Table of Contents

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

We employed the virus-dilution technique to quantify lytic virus production at nine sampling sites across three distinct habitats (vent, sub-vent, non-vent) along the East Pacific Rise (9°N 50’N and 104°W) in the Tica vent area during expedition FKt230627 aboard R/V Falkor(too). The virus-dilution technique leverages the density-dependent nature of viral infection. In this approach, viruses are largely removed from the sample, and the remaining prokaryotic cells are incubated in virus-free sample water. Due to the extreme dilution of viruses in the sample, new viral infections are prevented during the incubation period. Consequently, any observed increase in viral abundance is attributable to viral infection processes that were already active prior to sample collection (Köstner et al. 2017).

Samples were collected using a suction-sampler operated onboard the remotely-operated vehicle (ROV) SuBastian. Immediately after the ROV was brought on board, a total of 4 L per sample were transferred into two 2L Nalgene flasks subsequently used for setting-up the experimental incubations by sequential filtration. First, the sample was filtered over membrane filters with a pore-size of 3 µm (Isopore, TSTP04700, Millipore) to remove larger particles. Subsequently, prokaryotic cells within the filtrate of this first filtration step were concentrated using a tangential-flow filtration device with a pore-size of 0.22 µm (Vivaflow 200, PES membrane, Sartorius) until the volume of the retentate approached ~200 mL. The retentate from this second filtration step was subjected to another tangential-flow ultrafiltration device with a molecular weight cut-off of 100 kDa (Vivaflow 200, PES membrane, Sartorius) to remove the viruses, yielding a virus-free ultrafiltrate to be used as culture medium in the experimental incubations.

For each sample, duplicate experimental incubations were prepared by mixing 3 mL of prokaryotic concentrate with 47 mL of virus-free water from the same sample, yielding a final volume of 50 mL per incubation. Immediately afterward, 1.5 mL subsamples were taken to enumerate prokaryotes and viruses (see below). Subsequently, for each duplicate incubation, five 2.5 mL glass vials were filled with the prepared dilution of prokaryotic concentrate in virus-free filtrate, tightly sealed, and placed into pressure chambers (Klose et al. 2015, one chamber per duplicate incubation). The experiments were incubated at 250 bar pressure (approximately 2500 m depth), in the dark, and at in situ temperature for 30 hours. Every ~6 hours, the chambers were depressurized to remove one glass vial per duplicate incubation to collect 1.5 mL of water for subsequent enumeration of prokaryotes and viruses (see below). Immediately after subsampling, the chambers containing the remaining glass vials were re-pressurized to 250 bar and incubated in the dark and at in situ temperature until the next subsampling or until the experiments concluded after 30 hours. To assess the impact of pressure on virus production rates, virus-dilution experiments were also conducted at surface pressure conditions at specific sampling sites.

Samples for the enumeration of prokaryotic cells and viruses were immediately fixed with glutaraldehyde at a final concentration of 0.5% at room temperature for 10 minutes, flash-frozen in liquid nitrogen, and stored at -80°C until analysis by flow cytometry (Brussaard et al. 2010). Upon thawing samples were stained with SYBR Green I for 10 minutes at room temperature for prokaryotes or at 80°C for viruses. The samples were then analyzed using a FACSAria III flow cytometer (Becton Dickinson), and prokaryotes and viruses were enumerated on cytograms of side scatter versus green fluorescence (Brussaard et al. 2010).

BCO-DMO Data Manager Processing Notes:
* Data from source file BCODMOData.xls Sheet 1 were imported into the BCO-DMO data system with values "/N" identified as missing data identifiers. This table was attached to this dataset as 936224_v1_virus-production-hydrothermal-vent.csv

** Missing data in the BCO-DMO system displays as a blank (null values) by default and will vary depending upon the file format downloaded (blank in csv files, NaN in .mat matlab files, etc).

* Parameters (column names) were renamed to comply with BCO-DMO naming conventions. See https://www.bco-dmo.org/page/bco-dmo-data-processing-conventions

* Date format converted to ISO 8601 date format
* Time format converted to ISO 8601 time format
* timestamp with timezone column added with UTC time (ISO_DateTime_UTC column) from local data and times provided as UTC-6 (local time zone specified by data provider).

* columns "prokaryotes" and "viruses" were rounded to two decimal places to match the displayed format shown in Excel.

Since the discovery of deep-sea hydrothermal vents in 1977, scientists realize that life exists above and below the seafloor. The extent to which they are interconnected, however, remains poorly understood. We propose to characterize subsurface biosphere diversity, from viruses to animals, at deep-sea hydrothermal vents to elucidate the nature and extent of connectivity between the surficial and subsurface biospheres. We will test the hypotheses that 1) eukaryotic life is also an integral component of the subseafloor biosphere; 2) the subseafloor is inhabited by both cosmopolitan and endemic protists and fungi; and 3) the subseafloor habitats harbor larvae -and perhaps adult life stages- of vent endemic animals. This research will transform our understanding of the ecology/evolution of subseafloor habitats and our thinking about animal recruitment at deep-sea vents. Consistent with SOI's mission, we will expand our knowledge of limits of eukaryotic life. During a three-week cruise with R/V Falkor and ROV SuBastian to "East Pacific Rise" vents at the 9º50'N region, we will use in situ and lab experiments, molecular identification with amplicon sequencing, Illumina NextSeq 500 technology, 2bRAD population genetic analyses, and RT qPCR for functional analyses. Our data management plan includes deposition of metadata/samples to long-term repositories. Outreach includes inquiry-learning telepresence programs for children from USA and Europe.