Seawater was collected in 12-L polyvinylchloride bottles affixed to a 24-bottle rosette sampler, equipped with a Sea-Bird 911+ conductivity, temperature, and pressure profiler. Rates of size-fractionated (>3 μm and 0.2-3 um) and group-specific particulate 14C-based primary production were measured at six discrete depths (5, 25, 45, 75, 100, and 125 m) throughout the euphotic zone. Seawater samples from each depth were subsampled into triplicate 30-mL polycarbonate centrifuge tubes (Nalgene (TM) Oak Ridge) from a pre-dawn cast, inoculated under subdued light with 70 uL of NaH14CO3- (final activity = ~0.14 MBq/mL; MP Biomedicals 17441H), then incubated over the full photoperiod (~12-14 hours) in white mesh bags affixed to a floating in situ array at the corresponding depths where the water was collected. At the end of the incubation period (after sundown), each polycarbonate tube was sampled for size-fractionated and group-specific rates of 14C primary productivity. Aliquots (25 µl) were subsampled from each tube and stored in 20-mL glass scintillation vials containing 500 µL of B–phenylethylamine to determine the total activity of 14C added to each sample. Next, 5 mL of each sample was preserved in cryotubes containing 30 uL of 16% (final concentration 0.24% w/v) microscopy-grade paraformaldehyde (PFA, Alfa Aesar 43368), flash-frozen in liquid nitrogen, and stored at -80C for subsequent flow cytometric sorting. The remaining sample volume (~25 mL) was vacuum-filtered first onto a 25-mm diameter 3-μm pore size polycarbonate membrane (Millipore IsoporeTM), then the filtrate was vacuum-filtered onto a 25-mm diameter 0.2-um pore size polycarbonate membrane filter (GE Osmonics). After filtration, each filter was placed into a 20-mL glass scintillation vial and stored at -20C until analyzed back at the shore-based laboratory. Upon return to shore, vials were uncapped, 1 mL of 2 M hydrochloric acid was added to each filter, and vials were vented for at least 24 hours to remove remaining inorganic 14C. After venting, 10 mL of Ultima Gold liquid scintillation cocktail was added to each vial, and vials were placed in a liquid scintillation counter (Packard TRI-Carb 4640) for the determination of 14C activities. Group-specific rates of 14C-assimilation by Prochlorococcus, Synechococcus, and PPE were determined by measuring the amount of 14C assimilated into populations sorted using the BD InfluxTM (100-μm nozzle tip, 1X BioSure® sheath solution). Calibration of the # of cells sorted was conducted at the beginning of each sorting session; a known number of fluorescent microspherical beads (1 um, Fluoresbrite, Polysciences) were gated through the data acquisition software Spigot, sorted onto a slide, and checked for accuracy under the microscope. The ‘1.0 drop purity’ setting in the Spigot software was utilized as a conservative way of ensuring accuracy of the types of cells sorted into two 6.5-mL HDPE scintillation vials with the ‘two tube sort’ setting. Beads were included with the samples for size reference, and 0.2 x 103 to 4 x 103 beads were sorted for the determination of background levels of radioactivity (both organic 14C in the seawater and 14C absorbed to the beads). Picophytoplankton cells were triggered on forward scatter (FSC) and enumerated based on FSC and side scatter, chlorophyll-based red fluorescence (692 +- 20 nm), and phycoerythrin-based orange fluorescence (585 +- 20 nm) following excitation with two lasers, 488 nm (200 mW) and 457 nm (300 mW).
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