Collection

A subset of samples from a prior year's expedition to Axial Seamount (TN405) was also included in the analysis. During TN405 (July 2022), one ROV Jason dive collected diffuse vent fluid using the Hydrothermal Fluid and Particle Sampler (labeled "HFS" in the dataset; designed by David Butterfield) and seawater from the plume was collected via Niskin bottles during CTD casts. TN405 was interrupted and TN420 represents the majority of samples associated with this project.

Collection of diffuse vent fluid and nearby seawater (background) was conducted using the Universal Fluid Obtainer (UFO) and the SUPR sampler. The UFO was the primary device used for fluid sampling for 3 ROV dives (J2-1500 - J2-1502), while the SUPR was the primary mode of fluid collection for 4 ROV dives (J2-1503 - J2-1506). No fluid sampler was operational during the first dive (J2-1499). For the first 4 sites (J2-1499, J2-1501, J2-1502, and J2-1503), 3 Niskin bottles mounted on ROV Jason were fired a few meters above active venting fluid.

The UFO has 8 total ports for fluid collection. For each dive, 4 L bags were attached to 6 ports and sterivex filters (0.22 µm pore size) were connected to the final 2 ports. ROV Jason temperature probe would identify a suitable spot, based on consistent fluid flow and temperature (we targeted 20-80°C fluid), and then would use an intake wand connected to the UFO in the other manipulator. For sterivex filters, once they were recovered from the UFO manifold, they were preserved with RNAlater and stored at -80°C.

The SUPR sampler was typically deployed with 10 2 L bottles and 4 filter holders, where the in situ filters used were either 0.2 µm PES or 0.7 µm nominal GFF filters. The intake for the SUPR sampler includes a built in temperature probe, in order to simultaneously monitor fluid temperature while collecting vent fluid. Each filter holder used on the SUPR included a built-in reservoir for RNAlater; RNAlater would ‘weep’ onto the filter as fluid was pulled through the filter and holder.

Sample processing

For all samples, RNA was extracted and amplified similarly to the protocol described in Hu et al. (2018). Frozen filters were thawed and placed into sterile 15-ml falcon tubes with sterile forceps, 1-2 mL of RLT+ buffer (with β-Mercaptoethanol, Qiagen, Valencia, CA, USA) and RNase-free silica beads was added to each tube. Falcon tubes were bead-beaten by vortexing vigorously for 5 minutes. The original sample collection tubes with RNAlater were centrifuged to pellet any cellular material left in the RNAlater; the RNAlater was removed and replaced with 500-ul of RLT+ buffer (with β-Mercaptoethanol). This was vortexed and added to the 15-ml falcon tube. RNA was extracted with the RNAeasy kit (Qiagen #74104) with the in-line genomic DNA removal step (RNase-free DNase reagents, Qiagen #79254). RNA concentrations were determined using the Ribogreen protocol. Extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (iScript Select cDNA Synthesis, BioRad, #1708896, Hercules, CA); the concentration of RNA was normalized for the cDNA synthesis reaction (input –ng of RNA). Primers targeting the V4 hypervariable region of the 18S rRNA gene (Stoeck et al. 2010; Hu et al. 2015) were used in PCR reactions, which consisted of a final concentration of 1X Q5 High Fidelity Master Mix (NEB #M0492S, Ipswich, MA), 0.5 μM each of forward and reverse primers, and 1 ng of genetic material. The PCR thermal protocol started with an initial activation step (Q5 specific) of 98°C for 2 min, followed with 10 cycles of 98°C for 10 s, 53°C for 30 s, 72°C for 30 s, and 15 cycles of 98°C for 10 s, 48°C for 30 s, and 72°C for 30 s, and a final extension of 72°C for 2 min (modified from Rodriquez Martinez et al. 2012). The original extract total RNA was also PCR amplified to ensure no genomic DNA was present in the sample. PCR products were checked by confirming the presence of an ~400 bp product on an agarose gel. In cases with no amplification, the PCR reaction was repeated with a higher concentration of cDNA (1.5-2 ng). If this did not yield the expected PCR product, the reaction was repeated with an additional 5 cycles. PCR products were sent to Georgia Genomics and Bioinformatics Core (GGBC) and sequencings using the Illumina NextSeq2000 platform.