Table of Contents

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

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. 

- Imported "AxialSeamount_2023_metadata_BCO-DMO.xlsx" into the BCO-DMO system
- Converted date to ISO format YYYY-MM-DD
- Renamed fields to remove spaces and special characters
- Renamed "sample_name" to "site_name" after consulting with the submitter
- Replaced "6202024" and "7162024" values with "not applicable" in the "depth_category" parameter after consulting with submitter
- Removed "SPUID" and "sample_identifier" to reduce duplication after consulting with submitter
- Exported file as "969102_v1_microbial_diversity_axial_seamount.csv"

NSF Award Abstract:

Microorganisms are the ancestral forms of life on our planet and have been instrumental in shaping all of Earth?s environments into what they are today. In the ocean, microbial prokaryotes and eukaryotes form the foundation of marine food webs through their activity and interactions. Single-celled microbial eukaryotes (or protists) are some of the most important species on the planet, yet our understanding of how their activities influence and regulate the ocean ecosystem is poorly constrained. At deep-sea hydrothermal vents in particular, our understanding of microbial food web dynamics is incomplete without including the role of microbial eukaryotes. This project provides quantification of phagotrophic protistan grazing on the microbial communities inhabiting the highly productive diffuse vent mixing zone at hydrothermal vents, where the vent fluid-seawater interface promotes an increase in biological activity compared to the surrounding deep seawater. The results are contributing novel insights into the diversity and metabolic activities of the microbial eukaryotic community at vent fluid-seawater interfaces, establish the extent to which microbial eukaryotes impact primary production in the deep ocean by quantifying predation pressure, and estimate the amount of carbon transferred from primary producers to larger organisms. The investigators are training community college students from Cape Cod Community College by involving them in laboratory research through summer internships. The goal is to promote science, technology, engineering and math literacy among community college students through hands-on research experiences, peer-to-peer mentoring, and professional development opportunities, while also encouraging students to transfer to a four-year university, obtain a degree in a STEM subject, and continue on in a STEM field.

Grazing by microbial eukaryotes is a significant source of mortality for microbes in the oceans, thus influencing the composition of communities and serving as a major route for remineralization of organic material to all organisms. This project is quantifying the in situ rates of eukaryotic grazing on prokaryotic communities at hot spots of primary productivity in the deep sea and characterize the diversity of microbial eukaryotes, their abundance, and metabolic activities at the seafloor. The focus of the effort is at the underwater volcano Axial Seamount, home of the Ocean Observatories Initiative (OOI) Regional Cabled Array with well-characterized low-temperature diffusely venting fluids.

The two objectives of the work are to:

1. Quantify the rates and impact of phagotrophic microbial eukaryote grazing on prokaryotic communities at the seafloor in low-temperature diffuse fluid mixing zones.

2. Characterize microbial eukaryotic diversity, abundance, and metabolic gene expression at the seafloor in low-temperature diffuse fluid mixing zones.

The modular Microbial Sampler-Submersible Incubation Device (MS-SID) is being used for in situ seafloor tracer incubations and compared to shipboard incubations for the proposed grazing studies. This combination of technology provides detailed and quantitative assessments of protistan communities and establishes the extent to which microbial eukaryotes impact primary production in the deep ocean. Expected outcomes include the quantification of protistan grazing rates on bacterial and archaeal communities within the seafloor mixing zone, comparison of predation pressure at the vent-seawater interface and surrounding deep ocean water, as well as identification of key bacteriovores, fungi, and other protists and associated major active metabolic pathways within the hydrothermal mixing zones. The project is providing a significant advance in the understanding of the microbial loop in the deep ocean as current depictions of microbial ecology at hydrothermal vent sites do not typically include the role of microbial eukaryotes.

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.