Genomic Insights Into Chemosynthetic Symbiosis in a Deep-Sea Hydrothermal Vent Mussel

Background Symbiosis with chemosynthetic bacteria has allowed many invertebrates to flourish in ‘extreme’ deep-sea chemosynthesis-based ecosystems, such as hydrothermal vents and cold seeps. Bathymodioline mussels are considered as models of deep-sea animal-bacteria symbiosis, but the diversity of molecular mechanisms governing host-symbiont interactions remains understudied owing to the lack of hologenomes. In this study, we adopted a total hologenome approach in sequencing the hydrothermal vent mussel Bathymodiolus marisindicus and the endosymbiont genomes combined with a transcriptomic and proteomic approach that explore the mechanisms of symbiosis. Results Here, we provide the first coupled mussel-endosymbiont genome assembly. Comparative genome analysis revealed that both Bathymodiolus marisindicus and its endosymbiont reshape their genomes through the expansion of gene families, likely due to chemosymbiotic adaptation. Functional differentiation of host immune-related genes and attributes of symbiont self-protection that likely facilitate the establishment of endosymbiosis. Hologenomic analyses offer new evidence that metabolic complementarity between the host and endosymbionts enables the host to compensate for its inability to synthesize some essential nutrients, and two pathways (digestion of symbionts and molecular leakage of symbionts) that can supply the host with symbiontderived nutrients. Results also showed that bacteriocin and abundant toxins of symbiont may contribute to the defense of the B. marisindicus holobiont. Moreover, an exceptionally large number of anti-virus systems were identified in the B. marisindicus symbiont, which likely work synergistically to efficiently protect their hosts from phage infection, indicating virus-bacteria interactions in intracellular environments of a deepsea vent mussel. Conclusions Our study provides novel insights into the mechanisms of symbiosis enabling deep-sea mussels to successfully colonize the special hydrothermal vent habitats.

A relict oasis of living deep-sea mussels Bathymodiolus and microbial-mediated seep carbonates at newly-discovered active cold seeps in the Gulf of Cádiz, NE Atlantic Ocean

Extensive beds of the deep-sea mussel Bathymodiolus mauritanicus (currently also known as Gigantidas mauritanicus) linked to active cold seeps related to fissure-like activity on Al Gacel mud volcano, Gulf of Cádiz, were filmed and sampled for the first time during the oceanographic expedition SUBVENT-2 aboard R/V Sarmiento de Gamboa. Al Gacel mud volcano is one of up to 80 fluid venting submarine structures (mud volcanoes and mud volcano/diapir complexes) identified in the Gulf of Cádiz as result of explosive venting of hydrocarbon-enriched fluids sourced from deep seated reservoirs. This mud volcano is a cone-shaped edifice, 107 m high, 944 m in diameter constituted by mud breccias and, partially covered by pavements of seep carbonates. Extensive beds of this deep-sea mussel were detected at the northern flank at 810–815 m water depth associated with bacterial mats around intermittent buoyant vertical bubble methane plumes. High methane concentrations were measured in the water column above living mussel beds. Other chemosymbiotic species (Siboglinum sp., Solemya elarraichensis, Isorropodon sp., Thyasira vulcolutre and Lucinoma asapheus) were also found in different parts of Al Gacel mud volcano. Al Gacel mud volcano may currently represent one of the most active mud volcanoes in the Gulf of Cádiz, delivering significant amounts of thermogenic hydrocarbon fluids which contribute to foster the extensive chemosynthesis-based communities detected. This finding is of paramount importance for linking extremophile bivalve populations along the North Atlantic, including cold seeps of the Gulf of México, hydrothermal vents of the Mid-Atlantic Ridge and now, detailed documented at the Gulf of Cádiz.

Microbial Eukaryotes Associated With Sediments in Deep-Sea Cold Seeps

Microbial eukaryotes are key components of the marine food web, but their distribution in deep-sea chemosynthetic ecosystems has not been well studied. Here, high-throughput sequencing of the 18S rRNA gene and network analysis were applied to investigate the diversity, distribution and potential relationships between microbial eukaryotes in samples collected from two cold seeps and one trough in the northern South China Sea. SAR (i.e., Stramenopiles, Alveolata, and Rhizaria) was the predominant group in all the samples, and it was highly affiliated to genotypes with potential symbiotic and parasitic strategies identified from other deep-sea extreme environments (e.g., oxygen deficient zones, bathypelagic waters, and hydrothermal vents). Our findings indicated that specialized lineages of deep-sea microbial eukaryotes exist in chemosynthetic cold seeps, where microbial eukaryotes affiliated with parasitic/symbiotic taxa were prevalent in the community. The biogeographic pattern of the total community was best represented by the intermediate operational taxonomic unit (OTU) category, whose relative abundance ranged 0.01–1% within a sample, and the communities of the two cold seeps were distinct from the trough, which suggests that geographical proximity has no critical impact on the distribution of deep-sea microbial eukaryotes. Overall, this study has laid the foundations for future investigations regarding the ecological function and in situ trophic relationships of microbial eukaryotes in deep-sea ecosystems.

Seismic Diffraction Analysis of a Fluid Escape Pipe beneath the Submarine Gas Bubble Plume in the Haima Cold Seep Area

Pipe structures are considered as fluid conduits beneath cold seeps. These structures have been observed in many geological settings and are widely accepted as the most critical pathway for fluid migration. One of such pipe structures in the Haima cold seep region is investigated herein. The pipe structure extends from below the BSR and reaches the seafloor. It is characterized by a string of events with short and strong seismic amplitudes, similar to the string of bead reflections (SBRs) associated with small-scale caves in carbonate reservoirs. This leads to the hypothesis that multiple small-scale bodies exist within the pipe structure. We test this hypothesis by analysis of diffraction waves and numerical seismic modeling. Travel time pattern analysis indicates that the diffractors within the pipe structure caused the rich diffraction waves on the shot records, and the reversed polarity indicates that the diffractors have a lower impedance than the surrounding sediments. These low-impedance bodies are interpreted as gas pockets within the pipe structures. Based on these interpretations, a conceptual model is proposed to describe the fluid migration process within the pipe. Briefly, we propose that gas pockets within the pipe structure could be analogue to the magma chambers located beneath volcanoes and this may provide a new insight into how gases migrate through the pipe structure and reach the seafloor.

Production of Labile Protein-Like Dissolved Organic Carbon Associated With Anaerobic Methane Oxidization in the Haima Cold Seeps, South China Sea

Cold seeps where methane-rich fluids escape from the seafloor generally support enormous biomass of chemosynthetic organisms and associated fauna. In addition to transporting a great amount of methane toward the seafloor, cold seeps also contribute to the aged, dissolved organic carbon (DOC) pool in the deep ocean. Here, two sediment cores from the “Haima cold seeps,” northern South China Sea and a nearby reference core were analyzed for pore-water sulfate and DOC concentrations, δ13C of DOC, and optical properties of dissolved organic matter (DOM). High DOC concentrations (0.9–3.7 mM) accompanied by extremely low δ13C values (−43.9 to −76.2‰) suggest the conversion of methane into sedimentary DOC pool in the seep sediments. Parallel factor analysis (PARAFAC) of the fluorescence excitation-emission matrices shows higher fluorescent intensities of labile protein-like components (C2 and C4) and lower fluorescent intensities of refractory humic-like components (C1 and C3) in the seep cores compared to the reference core. The intensity of C2 is positively correlated with DOC concentrations and δ13C-DOC in the seep sediments, suggesting that the labile protein-like DOM was produced by the anaerobic oxidation of methane (AOM). Moreover, low humification index (HIX) and high biological index (BIX) values also indicate intensified production of relatively labile DOM with lower degradation degree in the seep cores compared to the reference core. Hence, we highlight that methane-derived DOC may serve as important carbon and energy sources for heterotrophic microbial communities due to its relatively labile nature.

A Deep Learning Model to Recognize and Quantitatively Analyze Cold Seep Substrates and the Dominant Associated Species

Characterizing habitats and species distribution is important to understand the structure and function of cold seep ecosystems. This paper develops a deep learning model for the fast and accurate recognition and classification of substrates and the dominant associated species in cold seeps. Considering the dense distribution of the dominant associated species and small objects caused by overlap in cold seeps, the feature pyramid network (FPN) embed into the faster region-convolutional neural network (R-CNN) was used to detect large-scale changes and small missing objects without increasing the number of calculations. We applied three classifiers (Faster R-CNN + FPN for mussel beds, lobster clusters and biological mixing, CNN for shell debris and exposed authigenic carbonates, and VGG16 for reduced sediments and muddy bottom) to improve the recognition accuracy of substrates. The model’s results were manually verified using images obtained in the Formosa cold seep during a 2016 cruise. The recognition accuracy of the two dominant species, e.g., Gigantidas platifrons and Munidopsidae could be 70.85 and 56.16%, respectively. Seven subcategories of substrates were also classified with a mean accuracy of 74.87%. The developed model is a promising tool for the fast and accurate characterization of substrates and epifauna in cold seeps, which is crucial for large-scale quantitative analyses.

When Imagery and Physical Sampling Work Together: Toward an Integrative Methodology of Deep-Sea Image-Based Megafauna Identification

Imagery has become a key tool for assessing deep-sea megafaunal biodiversity, historically based on physical sampling using fishing gears. Image datasets provide quantitative and repeatable estimates, small-scale spatial patterns and habitat descriptions. However, taxon identification from images is challenging and often relies on morphotypes without considering a taxonomic framework. Taxon identification is particularly challenging in regions where the fauna is poorly known and/or highly diverse. Furthermore, the efficiency of imagery and physical sampling may vary among habitat types. Here, we compared biodiversity metrics (alpha and gamma diversity, composition) based on physical sampling (dredging and trawling) and towed-camera still images (1) along the upper continental slope of Papua New Guinea (sedimented slope with wood-falls, a canyon and cold seeps), and (2) on the outer slopes of the volcanic islands of Mayotte, dominated by hard bottoms. The comparison was done on selected taxa (Pisces, Crustacea, Echinoidea, and Asteroidea), which are good candidates for identification from images. Taxonomic identification ranks obtained for the images varied among these taxa (e.g., family/order for fishes, genus for echinoderms). At these ranks, imagery provided a higher taxonomic richness for hard-bottom and complex habitats, partially explained by the poor performance of trawling on these rough substrates. For the same reason, the gamma diversity of Pisces and Crustacea was also higher from images, but no difference was observed for echinoderms. On soft bottoms, physical sampling provided higher alpha and gamma diversity for fishes and crustaceans, but these differences tended to decrease for crustaceans identified to the species/morphospecies level from images. Physical sampling and imagery were selective against some taxa (e.g., according to size or behavior), therefore providing different facets of biodiversity. In addition, specimens collected at a larger scale facilitated megafauna identification from images. Based on this complementary approach, we propose a robust methodology for image-based faunal identification relying on a taxonomic framework, from collaborative work with taxonomists. An original outcome of this collaborative work is the creation of identification keys dedicated specifically to in situ images and which take into account the state of the taxonomic knowledge for the explored sites.

Distinct Bottom-Water Bacterial Communities at Methane Seeps With Various Seepage Intensities in Haima, South China Sea

Methane seeps are chemosynthetic ecosystems in the deep-sea environment. Microbial community structures have been extensively studied in the seepage-affected sediments and investigation in the water column above the seeping sites is still lacking. In this study, prokaryotic communities in the bottom water about 50 cm from the seabed at methane seeps with various seepage intensities in Haima, South China Sea were comparatively studied by using 16S ribosomal RNA gene sequencing. These sites were assigned based on their distinct methane content levels and seafloor landscapes as the non-seepage (NS) site, low-intensity seepage (LIS) site, and high-intensity seepage (HIS) site. The abundances of the dominant phyla Proteobacteria, Bacteroidetes, and Actinobacteria differed significantly between NS and the two seepage sites (p < 0.05). Alpha diversity differed among the three sites with the HIS site showing the lowest community diversity. Principal component analysis revealed highly divergent bacterial community structures at three sites. Many environmental variables including temperature, alkalinity, pH, methane, dissolved organic carbon (DOC), and inorganic nutrients were measured. Redundancy analysis indicated that methane content is the key environmental factor driving bacterial community variation (p = 0.001). Linear discriminant analysis effect size analysis identified various differentially enriched genera at the LIS and HIS sites. Phylogenetic analysis revealed close phylogenetic relationship among the operational taxonomic units of these genera with known oil-degrading species, indicating oil seepage may occur at the Haima cold seeps. Co-occurrence networks indicated that the strength of microbial interactions was weakest at the HIS site. This study represents a comprehensive comparison of microbial profiles in the water column of cold seeps in the SCS, revealing that the seepage intensity has a strong impact on bacterial community dynamics.

Review of Methane Seepages in the Okinawa Trough: Progress and Outlook

It has been two decades since the cold seeps were firstly found in the Okinawa Trough (OT). The scientific cruises and the geological surveys since then have unveiled the currently active submarine methane seeps and significantly improved the understanding of methane seeps in the back-arc basin of the OT. In this paper, we review the up-to-date progress of the research of methane seepages then put forward the promising, yet challenging, outlook by listing the unsolved questions of the cold seeps in the OT. Multiple approaches and techniques, including seismic and echo-sounder recording, dredging, gravity-piston and ROV coring, seafloor drilling, and isotopic and microarray-based genomic analysis, have been used to reveal the geological processes responsible for the seeping activities and the biogeochemical processes related to them. The geophysical signature associated with gas seeps mainly includes the acoustic turbidity in the subsurface, the anomaly of the backscattering intensity at the seabed, and the gas plumes observed in the water column. Pore water and methane-derived authigenic carbonate archive the intensification of methane seepage and the paleoenvironment changes at different time scales. The methane feeding of the seeps in the OT was generated mostly via the microbially mediated process and has an origin mixed by thermogenic hydrocarbon gas in the middle OT. Sulfate-driven and Fe-driven anaerobic oxidations of methane are suggested to be the key biogeochemical processes, which would shape the material cycling in the seeping environment. The future research on the cold seeps in the OT is worth looking forward to due to its geographic and potential geologic links with the nearby hydrothermal activities. Multidisciplinary studies are expected to concentrate on their link with the undiscovered gas hydrates, the amount of methane transferring into the oceans and its impact on the climatic change, and the evolution of the seeping activities accompanied by the biogeochemical processes.