep species and tiny mussels from organic substrates, it was hypothesized that they gradually evolved from shallow to deeper environments, and specialized in decaying organic remains, then in seeps, and finally colonized deep‐sea vents. Here, we present a multigene phylogeny that reveals that most of the genera are polyphyletic and/or paraphyletic. The robustness of the phylogeny allows us to revise the genus‐level classification. Organic remains are robustly supported as the ancestral habitat for Bathymodiolinae. However, rather than a single step toward colonization of vents and seeps, recurrent habitat shifts from organic substrates to vents and seeps occurred during evolution, and never the reverse. This new phylogenetic framework challenges the gradualist scenarios “from shallow to deep.” Mussels from organic remains tolerate a large range of ecological conditions and display a spectacular species diversity contrary to vent mussels, although such habitats are yet underexplored compared to vents and seeps. Overall, our data suggest that for deep‐sea mussels, the high specialization to vent habitats provides ecological success in this harsh habitat but also brings the lineage to a kind of evolutionary dead end.     

Environmental acquisition of thiotrophic endosymbionts by deep-sea mussels of the genus bathymodiolus

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally "leaky" system of vertical transmission could not be excluded.     

Dispersal barriers and isolation among deep-sea mussel populations (Mytilidae: Bathymodiolus) from eastern Pacific hydrothermal vents

Deep-sea hydrothermal vent species are widely dispersed among habitat islands found along the global mid-ocean ridge system. We examine factors that affect population structure, gene flow and isolation in vent-endemic mussels of the genus Bathymodiolus from the eastern Pacific Ocean. Mussels were sampled from localities including the Galapagos Rift (GAR, 0 degrees 48' N; 86 degrees 10' W) and the East Pacific Rise (EPR, 13 degrees N to 32 degrees S latitude) across a maximum distance of 4900 km. The sampled range crossed a series of topographical features that interrupt linear aspects of the ridge system, and it encompassed regions of strong cross-axis currents that could impede along-axis dispersal of mussel larvae. Examinations of mitochondrial DNA sequences and allozyme variation revealed significant barriers to gene flow along the ridge axis. All populations from the GAR and EPR from 13 degrees N to 11 degrees S were homogeneous genetically and appeared to experience unimpeded high levels of interpopulational gene flow. In contrast, mussels from north and south of the Easter Microplate were highly divergent (4.4%), possibly comprising sister-species that diverged after formation of the microplate approximately 4.5 Ma. Strong cross-axis currents associated with inflated bathymetry of the microplate region may reinforce isolation across this region.     

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.     

Microbial community structure of hydrothermal deposits from geochemically different vent fields along the Mid-Atlantic Ridge

To evaluate the effects of local fluid geochemistry on microbial communities associated with active hydrothermal vent deposits, we examined the archaeal and bacterial communities of 12 samples collected from two very different vent fields: the basalt-hosted Lucky Strike (37°17'N, 32°16.3'W, depth 1600-1750 m) and the ultramafic-hosted Rainbow (36°13'N, 33°54.1'W, depth 2270-2330 m) vent fields along the Mid-Atlantic Ridge (MAR). Using multiplexed barcoded pyrosequencing of the variable region 4 (V4) of the 16S rRNA genes, we show statistically significant differences between the archaeal and bacterial communities associated with the different vent fields. Quantitative polymerase chain reaction (qPCR) assays of the functional gene diagnostic for methanogenesis (mcrA), as well as geochemical modelling to predict pore fluid chemistries within the deposits, support the pyrosequencing observations. Collectively, these results show that the less reduced, hydrogen-poor fluids at Lucky Strike limit colonization by strict anaerobes such as methanogens, and allow for hyperthermophilic microaerophiles, like Aeropyrum. In contrast, the hydrogen-rich reducing vent fluids at the ultramafic-influenced Rainbow vent field support the prevalence of methanogens and other hydrogen-oxidizing thermophiles at this site. These results demonstrate that biogeographical patterns of hydrothermal vent microorganisms are shaped in part by large scale geological and geochemical processes.     

Molecular diet analysis in mussels and other metazoan filter feeders and an assessment of their utility as natural eDNA samplers

Molecular gut content analysis is a popular tool to study food web interactions and has recently been suggested as an alternative source for DNA-based biomonitoring. However, the overabundant consumer's DNA often outcompetes that of its diet during PCR. Lineage-specific primers are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. Here, we designed an amplicon sequencing assay to monitor the eukaryotic diet of mussels and other metazoan filter feeders and explore the utility of mussels as natural eDNA samplers to monitor planktonic communities. We designed several lineage-specific rDNA primers with broad taxonomic suitability for eukaryotes. The primers were tested using DNA extracts of different limnic and marine mussel species and the results compared to eDNA water samples collected next to the mussel colonies. In addition, we analysed several 25-year time series samples of mussels from German rivers. Our primer sets efficiently prevent the amplification of mussels and other metazoans. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also show the utility of a reversed version of our primers, which prevents amplification of nonmetazoan taxa from complex eukaryote community samples, by enriching fauna associated with the marine brown algae Fucus vesiculosus. Our protocol will enable large-scale dietary analysis in metazoan filter feeders, facilitate aquatic food web analysis and allow surveying of aquacultures for pathogens. Moreover, we show that mussels and other aquatic filter feeders can serve as complementary DNA source for biomonitoring.     

Environmental Acquisition of Thiotrophic Endosymbionts by Deep-Sea Mussels of the Genus Bathymodiolus

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally “leaky” system of vertical transmission could not be excluded.     

Global depression in gene expression as a response to rapid thermal changes in vent mussels

Hydrothermal vent mussels belonging to the genus Bathymodiolus are distributed worldwide and dominate communities at shallow Atlantic hydrothermal sites. While organisms inhabiting coastal ecosystems are subjected to predictable oscillations of physical and chemical variables owing to tidal cycles, the vent mussels sustain pronounced temperature changes over short periods of time, correlated to the alternation of oxic/anoxic phases. In this context, we focused on the short-term adaptive response of mussels to temperature change at a molecular level. The mRNA expression of 23 genes involved in various cell functions of the vent mussel Bathymodiolus azoricus was followed after heat shocks for either 30 or 120 min, at 25 and 30°C over a 48 h recovery period at 5°C. Mussels were genotyped at 10 enzyme loci to explore a relationship between natural genetic variation, gene expression and temperature adaptation. Results indicate that the mussel response to increasing temperature is a depression in gene expression, such a response being genotypically correlated at least for the Pgm-1 locus. This suggests that an increase in temperature could be a signal triggering anaerobiosis for B. azoricus or this latter alternatively behaves more like a ‘cold’ stenotherm species, an attribute more related to its phylogenetic history, a cold seeps/wood fall origin.     

Food quality controls reproduction of the zebra mussel (Dreissena polymorpha)

Species such as Dreissena polymorpha sometimes contribute substantially in the transfer of primary to secondary production. During the ontogenetic cycle, the reproductive investment of adult mussels is one of the main parameters that affect recruitment success. We studied how food quality and temperature affect the reproductive investment in term of egg mass of D. polymorpha in a lake by sampling mussels monthly from 4 m and 15 m depths. Temperature affected reproduction directly and also indirectly through the food. To assess whether temperature and/or food conditions led to the differences observed in mussels sampled from the two depths, mussels were reared in the laboratory under two different temperature regimes for 3 months, simulating the temperature of the lake at 4 m and 15 m depth. Possible effects of food quality were tested at each temperature using four diets differing in fatty acid composition. Temperature played an important role as a trigger for spawning, and the type of diet clearly affected the reproductive investment. When the heterokont chromophyte alga Nannochloropsis limnetica, which is rich in polyunsaturated fatty acids (PUFAs) and long-chained PUFAs (>C18), was fed to mussels, an increased egg mass was obtained. This result was in contrast to that found when the green alga Scenedesmus obliquus and the cyanobacterium Aphanothece sp., both of which are deficient in long-chained PUFAs, were offered as food to the mussels. Such a PUFA-dependent food quality may affect reproduction in lakes. Food quality effects vary seasonally in a lake and may be most important in summer, when low-food-quality green algae and cyanobacteria are abundant. The low biochemical quality of these blooms may affect at least the later period of gametogenesis of D. polymorpha, which reproduces from June to August.     

High amounts of sulphur-amino acids in three symbiotic mytilid bivalves from deep benthic communities

Three sulphur-amino acids, taurine, hypotaurine and thiotaurine, are shown to exist in deep-sea symbiotic mussels. Their relative and absolute importance differs in the three species of mussels studied and seems to be related to the metabolism of the symbionts. Thiotaurine only occurs in high concentration in thiotrophic species, whereas hypotaurine and taurine are found in all the species studied. Differences in sulphur-amino acid composition may also depend on the physiological condition of the organism or on environmental features. These compounds were previously found in Riftia pachyptila, Calyptogena phaseoliformis and in two hydrothermal vent mussels from the western Pacific. Given their abundance, they may play an essential role in these symbioses.     

Novel and diverse integron integrase genes and integron-like gene cassettes are prevalent in deep-sea hydrothermal vents

The lack of information about mobile DNA in deep-sea hydrothermal vents limits our understanding of the phylogenetic diversity of the mobile genome of bacteria in these environments. We used culture-independent techniques to explore the diversity of the integron/mobile gene cassette system in a variety of hydrothermal vent communities. Three samples, which included two different hydrothermal vent fluids and a mussel species that contained essentially monophyletic sulfur-oxidizing bacterial endosymbionts, were collected from Suiyo Seamount, Izu-Bonin, Japan, and Pika site, Mariana arc. First, using degenerate polymerase chain reaction (PCR) primers, we amplified integron integrase genes from metagenomic DNA from each sample. From vent fluids, we discovered 74 new integrase genes that were classified into 11 previously undescribed integron classes. One integrase gene was recorded in the mussel symbiont and was phylogenetically distant from those recovered from vent fluids. Second, using PCR primers targeting the gene cassette recombination site (59-be), we amplified and subsequently identified 60 diverse gene cassettes. In multicassette amplicons, a total of 13 59-be sites were identified. Most of these sites displayed features that were atypical of the features previously well conserved in this family. The Suiyo vent fluid was characterized by gene cassette open reading frames (ORFs) that had significant homologies with transferases, DNA-binding proteins and metal transporter proteins, while the majority of Pika vent fluid gene cassettes contained novel ORFs with no identifiable homologues in databases. The symbiont gene cassette ORFs were found to be matched with DNA repair proteins, methionine aminopeptidase, aminopeptidase N, O-sialoglycoprotein endopeptidase and glutamate synthase, which are proteins expected to play a role in animal/symbiont metabolism. The success of this study indicates that the integron/gene cassette system is common in deep-sea hydrothermal vents, an environment type well removed from anthropogenic disturbance.     

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