Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Related Publications
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

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 the most sandwiches were brought to the surface before preservation, 1 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).

- Imported "RR2102_colonist_counts.csv" into BCO-DMO system
- Converted deployment, purse recovery, and recovery dates into ISO 8601 format: YYYY-MM-DD HH:MM
- Renamed fields to conform with BCO-DMO naming conventions
- Saved the file as "949181_v1_colonization_exp_tica_vent.csv"

Scientific names in the supplemental files were checked using World Register of Marine Species (WoRMS) Taxon Match. All scientific names in the data are valid and accepted names as of 2025-01-23.

Both "RR2102_morphotype_guide.pdf" and "Morphcode_info.csv" have been attached as supplemental files. The file "RR2102_morphotype_guide.pdf" provides a visual guide of the morphotypes and the "Morphcode_info.csv" file provides the textual information in a structured table.

NSF Award Abstract:
Over four decades of research have shown that tiny free-swimming offspring of the unique inhabitants of hydrothermal vents can disperse effectively between their specialized habitats. Yet, we know almost nothing about how these larval animals complete the journey by locating and settling down in suitable locations. This question remains one of the key unresolved puzzles in the ecology of the deep sea and is becoming increasingly important to solve as hydrothermal vents are becoming threatened by human impacts. The investigators suggest that the films of bacteria that first form at vents are good signposts for settlement of larvae because they indicate that the hydrothermal vents are suitable for life. This project uses a combined program of field experiments, cutting-edge molecular biology techniques, and shipboard experiments with hydrothermal-vent larvae and cultured bacterial films. The project also connects undergraduate research interns at a primarily undergraduate institution (Western Washington University) with undergraduate research interns at two research institutions (Rutgers and Woods Hole Oceanographic Institution) while working on the project at sea together. Finally, the team is producing a science-in-action documentary filled with ocean science and exploration intended for television distribution and museum screenings. The investigators are using footage of the deep-sea vents, shipboard and diving operations, and laboratory work to create a documentary that highlights the foundation of scientific research—hypothesis-driven research, the application of the scientific method, and the importance of critical thinking—all in the framework of the study of an exciting, but threatened, ecosystem.

Hydrothermal vents are particularly tractable systems in which to study questions about the roles of biofilms in larval settlement because biofilms at vents are relatively low-complexity; vent animals are strictly dependent on vent microbes, often through symbiotic partnerships acquired after settlement; and environmental variations are present within the range of a common larval pool. Moreover, decades of research on settlement in model organisms give us good insight into biofilm cues; there is solid foundational understanding about colonization patterns at vents; we now have excellent tools to collect, identify, and culture vent larvae and microbes; and modern environmental "-omics" techniques are a good tool to characterize biological cues produced by biofilms. The project provides an unprecedented, quantitative look into the role of microbial biofilms in structuring larval settlement at hydrothermal vents, achieved only through the close collaboration of microbial and larval ecologists. The combined field program of short-term settlement experiments, microbial "-omics" work, and subsequent shipboard settlement experiments allows the investigative team to use field experiments to statistically model the factors that best predict larval settlement in the field, then test those predictions with shipboard experiments that decouple covarying conditions. This extensive characterization of putative larval settlement cues and their relationship to colonization success in heterogeneous vent habitat niches will contribute to a broader understanding of colonization success across diverse marine ecosystems. Understanding the role that the initial settlement of larvae plays in the recovery and resilience of hydrothermal-vent ecosystems is critical to developing informed management plans for deep-sea mining.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.