Study site and experiment details

A short-term in-situ colonization experiment was conducted at Tica Vent (9°50.3987 N, 104°17.4970 W) in the 9°50’ N region of the EPR. Colonization surfaces, comprised of 6 stacked polycarbonate plates separated by spacers and termed “sandwiches” (described by Mullineaux et al., 2010 and Dykman et al., 2021), were deployed by the deep submergence vehicles HOV Alvin and ROV Jason over two cruises; one aboard the R/V Atlantis in December 2019 and the other on the R/V Roger Revelle in March-April 2021.

During the first deployment in December 2019, 12 sandwiches were covered in 200 µm nylon mesh bags termed “purses” to prevent animal colonization while allowing for microbial biofilm development. The purses were custom designed with handles and completely enclosed around the sandwich with hook and loop fasteners so that they could be removed in-situ by vehicle manipulators (L. Lavigne, Anacortes, WA). Four replicate “pursed sandwiches” were placed within each of three biogenic zones (Alvinella-dominated, Riftia-dominated, and mussel-dominated) that span a gradient of temperature and chemical conditions. The colonization experiment was initiated when the ROV Jason revisited the pursed sandwiches approximately 15 months later. Several pursed sandwiches (3 from the Alvinellid zone and 2 from the Riftia zone) were lost due to overgrowth by animal communities, resulting in the discovery of 7 of the initial 12 pursed sandwiches. For the 7 pursed sandwiches that were discovered, purses were removed, and the established biofilm sandwiches were placed back in the location they had been found. Fresh sandwiches (no previously developed biofilm) were deployed next to each of the 7 established biofilm sandwiches to act as paired controls. The sandwiches were left on the seafloor for approximately 15 days to allow for animal colonization. After which, they were recovered into separate sealed collection compartments on the ROV Jason.

Although most sandwiches were brought to the surface before preservation, one established biofilm sandwich from each zone (including the only established biofilm sandwich in the Alvinellid zone) was recovered into individual cylinders specifically designed for immediate preservation of samples in-situ (Analytical Instrument Systems, Ringoes, NJ) that were filled with an RNA stabilization solution (25 mM sodium citrate, 10 mM EDTA, 70 g ammonium sulfate per 100 mL solution, adjusted to pH 7.0 with sulfuric acid); preservation in the RNA stabilization buffer makes these samples available for other work not reported here. A temperature probe held at the base of each sandwich for approximately 1-2 minutes was used to record the temperature maximum at deployments and recoveries of all sandwiches.

Shipboard preservation and processing of sandwiches

Upon vehicle recovery, containers holding sandwiches were transported to a cold room (4 °C). Attached weights and polypropylene handles were removed and the zipties holding each sandwich together were cut to remove the top sandwich plate. Any animals visible on the top plate were gently removed and placed into a separate container filled with RNA preservative. The top plate was then placed into a plastic bag with enough RNA preservative to cover the entire plate and frozen at -80 °C until further processing for 16S rRNA gene sequencing (not included in the dataset here). The remaining parts of the sandwich were placed into separate plastic bags, submerged in RNA preservative, and stored at 4 °C until further processing for animal colonization. To collect any fauna that had fallen off the sandwiches, seawater or RNA preservative in the sandwich collection compartments was poured and rinsed over a 63 µm mesh sieve, then stored in RNA preservative at 4 °C until further processing.

Characterizing animal colonist communities

At Western Washington University’s Shannon Point Marine Center (SPMC) faunal colonists from each sandwich were observed and counted under a stereo microscope (Olympus) at 4x magnification. Both sides of each sandwich plate were carefully inspected under a microscope and attached organisms were removed and counted. Additionally, any organisms in the RNA stabilization solution in the containers holding the sandwich plates and sieved sandwich compartment contents were sorted and counted. Individual colonists were grouped into morphotypes based on visual features while any damaged organisms that could not be assigned to a morphotype group were grouped into a broad unknown taxonomic group (e.g., unknown polychaete, unknown gastropod, etc.). Representative organisms from morphotype groups were imaged on either a stereo microscope (Leica; magnification range 0.8-10x) or a compound microscope (Leica; 20x magnification) for possible further taxonomic identification and to create a morphotype guide (See supplemental files: RR2102_morphotype_guide.pdf).