Diverse DNA modification in marine prokaryotic and viral communities.

DNA chemical modifications, including methylation, are widespread and play important roles in prokaryotes and viruses. However, current knowledge of these modification systems is severely biased towards a limited number of culturable prokaryotes, despite the fact that a vast majority of microorganisms have not yet been cultured. Here, using single-molecule real-time sequencing, we conducted culture-independent 'metaepigenomic' analyses (an integrated analysis of metagenomics and epigenomics) of marine microbial communities. A total of 233 and 163 metagenomic-assembled genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTase genes were not genetically linked with the endonuclease genes predicted to be involved in defense mechanisms against extracellular DNA. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, 5 of which showed novel specificities and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative specificities in MTases that are highly conserved in Alphaproteobacteria, which may enhance our understanding of the co-evolutionary history of the methylation systems and the genomes. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses in nature.

Potential Interactions between Clade SUP05 Sulfur-Oxidizing Bacteria and Phages in Hydrothermal Vent Sponges.

In deep-sea hydrothermal vent environments, sulfur-oxidizing bacteria belonging to the clade SUP05 are crucial symbionts of invertebrate animals. Marine viruses, as the most abundant biological entities in the ocean, play essential roles in regulating the sulfur metabolism of the SUP05 bacteria. To date, vent sponge-associated SUP05 and their phages have not been well documented. The current study analyzed microbiomes of Haplosclerida sponges from hydrothermal vents in the Okinawa Trough and recovered the dominant SUP05 genome, designated VS-SUP05. Phylogenetic analysis showed that VS-SUP05 was closely related to endosymbiotic SUP05 strains from mussels living in deep-sea hydrothermal vent fields. Homology and metabolic pathway comparisons against free-living and symbiotic SUP05 strains revealed that the VS-SUP05 genome shared many features with the deep-sea mussel symbionts. Supporting a potentially symbiotic lifestyle, the VS-SUP05 genome contained genes involved in the synthesis of essential amino acids and cofactors that are desired by the host. Analysis of sponge-associated viral sequences revealed putative VS-SUP05 phages, all of which were double-stranded viruses belonging to the families Myoviridae, Siphoviridae, Podoviridae, and Microviridae Among the phage sequences, one contig contained metabolic genes (iscR, iscS, and iscU) involved in iron-sulfur cluster formation. Interestingly, genome sequence comparison revealed horizontal transfer of the iscS gene among phages, VS-SUP05, and other symbiotic SUP05 strains, indicating an interaction between marine phages and SUP05 symbionts. Overall, our findings confirm the presence of SUP05 bacteria and their phages in sponges from deep-sea vents and imply a beneficial interaction that allows adaptation of the host sponge to the hydrothermal vent environment.IMPORTANCE Chemosynthetic SUP05 bacteria dominate the microbial communities of deep-sea hydrothermal vents around the world, SUP05 bacteria utilize reduced chemical compounds in vent fluids and commonly form symbioses with invertebrate organisms. This symbiotic relationship could be key to adapting to such unique and extreme environments. Viruses are the most abundant biological entities on the planet and have been identified in hydrothermal vent environments. However, their interactions with the symbiotic microbes of the SUP05 clade, along with their role in the symbiotic system, remain unclear. Here, using metagenomic sequence-based analyses, we determined that bacteriophages may support metabolism in SUP05 bacteria and play a role in the sponge-associated symbiosis system in hydrothermal vent environments.

Psychrobium conchae gen. nov., sp. nov., a psychrophilic marine bacterium isolated from the Iheya North hydrothermal field.

A novel psychrophilic, marine, bacterial strain designated BJ-1(T) was isolated from the Iheya North hydrothermal field in the Okinawa Trough off Japan. Cells were Gram-negative, rod-shaped, non-spore-forming, aerobic chemo-organotrophs and motile by means of a single polar flagellum. Growth occurred at temperatures below 16 °C, with the optimum between 9 and 12 °C. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the closest relatives of strain BJ-1(T) were Shewanella denitrificans OS-217(T) (93.5% similarity), Shewanella profunda DSM 15900(T) (92.9%), Shewanella gaetbuli TF-27(T) (92.9%), Paraferrimonas sedimenticola Mok-106(T) (92.1%) and Ferrimonas kyonanensis Asr22-7(T) (91.7%). The major respiratory quinone was Q-8. The predominant fatty acids were C(16:1)ω7c and C(16:0). The G+C content of the novel strain was 40.5 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, it is proposed that strain BJ-1(T) represents a novel species in a new genus, for which the name Psychrobium conchae gen. nov., sp. nov. is proposed. The type strain of Psychrobium conchae is BJ-1(T) ( =JCM 30103(T) =DSM 28701(T)).

Millisecond-scale behaviours of plankton quantified in vitro and in situ using the Event-based Vision Sensor.

The Event-based Vision Sensor (EVS) is a bio-inspired sensor that captures detailed motions of objects, aiming to become the 'eyes' of machines like self-driving cars. Compared to conventional frame-based image sensors, the EVS has an extremely fast motion capture equivalent to 10,000-fps even with standard optical settings, plus high dynamic ranges for brightness and also lower consumption of memory and energy. Here, we developed 22 characteristic features for analysing the motions of aquatic particles from the EVS raw data and tested the applicability of the EVS in analysing plankton behaviour. Laboratory cultures of six species of zooplankton and phytoplankton were observed, confirming species-specific motion periodicities up to 41 Hz. We applied machine learning to automatically classify particles into four categories of zooplankton and passive particles, achieving an accuracy up to 86%. At the in situ deployment of the EVS at the bottom of Lake Biwa, several particles exhibiting distinct cumulative trajectory with periodicities in their motion (up to 16 Hz) were identified, suggesting that they were living organisms with rhythmic behaviour. We also used the EVS in the deep sea, observing particles with active motion and periodicities over 40 Hz. Our application of the EVS, especially focusing on its millisecond-scale temporal resolution and wide dynamic range, provides a new avenue to investigate organismal behaviour characterised by rapid and periodical motions. The EVS will likely be applicable in the near future for the automated monitoring of plankton behaviour by edge computing on autonomous floats, as well as quantifying rapid cellular-level activities under microscopy.

Microbial decomposition of biodegradable plastics on the deep-sea floor.

Microbes can decompose biodegradable plastics on land, rivers and seashore. However, it is unclear whether deep-sea microbes can degrade biodegradable plastics in the extreme environmental conditions of the seafloor. Here, we report microbial decomposition of representative biodegradable plastics (polyhydroxyalkanoates, biodegradable polyesters, and polysaccharide esters) at diverse deep-sea floor locations ranging in depth from 757 to 5552 m. The degradation of samples was evaluated in terms of weight loss, reduction in material thickness, and surface morphological changes. Poly(L-lactic acid) did not degrade at either shore or deep-sea sites, while other biodegradable polyesters, polyhydroxyalkanoates, and polysaccharide esters were degraded. The rate of degradation slowed with water depth. We analysed the plastic-associated microbial communities by 16S rRNA gene amplicon sequencing and metagenomics. Several dominant microorganisms carried genes potentially encoding plastic-degrading enzymes such as polyhydroxyalkanoate depolymerases and cutinases/polyesterases. Analysis of available metagenomic datasets indicated that these microorganisms are present in other deep-sea locations. Our results confirm that biodegradable plastics can be degraded by the action of microorganisms on the deep-sea floor, although with much less efficiency than in coastal settings.

Deep Subseafloor Biogeochemical Processes and Microbial Populations Potentially Associated with the 2011 Tohoku-oki Earthquake at the Japan Trench Accretionary Wedge (IODP Expedition 343).

Post-mega-earthquake geochemical and microbiological properties in subseafloor sediments of the Japan Trench accretionary wedge were investigated using core samples from Hole C0019E, which was drilled down to 851| |m below seafloor (mbsf) at a water depth of 6,890 m. Methane was abundant throughout accretionary prism sediments; however, its concentration decreased close to the plate boundary decollement. Methane isotope systematics indicated a biogenic origin. The content of mole-cular hydrogen (H2) was low throughout core samples, but markedly increased at specific depths that were close to potential faults predicted by logging-while-drilling ana-lyses. Based on isotopic systematics, H2 appeared to have been abundantly produced via a low-temperature interaction between pore water and the fresh surface of crushed rock induced by earthquakes. Subseafloor microbial cell density remained constant at approximately 105| |cells| |mL-1. Amplicon sequences revealed that predominant members at the phylum level were common throughout the units tested, which also included members frequently found in anoxic subseafloor sediments. Metabolic potential assays using radioactive isotopes as tracers revealed homoacetogenic activity in H2-enriched core samples collected near the fault. Furthermore, homoacetogenic bacteria, including Acetobacterium carbinolicum, were isolated from similar samples. Therefore, post-earthquake subseafloor microbial communities in the Japan Trench accretionary prism appear to be episodically dominated by homoacetogenic populations and potentially function due to the earthquake-induced low-temperature generation of H2. These post-earthquake microbial communities may eventually return to the steady-state communities dominated by oligotrophic heterotrophs and hydrogenotrophic and methylotrophic methanogens that are dependent on refractory organic matter in the sediment.

Noble gases and nitrogen in samples of asteroid Ryugu record its volatile sources and recent surface evolution.

The near-Earth carbonaceous asteroid (162173) Ryugu is expected to contain volatile chemical species that could provide information on the origin of Earth's volatiles. Samples of Ryugu were retrieved by the Hayabusa2 spacecraft. We measured noble gas and nitrogen isotopes in Ryugu samples and found that they are dominated by presolar and primordial components, incorporated during Solar System formation. Noble gas concentrations are higher than those in Ivuna-type carbonaceous (CI) chondrite meteorites. Several host phases of isotopically distinct nitrogen have different abundances among the samples. Our measurements support a close relationship between Ryugu and CI chondrites. Noble gases produced by galactic cosmic rays, indicating a ~5 million year exposure, and from implanted solar wind record the recent irradiation history of Ryugu after it migrated to its current orbit.

Species assemblage networks identify regional connectivity pathways among hydrothermal vents in the Northwest Pacific.

The distribution of species among spatially isolated habitat patches supports regional biodiversity and stability, so understanding the underlying processes and structure is a key target of conservation. Although multivariate statistics can infer the connectivity processes driving species distribution, such as dispersal and habitat suitability, they rarely explore the structure. Methods from graph theory, applied to distribution data, give insights into both connectivity pathways and processes by intuitively formatting the data as a network of habitat patches. We apply these methods to empirical data from the hydrothermal vent habitats of the Northwest Pacific. Hydrothermal vents are "oases" of biological productivity and endemicity on the seafloor that are imminently threatened by anthropogenic disturbances with unknown consequences to biodiversity. Here, we describe the structure of species assemblage networks at hydrothermal vents, how local and regional parameters affect their structure, and the implications for conservation. Two complementary networks were formed from an extensive species assemblage dataset: a similarity network of vent site nodes linked by weighted edges based on their pairwise assemblage similarity and a bipartite network of species nodes linked to vent site nodes at which they are present. Using these networks, we assessed the role of individual vent sites in maintaining network connectivity and identified biogeographic sub-regions. The three sub-regions and two outlying sites are separated by their spatial arrangement and local environmental filters. Both networks detected vent sites that play a disproportionately important role in regional pathways, while the bipartite network also identified key vent sites maintaining the distinct species assemblages of their sub-regions. These regional connectivity pathways provide insights into historical colonization routes, while sub-regional connectivity pathways are of value when selecting sites for conservation and/or estimating the multivent impacts from proposed deep-sea mining.

First asteroid gas sample delivered by the Hayabusa2 mission: A treasure box from Ryugu.

The Hayabusa2 spacecraft returned to Earth from the asteroid 162173 Ryugu on 6 December 2020. One day after the recovery, the gas species retained in the sample container were extracted and measured on-site and stored in gas collection bottles. The container gas consists of helium and neon with an extraterrestrial 3He/4He and 20Ne/22Ne ratios, along with some contaminant terrestrial atmospheric gases. A mixture of solar and Earth's atmospheric gas is the best explanation for the container gas composition. Fragmentation of Ryugu grains within the sample container is discussed on the basis of the estimated amount of indigenous He and the size distribution of the recovered Ryugu grains. This is the first successful return of gas species from a near-Earth asteroid.

Deep-sea water displacement from a turbidity current induced by the Super Typhoon Hagibis.

Turbidity currents are the main drivers behind the transportation of terrestrial sediments to the deep sea, and turbidite deposits from such currents have been widely used in geological studies. Nevertheless, the contribution of turbidity currents to vertical displacement of seawater has rarely been discussed. This is partly because until recently, deep-sea turbidity currents have rarely been observed due to their unpredictable nature, being usually triggered by meteorological or geological events such as typhoons and earthquakes. Here, we report a direct observation of a deep-sea turbidity current using the recently developed Edokko Mark 1 monitoring system deployed in 2019 at a depth of 1,370 m in Suruga Bay, central Japan. A turbidity current occurred two days after its probable cause, the Super Typhoon Hagibis (2019), passed through Suruga Bay causing devastating damage. Over aperiod of 40 hours, we observed increased turbidity with turbulent conditions confirmed by a video camera. The turbidity exhibited two sharp peaks around 3:00 and 11:00 on October 14 (Japan Standard Time). The temperature and salinity characteristics during these high turbidity events agreed with independent measurements for shallow water layers in Suruga Bay at the same time, strongly suggesting that the turbidity current caused vertical displacement in the bay's water column by transporting warmer and shallower waters downslope of the canyon. Our results add to the previous few examples that show meteorological and geological events may have significant contributions in the transportation of shallower seawater to the deep sea. Recent technological developments pertaining to the Edokko Mark 1 and similar devices enable straightforward, long-term monitoring of the deep-seafloor and will contribute to the understanding of similar spontaneous events in the deep ocean.

Using Soundscapes to Assess Deep-Sea Benthic Ecosystems.

Targets of deep-sea mining commonly coincide with biodiversity hotspots, such as hydrothermal vents. The resilience of these ecosystems relies on larval dispersal, which may be directed by habitat-specific soundscapes. We urge for a global effort to implement soundscape as a conservation tool to assess anthropogenic disruption to deep-sea benthic ecosystems.

Response of N2O production rate to ocean acidification in the western North Pacific.

Ocean acidification induced by the increase of anthropogenic CO2 emissions has a profound impact on marine organisms and biogeochemical processes.1 The response of marine microbial activities to ocean acidification might play a crucial role in the future evolution of air-sea fluxes of biogenic gases such as nitrous oxide (N2O), a strong greenhouse gas and the dominant stratospheric ozone-depleting substance.2 Here, we examine the response of N2O production from nitrification to acidification in a series of incubation experiments conducted in subtropical and subarctic western North Pacific. The experiments show that, when pH was reduced, the N2O production rate during nitrification measured at subarctic stations increased significantly whereas nitrification rates remained stable or decreased. Contrary to what was previously thought, these results suggest that the effect of ocean acidification on N2O production during nitrification and nitrification rates are likely uncoupled. Collectively these results suggest that, if seawater pH continues to decline at the same rate, ocean acidification could increase the marine N2O production during nitrification in subarctic North Pacific by 185 to 491% by the end of the century.

Evaluation of nutrient and energy sources of the deepest known serpentinite-hosted ecosystem using stable carbon, nitrogen, and sulfur isotopes.

The Shinkai Seep Field (SSF) in the southern Mariana forearc discovered in 2010 is the deepest (~5,700 m in depth) known serpentinite-hosted ecosystem dominated by a vesicomyid clam, Calyptogena (Abyssogena) mariana. The pioneering study presumed that the animal communities are primary sustained by reducing fluid originated from the serpentinization of mantle peridotite. For understanding the nutrient and energy sources for the SSF community, this study conducted four expeditions to the SSF and collected additional animal samples such as polychaetes and crustaceans as well as sediments, fragments of chimneys developing on fissures of serpentinized peridotite, seeping fluid on the chimneys, and pore water within the chimneys. Geochemical analyses of seeping fluids on the chimneys and pore water of the chimneys revealed significantly high pH (~10) that suggest subseafloor serpentinization controlling fluid chemistry. Stable isotope systematics (carbon, nitrogen, and sulfur) among animals, inorganic molecules, and environmental organic matter suggest that the SSF animal community mostly relies on the chemosynthetic production while some organisms appear to partly benefit from photosynthetic production despite the great depth of SSF.

Deep-biosphere methane production stimulated by geofluids in the Nankai accretionary complex.

Microbial life inhabiting subseafloor sediments plays an important role in Earth's carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (102 to 103 cells cm-3) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated.

Unanticipated discovery of two rare gastropod molluscs from recently located hydrothermally influenced areas in the Okinawa Trough.

BACKGROUND: The deep-sea hydrothermal vent is one of the most 'extreme' environments in the marine realm. Few species are capable of inhabiting such ecosystems, despite extremely high productivity there supported by microbial chemosynthesis, leading to high biomass and low species richness. Although gastropod molluscs are one of the main constituents of megafaunal communities at vent ecosystems, most species belong to several typical families (e.g., Provannidae, Peltospiridae, Lepetodrilidae) specialised and adapted to life at vents. METHODS: During recent surveys of Okinawa Trough hydrothermal vent systems, two snails atypical of vent ecosystems were unexpectedly found in newly discovered hydrothermally influenced areas. Shell and radular characteristics were used to identify the gastropods morphologically. RESULTS: One species was a vetigastropod, the calliostomatid Tristichotrochus ikukoae (Sakurai, 1994); and the other was a caenogastropod, the muricid Abyssotrophon soyoae (Okutani, 1959). Both gastropods were previously only known from regular non-chemosynthetic deep-sea and very rare-only two definitive published records exist for T. ikukoae and three for A. soyoae. The radula formula of Tristichotrochus ikukoae is accurately reported for the first time and based on that it is returned to genus Otukaia. For both species, barcode sequences of the cytochrome c oxidase I (COI) gene were obtained and deposited for future references. DISCUSSION: These new records represent the second record of calliostomatids from vents (third from chemosynthetic ecosystems) and the third record of muricids from vents (tenth from chemosynthetic ecosystems), and extend the distribution of both species to the southwest. Neither family has been recorded at chemosynthetic ecosystems in the western Pacific. Both were from weakly diffuse flow areas not subject to high temperature venting but were nevertheless associated with typical vent-reliant taxa such as Lamellibrachia tubeworms and Bathymodiolus mussels. These new records show that these species are capable of tolerating environmental stress associated with weak hydrothermally influenced areas, despite not being vent endemic species, adding to the list of known vent/non-vent species intersections. This signifies that such weakly influenced areas may provide key habitats for them, and that such areas may play a role in the evolution of biological adaptations to 'extreme' chemosynthetic ecosystems.

Leaching of Metals and Metalloids from Hydrothermal Ore Particulates and Their Effects on Marine Phytoplankton.

Seafloor massive sulfide deposits have attracted much interest as mineral resources. Therefore, the potential environmental impacts of full-scale mining should be considered. In this study, we focused on metal and metalloid contamination that could be triggered by accidental leakage and dispersion of hydrothermal ore particulates from mining vessels into surface seawater. We determined the leaching potential of metals and metalloids from four hydrothermal ores collected from the Okinawa Trough into aerobic seawater and then evaluated the toxic effects of ore leachates on a phytoplankton species, Skeletonema marinoi-dohrnii complex, which is present ubiquitously in the ocean. Large amounts of metals and metalloids were released from the ground hydrothermal ores into seawater within 5 min under aerobic conditions. The main components of leachates were Zn + Pb, As + Sb, and Zn + Cu, which were obtained from the Fe-Zn-Pb-rich and Zn-Pb-rich zero-age, Ba-rich, and Fe-rich ores, respectively. The leachates had different chemical compositions from those of the ore. The rapid release and difference in chemical compositions between the leachates and the ores indicated that substances were not directly dissolved from the sulfide-binding mineral phase but from labile phases mainly on the adsorption-desorption interface of the ores under these conditions. All ore leachates inhibited the growth of S. marinoi-dohrnii complex but with different magnitudes of toxic effects. These results indicate that the fine particulate matter of hydrothermal ores is a potential source of toxic contamination that may damage primary production in the ocean. Therefore, we insist on the necessity for the prior evaluation of toxic element leachability from mineral ores into seawater to minimize mining impacts on the surface environment.

Defining boundaries for the distribution of microbial communities beneath the sediment-buried, hydrothermally active seafloor.

Subseafloor microbes beneath active hydrothermal vents are thought to live near the upper temperature limit for life on Earth. We drilled and cored the Iheya North hydrothermal field in the Mid-Okinawa Trough, and examined the phylogenetic compositions and the products of metabolic functions of sub-vent microbial communities. We detected microbial cells, metabolic activities and molecular signatures only in the shallow sediments down to 15.8 m below the seafloor at a moderately distant drilling site from the active hydrothermal vents (450 m). At the drilling site, the profiles of methane and sulfate concentrations and the δ13C and δD isotopic compositions of methane suggested the laterally flowing hydrothermal fluids and the in situ microbial anaerobic methane oxidation. In situ measurements during the drilling constrain the current bottom temperature of the microbially habitable zone to ~45 °C. However, in the past, higher temperatures of 106-198 °C were possible at the depth, as estimated from geochemical thermometry on hydrothermally altered clay minerals. The 16S rRNA gene phylotypes found in the deepest habitable zone are related to those of thermophiles, although sequences typical of known hyperthermophilic microbes were absent from the entire core. Overall our results shed new light on the distribution and composition of the boundary microbial community close to the high-temperature limit for habitability in the subseafloor environment of a hydrothermal field.

Origin of methane-rich natural gas at the West Pacific convergent plate boundary.

Methane emission from the geosphere is generally characterized by a radiocarbon-free signature and might preserve information on the deep carbon cycle on Earth. Here we report a clear relationship between the origin of methane-rich natural gases and the geodynamic setting of the West Pacific convergent plate boundary. Natural gases in the frontal arc basin (South Kanto gas fields, Northeast Japan) show a typical microbial signature with light carbon isotopes, high CH4/C2H6 and CH4/3He ratios. In the Akita-Niigata region - which corresponds to the slope stretching from the volcanic-arc to the back-arc -a thermogenic signature characterize the gases, with prevalence of heavy carbon isotopes, low CH4/C2H6 and CH4/3He ratios. Natural gases from mud volcanoes in South Taiwan at the collision zone show heavy carbon isotopes, middle CH4/C2H6 ratios and low CH4/3He ratios. On the other hand, those from the Tokara Islands situated on the volcanic front of Southwest Japan show the heaviest carbon isotopes, middle CH4/C2H6 ratios and the lowest CH4/3He ratios. The observed geochemical signatures of natural gases are clearly explained by a mixing of microbial, thermogenic and abiotic methane. An increasing contribution of abiotic methane towards more tectonically active regions of the plate boundary is suggested.

Deepest and hottest hydrothermal activity in the Okinawa Trough: the Yokosuka site at Yaeyama Knoll.

Since the initial discovery of hydrothermal vents in 1977, these 'extreme' chemosynthetic systems have been a focus of interdisciplinary research. The Okinawa Trough (OT), located in the semi-enclosed East China Sea between the Eurasian continent and the Ryukyu arc, hosts more than 20 known vent sites but all within a relatively narrow depth range (600-1880 m). Depth is a significant factor in determining fluid temperature and chemistry, as well as biological composition. However, due to the narrow depth range of known sites, the actual influence of depth here has been poorly resolved. Here, the Yokosuka site (2190 m), the first OT vent exceeding 2000 m depth is reported. A highly active hydrothermal vent site centred around four active vent chimneys reaching 364°C in temperature, it is the hottest in the OT. Notable Cl depletion (130 mM) and both high H2 and CH4 concentrations (approx. 10 mM) probably result from subcritical phase separation and thermal decomposition of sedimentary organic matter. Microbiota and fauna were generally similar to other sites in the OT, although with some different characteristics. In terms of microbiota, the H2-rich vent fluids in Neuschwanstein chimney resulted in the dominance of hydrogenotrophic chemolithoautotrophs such as Thioreductor and Desulfobacterium. For fauna, the dominance of the deep-sea mussel Bathymodiolus aduloides is surprising given other nearby vent sites are usually dominated by B. platifrons and/or B. japonicus, and a sponge field in the periphery dominated by Poecilosclerida is unusual for OT vents. Our insights from the Yokosuka site implies that although the distribution of animal species may be linked to depth, the constraint is perhaps not water pressure and resulting chemical properties of the vent fluid but instead physical properties of the surrounding seawater. The potential significance of these preliminary results and prospect for future research on this unique site are discussed.