Single-celled microbial eukaryotes inhabit deep-sea hydrothermal vent environments and play critical ecological roles in the vent-associated microbial food web. 18S rRNA amplicon sequencing of diffuse venting fluids from two geochemically-distinct hydrothermal vent fields was applied to investigate community diversity patterns among protistan assemblages. Piccard and Von Damm vent fields are situated 20 km apart at the Mid-Cayman Rise in the Caribbean Sea. We describe species diversity patterns with respect to hydrothermal vent field and sample type, identify putative vent endemic microbial eukaryotes, and test how vent fluid geochemistry may influence microbial community diversity. Individual vent fields supported distinct and highly diverse assemblages of protists that included potentially endemic or novel vent-associated strains. This data adds to our growing knowledge of the biogeography of deep-sea microbial eukaryotes.

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Deployments
• Project Information
• Program Information
• Funding

Samples and experiments were collected and executed during cruise AT42-22 aboard the RV Atlantis with ROV Jason in January-February 2020 at the Von Damm (2300 m; 18°23’N, 81°48’W) and Piccard (5000 m; 18°33’N, 81°43’W) hydrothermal fields located along the Mid-Cayman Rise.

Fluids for shipboard grazing experiments and biogeochemistry were obtained in 10L volume bags (Kynar, Keika Ventures; polyvinylidene fluoride) using the Hydrothermal Organic Geochemistry (HOG) sampler mounted on ROV Jason. Between 4-10 L of vent fluid was collected and filtered through a 47 mm polyethersulphone (PES) filter (Millipore) with a pore size of 0.2 µm and preserved with RNAlater (Ambion) at the seafloor for molecular analysis of microbial communities. 

Non-vent samples were collected from within the overlying non-buoyant hydrothermal plume at each site and from background seawater via CTD-mounted Niskin bottles. Plume samples were identified using in situ CTD sensors to detect the presence of hydrothermal influence in real-time (back-scatter and temperature) above each vent field. Background seawater samples were collected outside of the influence of the hydrothermal vent at approximately the same depth as the vent sites (~2350 m and ~4950 m).

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.

Website: https://www.darkenergybiosphere.org/award/probing-subseafloor-microbial-interactions-via-hydrothermal-vent-fluids-a-focus-on-protists/

Coverage: Axial Seamount Juan de Fuca Ridge NE Pacific 46 N 130 W, Gorda Ridge NE Pacific 41 N 127 W, Mid-Cayman Rise Caribbean Sea 18 N 82 W

Adjusted C-DEBI Award Description:

Highly reduced and thermally charged venting fluids from the subseafloor mix with surrounding seawater, creating a sharp geochemical gradient which promotes a hub of biological diversity at hydrothermal vent ecosystems. While studies of prokaryotic diversity at hydrothermal vent sites have highlighted the important roles microorganisms play in deep sea carbon cycling and offered a unique window into subseafloor microbial communities, depictions of deep-sea marine ecology and food webs are incomplete without characterization of single-celled microbial eukaryotes (protists). I propose to use culture-independent techniques (tag-sequencing and metatranscriptomics) to provide a thorough understanding of protistan biogeography in and near venting fluids, focusing on the vent fluid-seawater interface. Additionally, these qualitative analyses will be paired with quantitative experiments that measure protistan grazing pressure. Understanding trophic interactions within the protistan community is incredibly important, as these processes form the foundation of deep-sea marine food webs and mediate a significant amount of carbon transferred to higher trophic levels.

C-DEBI project link: https://www.darkenergybiosphere.org/award/probing-subseafloor-microbial-interactions-via-hydrothermal-vent-fluids-a-focus-on-protists/

Website: http://www.darkenergybiosphere.org

Coverage: Global

The mission of the Center for Dark Energy Biosphere Investigations (C-DEBI) is to explore life beneath the seafloor and make transformative discoveries that advance science, benefit society, and inspire people of all ages and origins.

C-DEBI provides a framework for a large, multi-disciplinary group of scientists to pursue fundamental questions about life deep in the sub-surface environment of Earth. The fundamental science questions of C-DEBI involve exploration and discovery, uncovering the processes that constrain the sub-surface biosphere below the oceans, and implications to the Earth system. What type of life exists in this deep biosphere, how much, and how is it distributed and dispersed? What are the physical-chemical conditions that promote or limit life? What are the important oxidation-reduction processes and are they unique or important to humankind? How does this biosphere influence global energy and material cycles, particularly the carbon cycle? Finally, can we discern how such life evolved in geological settings beneath the ocean floor, and how this might relate to ideas about the origin of life on our planet?

C-DEBI's scientific goals are pursued with a combination of approaches:
(1) coordinate, integrate, support, and extend the research associated with four major programs—Juan de Fuca Ridge flank (JdF), South Pacific Gyre (SPG), North Pond (NP), and Dorado Outcrop (DO)—and other field sites;
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