Sampling and Analytical Methodology:
Experiments were conducted between July 2011 and April 2013 during five research cruises to Station ALOHA (22.75°N, 158°W), the well-characterized study site of the Hawaii Ocean Time-series (HOT) program. Sampling occurred during four HOT cruises and one Center for Microbial Oceanography: Research and Education (C-MORE) cruise (termed HOE-DYLAN 5) aboard the R/V Kilo Moana. Seawater was collected in 12 L polyvinylchloride bottles affixed to a 24-bottle rosette sampler equipped with a Sea-Bird 911+ conductivity, temperature, and depth profiler. Nine 20-L polycarbonate carboys were filled with 25 m Station ALOHA seawater pre-filtered off the rosette sampler through a Nitex mesh (pore size ~202 μm) to exclude larger zooplankton. Of these, 3 carboys received additions of nitrate (target ~2.8 μM N final concentration as NaNO3) and three carboys received additions of ammonium (target ~2.8 μM N final concentration as NH4Cl). All carboys, including three ‘Control’ carboys, received additions of phosphate (target ~0.2 μM P final concentration as KH2PO4) and silicic acid (target ~2.8 μM Si final concentration as Na2SiO3) to achieve a final N:P:Si stoichiometric ratio (14:1:14). Carboys were incubated for 120 to 144 hours and subsampled at approximately daily scales throughout the experiment (Table 1). All sampling was conducted before sunrise in order to allow productivity rate measurements to span the full photoperiod.
Seawater samples (2 mL) for photosynthetic picoeukaryote cell abundance measurements were collected for each experiment into cryotubes (Corning) containing 30 µl of 16% paraformaldehyde for a final concentration of 0.24% (w/v), kept for 15 minutes in the dark, flash-frozen in liquid nitrogen, and stored at -80°C until analyzed. Photosynthetic picoeukaryote cells were distinguished using a BD InfluxTM flow cytometer (triggered on forward scatter) with the data acquisition software Spigot. Cells were enumerated based on forward scatter, side scatter, chlorophyll-based red fluorescence (692 ± 20 nm), and phycoerythrin-based orange fluorescence (585 ± 20 nm) on two lasers, 488 nm and 457 nm. Cell counts were determined using the data analysis software FlowJo 10.0.7.
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