Filters retrieved from ROV Jason and from the final time point of each grazing assay were processed identically. RNA was extracted from frozen filters (stored in RNAlater) as amplicon sequences originating from extracted RNA are more likely to represent metabolically active cells, rather than inactive cellular material that may have sunk from above. The filter was first separated from the RNAlater and distributed into tubes with a lysis buffer (Qiagen 1053393). The RNAlater was centrifuged for 15 minutes at 16,000 x g, and the supernatant was removed. Lysis buffer was added on top of any cellular materials collected, vortexed, and then combined with the filter. The filter and lysis buffer solution was vortexed thoroughly with RNAase-free silica beads. The lysis buffer was then separated from beads and filter material with a syringe and processed using the Qiagen RNeasy extract kit (Qiagen 74104), which included an inline RNAse-free DNase removal step (Qiagen 79256). Total RNA was reverse transcribed to cDNA and amplified with V4-specific primers. MiSeq 2 x 300 bp PE sequencing was performed at the Marine Biological Laboratory Bay Paul Centre Keck sequencing facility. Amplicon sequences were processed using QIIME2 (version 2021.4).

Following quality control, Amplicon Sequence Variants (or ASVs) were determined from the sequences and taxonomic assignment was done using the PR2 database (v 4.14). For this analysis, we focused primarily on the microeukaryotic population, removing sequences assigned to prokaryotes or Metazoa. ASVs were categorized as vent-only or cosmopolitan, based on their presence in only vent samples or throughout vent, plume, and background samples, respectively.