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.
Rates of size-fractionated (0.2-3 μm and >3 μm) primary production were assessed based on the assimilation of 14C-bicarbonate into particulate organic matter. Seawater was collected into 30 mL polycarbonate centrifuge tubes (Nalgene™ Oak Ridge) before sunrise, spiked under subdued light with 70 µL of NaH14CO3- (MP Biomedicals 17441H, stock concentration 2 mCi ml-1) to a final activity of ~0.14 MBq NaH14CO3- mL-1. The tubes were placed in white mesh bags in the same incubator as the experiment carboys over the full photoperiod (~12-14 hours). After sundown, 25 µL aliquots from each sample were collected and stored in 20 mL glass scintillation vials containing 500 µL of β–phenylethylamine to determine the total activity of 14C added to each sample. The remaining sample volume (~25 mL) was serially vacuum-filtered, first onto 25 mm diameter, 3 μm pore size polycarbonate membranes, then onto 25 mm diameter, 0.2 μm pore size membranes. After filtration, each filter was placed in 20 mL glass scintillation vials, to which 1 mL of 2 M hydrochloric acid was added and vented for at least 24 hours to remove adsorbed 14C-bicarbonate. Ten mL of Ultima Gold liquid scintillation cocktail was then added to each vial and placed in a liquid scintillation counter for the determination of 14C activities.
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.
For photosynthetic pigment analyses using high performance liquid chromatography (HPLC), seawater (2 L) was collected into brown, narrow-mouthed HDPE bottles and subsequently filtered using a peristaltic pump onto 25 mm diameter, GF/F filters. Filters were immediately flash-frozen in liquid nitrogen and stored at -80°C until analyzed. Photosynthetic pigments were extracted from the filters in 3 mL 100% acetone (HPLC grade) in culture tubes along with 50 µL canthaxanthin, an internal standard, and placed at 4oC for 24 hours. Chlorophyll and carotenoid pigments were separated on a Varian 9012 HPLC system and analyzed using SpectraSYSTEM Thermo Separation Products dual wavelength UV/VIS UV2000 and fluorescence FL2000 detectors. Pigment identifications were based on absorbance spectra, co-chromatography with standards, and relative retention time with a monovinyl Chl a standard and representative culture extracts, and Spectra-Physics WOW® software was used to calculate peak area.
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