Flow cytometry analyses

Duplicate 1.8-ml samples were fixed with paraformaldehyde (electron microscopy grade, 0.25% final concentration) for 15–20 min in the dark at room temperature, flash frozen in liquid nitrogen and preserved at -80°C until analysis. The sheath fluid consisted in a sodium chloride solution filtered in-line through a 0.22 µm Sterivex filter unit. Reference beads (Fluoresbrite, YG, 1-µm) were added to each sample to maintain proper alignment and focus of the instrument. Prochlorococcus, Synechococcus and pigmented protists (P-Protists) were discriminated in unstained samples based on their chlorophyll (red) fluorescence and forward scatter (FSC, size) signatures. The high phycoerythrin (orange) signal in Synechococcus was used to distinguish them from Prochlorococcus and P-Protists. Using a forward scatter detector with small particle option and focusing a 488 plus a 457 nm (200 and 300 mW solid state, respectively) laser into the same pinhole permitted the resolution of dim surface Prochlorococcus group from background noise (Duhamel et al., 2014). Additionally, non-pigmented protists (NP-Protists) were discriminated from P-Protists in SYBR Green stained samples using a chlorophyll (red) vs SYBR Green (green) fluorescence plot, as described in Christaki et al. (2011) and Bock et al. (2018).

Microscopy

Ten-ml aliquots of the formalin-fixed plankton samples were centrifuged to sediment the plankton, and the overlying supernatant removed leaving 0.5 to 1 ml. The sedimented pellet was gently resuspended in the remaining supernatant and stained with Lugol’s iodine. Samples of the thoroughly suspended stained sample (20 µl) were taken with a micropipette and deposited as a droplet in the base of an Uttermohl viewing chamber and examined using an inverted compound microscope with phase contrast optics. The contents of each 20-µl droplet was scanned field-by-field exhaustively at 400x, and each individual microplankton observed was measured in the micron range, using an ocular reticule, and assigned to a broad taxonomic morphogroup (e.g., coccolithophore, diatom, dinoflatellate). Examples of individuals within each morphogroup were photographed using a digital camera, and representative images of each morphogroup were assembled in a plate of micrographs illustrating the range of microplankton that were observed.

Alkaline phosphatase activity

Alkaline phosphatase activity (APA) was measured following a modification of the Dyhrman and Ruttenberg (2006) protocol. Seawater samples were collected onto two separate filters with 0.2- and 0.8-µm porosities, chosen to tease apart activity by the bacterioplankton (i.e. bacteria and cyanobacteria) and the protists enriched fractions (0.2–0. 8 μm and >0.8 µm, respectively). Filters were stored frozen at –20°C until analysis (<1 month). Samples were processed using the fluorogenic phosphatase substrate 4-methylumbelliferyl phosphate (MUF-P, Sigma-Aldrich) at saturating concentration (10 μmol l–1, Casey et al. (2009)). Fluorescence was measured at several time points within the linear range of the assay. A standard curve using 4-methylumbelliferone (MUF, Sigma-Aldrich) was used to calculate MUF-P hydrolysis rates (Duhamel et al 2011).