Culturing and experimental set-up:
Phytoplankton cultures used in this study were obtained from the Roscoff Culture Collection (RCC; Vaulot et al. 2004). Host cultures of Scrippsiella acuminata (RCC 1627) were maintained in 0.2 µm sterile-filtered autoclaved seawater that was enriched with f/2 minus silica (Guillard 1975). Hosts were transferred into fresh media every 7-10 d to maintain exponential growth. Two strains of Amoebophrya sp. parasite spores (RCC 4390 and 4401) were inoculated every 2-3 d with fresh and exponentially growing host culture at a ratio of 1:1 spore to host by volume to maintain infection. Parasite and host cultures were kept at 18 °C on a 12:12 hr light: dark cycle at 80-100 µmol photon m-2 s-1. Spore cultures were spot-checked each week on a Nikon eclipse TE300 microscope at 20x magnification.
To prepare for the infection experiment, fresh parasite spores were filtered through 10 µm mesh to separate the smaller spores (2-5 µm) from any remaining host cells (Chen et al. 2021). Spores were added separately (4401 and 4390) into healthy host cultures in triplicate 1.2-L bottles at a ratio of 1:1 spore to host based on their cell densities (Long et al. 2021). Several control treatments, including spore-only, host-only, f/2 media, and Milli-Q water, were also included in triplicate. Separate infection experiments were conducted for each parasite strain (Infection_experiment column in data file), with their own set of treatments and controls (Treatment column). Bottles were sampled daily for 4 d to capture a single infection cycle.
Flow cytometry:
Samples for flow cytometry were run daily to estimate changes in host and parasite spore abundances in the infected and control treatments. Two separate runs were performed for host and spores. For the hosts, 200 µl aliquots were sampled from triplicate bottles, added to a 96-well plate, and run live on a Guava easyCyte HT (Millipore) flow cytometer at low flow rate (0.24 µl s-1) for 3 min per well. Host populations were distinguished using predefined gates that were based on plots of forward scatter and red fluorescence (692 nm). For the spores, 198 µl aliquots were added to a 96-well plate, stained with 2 µl of 100x SYBR Green (Thermo Fisher), and allowed to incubate in the dark at room temperature for 20-30 min (Kayal et al. 2021). Parasite spore populations were detected based on forward scatter (proxy for cell size) and green fluorescence (512 nm) originating from SYBR-Green stained DNA (Kayal et al. 2021).
In addition, samples (1.8 mL) from each bottle were fixed with 1% glutaraldehyde, stored at 4 °C, and run on a Sony SH800Z sorting flow cytometer (Sony) within 4-6 months to provide an estimate on the prevalence of infection (% of hosts infected). Amoebophrya sp. emit a natural green autofluorescence (Long et al. 2021), and so, we used predefined gates of the hosts using red and green fluorescence vs. forward scatter to bin infected from healthy hosts. This resulted in distinct populations of likely infected hosts that were larger and had stronger fluorescence, which may signify active intracellular infection and growth via spores. The difference in cell abundance from predefined gates were used as a conservative estimate of host infection (% infected).
DOC analysis:
Bulk DOC samples (40 mL) were filtered per bottle at each time point through pre-combusted GF/F filters and stored in HDPE plastic bottles at -20 °C until analysis. DOC samples were analyzed using high temperature combustion on a TOC-V analyzer (Shimadzu) at the University of New Hampshire (Carlson et al. 2010). Bulk DOC samples were not run for the 4390 strain infection experiment.
Organism information:
TaxonomicName, Life Science Identifier (LSID), strain_identifier, organism_type
Scrippsiella acuminata, urn:lsid:marinespecies.org:taxname:1321853, RCC 1627, host
Amoebophrya sp., urn:lsid:marinespecies.org:taxname:109448, RCC 4390 and 4401, parasite
