Plankton Sampling and Analysis

Plankton was sampled from Rosette Bottles and by net sampler, including 200 $\mu$m Nansen and WP2 and other model nets, that were towed or deployed vertically. Etherotrophic (bacteria) and autothrophic picoplankton, nanoplankton, microphytoplankton and microzooplankton were sampled by Rosette Bottles. Microphyto and mesozoo plankton were also sampled by nets.

Samples (10 mL) taken to estimate picoplankton abundance (Pico) were fixed with a 2% final concentration borate-buffered formalin (pre-filtered through a 0.2-$\mu$m Acrodisc filter) and stored at +4.

Picoplankton abundances will be determined using epifluorescence microscopy after staining the cells with 4,6-diamidino-2-phenylindole (DAPI) PORTER1980 at 1 $\mu$g ml$^{-1}$ final concentration.

For microzooplankton (Micro) analyses, 2 L of water were collected and preserved with 4% formaldehyde solution. Subsamples (50-100 mL) will be examined in a settling chamber using an inverted microscope (x200) according to the Uthermöl method UTHERMOL1931, LUND1958.

Hydrolytic enzyme activity was measured with fluorogenic analogs of natural substrates HOPPE1993: methyl umbelliferyl-$\beta$-D-glucoside, methyl umbelliferyl-$\alpha$-D-glucoside, methyl umbelliferyl-$\beta$-D-galactoside, methyl umbelliferyl-$\beta$-D-galactoside (MUF, $\beta$-D-galactosidase). Hydrolysis rate was measured by incubation of 2.5 mL subsamples with 800, 400, 200, 100 and 50 $\mu$m substrates. Incubation was performed in the dark at in situ temperatures for 2-3 h. Fluorescence was measured at 380 nm excitation and 440 nm emission for ubstrates using a fluorometer (Shimadzu RF 1501). Standard solutions of MUF were used to calibrate the fluorometer.

Mesozooplankton samples were collected by vertical hauls, from the bottom to the surface, using a WP2 net (0.57 m diameter, 200 $\mu$m mesh). The samples were preserved in 4% borax-buffered formaldehyde until laboratory analysis, while a "fresh" aliquot was stocked in freezer at -20. Taxonomic and quantitative zooplankton determinations will be performed using a Zeiss stereomicroscope on a representative sub-samples or, for the rare species, on the total sample. Each sample will be poured into a beaker to allow a thorough mixing for random distribution of the organisms, and aliquots of the samples will be analysed until at least 1000 individuals will be counted. Not formaldehyde-fixed subsamples, deriving from origin samples, will be used for the determination of mesozooplankton biomass. The organic carbon content will be determined with a Perkin-Elmer 2400 CHN elemental analyser.