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).