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The shallow-water hydrothermal vent (HV) system off Kueishan Island lies at the end of the Okinawa Trough to the northeast of Taiwan. Near its submarine vent openings, aperiodic vent discharges generate a dynamic acidic (pH 5.5-8.1) and sulfidic (9-3000 μM) ecosystem. The dominant metazoan in this unique environment is the brachyuran vent crab, Xenograpsus testudinatus, which has developed robust metabolic strategies and highly adaptive acid-base regulatory mechanisms to maintain its physiological homeostasis. X. testudinatus is considered a holobiont, but the symbiotic mechanisms underlying acid and sulfur tolerance in the host-microbe system remain largely unclear. In this study, we used LoopSeq long-read sequencing of the full-length 16S rRNA gene to identify the bacterial communities present in the gills and carapace surface of X. testudinatus. The alpha diversity analysis, Venn diagram, and principal coordinate analysis (PCoA) indicated that the gills and carapace surface exhibit different bacterial constituents. Further measurements of relative abundance, coupled with functional predictions and fluorescence in situ hybridization (FISH), revealed a predominance of Sulfurovum sp. NBC37-1, a key bacterium that can perform sulfur and hydrogen oxidation to support denitrification processes. Consequently, our findings suggest that the symbiotic bacteria may play a critical role in conferring the extraordinary acid and sulfur tolerances of X. testudinatus, allowing the crustacean holobiont to thrive in its ecological niche within one of the most extreme marine habitats on Earth.

With large topographic gradients, rifted basins serve as efficient traps for particulate matter from adjacent lands and the ocean surface. However, the fate of organic carbon in the sediment, mostly unaltered by the hydrothermal activities known to occur in young rifts, remains poorly understood. In this study, we present an examination of diagenetic activities and carbon remineralization based on the first complete suite of pore-water data of sediment marginally affected by hydrothermal activities in the Southern Okinawa Trough (SOT). The sediment showed an oxygen penetration depth of 1 cm, consumption of NO3- in the top 1 cm, smeared profiles of Mn2+ and Fe2+ with the latter reaching up to 450 μmol L-1, and relatively unchanged SO42- concentrations with depth. Net production rates of dissolved species resolved from pore-water profiles provide an estimate of 1.68 ± 0.21 mmol C m-2 d-1 as the total carbon remineralization rate in the upper 30 cm sediment column, with aerobic carbon oxidation being the major pathway. The rate, one order of magnitude lower than that of the adjacent East China Sea, is attributed to the lower bottom-water temperature and carbon flux in the trough. The high carbon burial efficiency of SOT (68% of carbon reaching the seafloor and processed thereunder) reflects the combined effects of small mountain rivers and rifting-induced particle trapping.

The advanced instrumented GeoMICROBE sleds (Cowen et al., 2012) facilitate the collection of hydrothermal fluids and suspended particles in the subseafloor (basaltic) basement through Circulation Obviation Retrofit Kits (CORKs) installed within boreholes of the Integrated Ocean Drilling Program. The main components of the GeoMICROBE can be converted into a mobile pumping system (MPS) that is installed on the front basket of a submersible or remotely-operated-vehicle (ROV). Here, we provide details of a hydrothermal fluid-trap used on the MPS, through which a gastight sampler can withdraw fluids. We also applied the MPS to demonstrate the value of fixing samples at the seafloor in order to determine redox-sensitive dissolved iron concentrations and speciation measurements. To make the best use of the GeoMICROBE sleds, we describe a miniature and mobile version of the GeoMICROBE sled, which permits rapid turn-over and is relatively easy for preparation and operation. Similar to GeoMICROBE sleds, the Mobile GeoMICROBE (MGM) is capable of collecting fluid samples, filtration of suspended particles, and extraction of organics. We validate this approach by demonstrating the seafloor extraction of hydrophobic organics from a large volume (247L) of hydrothermal fluids. • We describe the design of a hydrothermal fluid-trap for use with a gastight sampler, as well as the use of seafloor fixation, through ROV- or submersible assisted mobile pumping systems. • We describe the design of a Mobile GeoMICROBE (MGM) that enhances large volume hydrothermal fluid sampling, suspended particle filtration, and organic matter extraction on the seafloor. • We provide an example of organic matter extracted and characterized from hydrothermal fluids via a MGM.

ABSTRACT Microbial life has been detected well into the igneous crust of the seafloor (i.e., the oceanic basement), but there have been no reports confirming the presence of viruses in this habitat. To detect and characterize an ocean basement virome, geothermally heated fluid samples (ca. 60 to 65°C) were collected from 117 to 292 m deep into the ocean basement using seafloor observatories installed in two boreholes (Integrated Ocean Drilling Program [IODP] U1362A and U1362B) drilled in the eastern sediment-covered flank of the Juan de Fuca Ridge. Concentrations of virus-like particles in the fluid samples were on the order of 0.2 × 105 to 2 × 105 ml−1 (n = 8), higher than prokaryote-like cells in the same samples by a factor of 9 on average (range, 1.5 to 27). Electron microscopy revealed diverse viral morphotypes similar to those of viruses known to infect bacteria and thermophilic archaea. An analysis of virus-like sequences in basement microbial metagenomes suggests that those from archaeon-infecting viruses were the most common (63 to 80%). Complete genomes of a putative archaeon-infecting virus and a prophage within an archaeal scaffold were identified among the assembled sequences, and sequence analysis suggests that they represent lineages divergent from known thermophilic viruses. Of the clustered regularly interspaced short palindromic repeat (CRISPR)-containing scaffolds in the metagenomes for which a taxonomy could be inferred (163 out of 737), 51 to 55% appeared to be archaeal and 45 to 49% appeared to be bacterial. These results imply that the warmed, highly altered fluids in deeply buried ocean basement harbor a distinct assemblage of novel viruses, including many that infect archaea, and that these viruses are active participants in the ecology of the basement microbiome. IMPORTANCE The hydrothermally active ocean basement is voluminous and likely provided conditions critical to the origins of life, but the microbiology of this vast habitat is not well understood. Viruses in particular, although integral to the origins, evolution, and ecology of all life on earth, have never been documented in basement fluids. This report provides the first estimate of free virus particles (virions) within fluids circulating through the extrusive basalt of the seafloor and describes the morphological and genetic signatures of basement viruses. These data push the known geographical limits of the virosphere deep into the ocean basement and point to a wealth of novel viral diversity, exploration of which could shed light on the early evolution of viruses.

Although fluids within the upper oceanic basaltic crust harbor a substantial fraction of the total prokaryotic cells on Earth, the energy needs of this microbial population are unknown. In this study, a nanocalorimeter (sensitivity down to 1.2 nW ml-1) was used to measure the enthalpy of microbially catalyzed reactions as a function of temperature in samples from two distinct crustal fluid aquifers. Microorganisms in unamended, warm (63°C) and geochemically altered anoxic fluids taken from 292 meters sub-basement (msb) near the Juan de Fuca Ridge produced 267.3 mJ of heat over the course of 97 h during a step-wise isothermal scan from 35.5 to 85.0°C. Most of this heat signal likely stems from the germination of thermophilic endospores (6.66 ? 104 cells ml-1 FLUID) and their subsequent metabolic activity at temperatures greater than 50°C. The average cellular energy consumption (5.68 pW cell-1) reveals the high metabolic potential of a dormant community transported by fluids circulating through the ocean crust. By contrast, samples taken from 293 msb from cooler (3.8°C), relatively unaltered oxic fluids, produced 12.8 mJ of heat over the course of 14 h as temperature ramped from 34.8 to 43.0°C. Corresponding cell-specific energy turnover rates (0.18 pW cell-1) were converted to oxygen uptake rates of 24.5 nmol O2 ml-1 FLUID d-1, validating previous model predictions of microbial activity in this environment. Given that the investigated fluids are characteristic of expansive areas of the upper oceanic crust, the measured metabolic heat rates can be used to constrain boundaries of habitability and microbial activity in the oceanic crust. [ABSTRACT FROM AUTHOR]