The investigation was carried out during TS42 cruise between 7 July and 18 August 2024 by RV Tan Suo Yi Hao with the full-ocean-depth human-occupied vehicle Fendouzhe, which was fitted with hydraulically powered manipulators on two swing arms. Under the guidance of operators in the human-occupied vehicle, the arms efficiently acquired the samples and stored them safely in a biological box and a geological box of the vehicle.
Processing of benthic fauna and sea floor video footage
Upon retrieval of the submersible, all collected specimens were promptly transferred from the biological collection box and slurp sampler to the shipboard laboratory. The specimens were then sorted into main taxonomic groups of different levels using visual inspection or under stereomicroscopes. Each organism was counted and preserved in pre-cooled, non-denatured 95% ethanol or in a 4% buffered formaldehyde solution depending on the taxon. Following initial preservation, certain taxonomic groups were further transferred to 70% ethanol for long-term storage.
Visual assessment of species identification, density and spatial structure of the macro-epifauna and mega-epifauna of the seep communities was carried out on the basis of the analysis of video footage recorded by two high-definition cameras mounted on the human-occupied vehicle. For each dive, between three and ten representative screenshots showing the densest cold-seep communities were selected from the video footage. The area of each image was estimated using the submersible’s laser scale, which projects two parallel laser points 10 cm apart onto the sea floor. This provided a reliable spatial reference for calculating the area of the sea floor captured in each image. A standardized quadrat (for example, 50 × 50 cm) was drawn near the laser dots by using this laser scale as a reference in the image. The calculated area was then converted into square metres for standardized density calculations. Animals visible in each quadrat were manually counted, and faunal density was expressed as the number of individuals per square metre. For each dive, density values from the selected images were used to calculate the mean faunal density, and the standard deviation was computed to quantify the variability among replicate images.
Phylogenetic analyses of the coxI gene sequences
Up to 0.5 cm3 of fauna tissue was cut into tiny pieces and subjected to DNA extraction using the PowerSoil DNA Isolation kit (MoBio Laboratories). The extracted DNA was quantified using a Qubit dsDNA HS Assay Kit with Qubit 2.0 fluorometer (Invitrogen). A metagenomic library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina v.4 and sequenced on the NovaSeq X Plus platform (Illumina) to generate 2 × 150 bp pair-ended reads. Raw sequencing reads were qualified using Fastp v.0.23.2 and assembly into contigs using MEGAHIT v.1.2.9. The coxI gene encoding cytochrome c oxidase subunit I of the fauna was retrieved from metagenomic contigs. The retrieved coxI was searched against the National Center for Biotechnology Information GenBank database for preliminary taxonomy identification.
Gas and pore-water sampling
During each dive, 6–12 sediment pushcores were collected using the manipulators of the submersible. Upon recovery, these pushcores were immediately transported to the ship’s cold room, which is kept at around 4 °C, to facilitate subsequent processing. Among them, one or two pushcores were allocated for gas concentration analysis onboard, with only subsamples exhibiting high methane concentrations being prepared for further carbon and deuterium isotope analysis. In addition, one or two pushcores were used to extract pore water samples for geochemical analysis.
Pore-water sampling was conducted using Rhizon samplers (as described in ref. 51). These samplers were inserted into the cores at 2-cm intervals and connected to 50-ml evacuated disposable syringes fitted with three-way Luer-lock stopcocks. The first approximately 1 ml of extracted pore water was discarded to remove any contaminants. Subsequently, around 15 ml of pore water was collected within a 2-h time frame. One portion of the pore water was preserved with a 20% zinc acetate solution for subsequent hydrogen sulfide analysis. The remaining pore water samples were transferred to 15-ml centrifuge tubes and frozen for subsequent ion analysis.
For the analysis of gas composition and isotopes, sediment samples were collected using a 2.5-ml cutoff plastic syringe, which was inserted through pre-drilled holes in the pushcore tube at depth intervals of 4 cm. A 2.5-ml sediment sample was then transferred to a 20-ml gas-tight glass vial, which was filled with 2 M NaOH solution and sealed with a crimp cap containing butyl rubber stoppers. The vials were vigorously shaken and stored in an upside-down position at 4 °C until analysis, which was conducted onboard in a single day. Before analysis, the vials were shaken again, and 2 ml of the NaOH solution was replaced with helium gas to create a headspace. The headspace gas from the push core was also directly extracted using a 20-ml syringe equipped with a three-way stopcock and was immediately transferred to a 12-ml vacuum Labco vial for further analysis.
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