Dinoflagellate culturing: Alexandrium fundyense (strain CCMP 1911) was obtained from the National Center for Marine Algae and Microbiota (NCMA). The strain was originally isolated from Sequim Bay, Washington, USA. Heterocapsa rotundata (strain K-0483), obtained from the Scandanavian Culture Collection of Algae and Protozoa, was isolated from the southern Kattegat Sea near Denmark. Dinoflagellate cultures were maintained in f/2 medium without added silicate at 15°C under a 12L:12D light cycle and transferred every two to three weeks into new media. Growth irradiance for A. fundyense was 53 µmol photons m-2 s-1; growth irradiance for H. rotundata was 12 µmol photons m-2 s-1.
Cell concentration and cell volume: Cell concentration (cells mL-1) and cell volume (µm3 cell-1) estimates for H. rotundata were obtained from live samples measured with a Beckman Coulter Z2 Particle Count and Size Analyzer with Z2 AccuComp software. For A. fundyense, samples were preserved in acid Lugol’s solution (final concentration 2%). Cells were counted in a 1 ml Sedgewick Rafter chamber using light microscopy; cell volume was estimated from length and width of cells (n = 42 or 83 cells per sample) measured with Leica Application Suite X image analysis software and assuming cell shape was approximated by an oblate ellipsoid.
Chlorophyll-a: Chl-a concentrations were measured by filtering samples through 0.7 µm effective pore size 25 mm glass fiber filters. Pigments were then extracted in 90% acetone for 24 h (dark, -20°C) and fluorescence was measured on a Turner 10-AU fluorometer using the acidification method. Concentrations are reported per cell and per unit cell volume.
Dissolved and particulate dimethylsulfoniopropionate (DMSP): DMSP samples (4 ml) were gravity-filtered through precombusted 0.7 µm effective pore size 25 mm glass fiber filters so as not to rupture the cells. For measurement of particulate (intracellular) DMSP (DMSPp), filters were placed into sealed 20-ml glass vials containing 3 ml of 5 N NaOH. For measurement of DMSP in the dissolved (extracellular) phase (DMSPd), the first 4.5 mL of each sample’s filtrate were caught in a 5 ml polystyrene culture tube, which was capped and stored at -80°C. Later, DMSPd samples were thawed and sparged with N2 gas for 1 min to remove any dimethyl sulfide (DMS) already present. Each sparged sample (4 ml) was then dispensed into a 20-ml vial containing 1 ml of 5 N NaOH, and sealed. Upon being sealed, prepared vials for either analysis were allowed to equilibrate for at least 24 h, allowing for the 1:1 conversion of DMSP to gaseous DMS, detectable via gas chromatography. Standards for DMSPp were prepared from pre-diluted DMSP solutions at the same time that samples were filtered and sealed into vials, while DMSPd standards were made at the same time that samples were sparged and sealed into vials. Samples and standards were analyzed using a Shimadzu Gas Chromatograph 14-A equipped with a flame photometric detector and a Supelco packed Chromosil 330 column. The chromatograph was operated isothermally at 90°C with flow rates of hydrogen, air, and helium (carrier gas) at 50, 60, and 150 kPa, respectively. DMSPp samples and standards were measured via direct injection while DMSPd samples and standards were measured with a headspace sweep (He flow rate 40 kPa). DMSPp concentrations are reported per cell and per unit cell volume. DMSPd concentrations are reported per cell and per unit seawater volume.
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