Dataset: oxidative stress TEP
Deployment: lab_Thornton

Growth of the phytoplankton
The diatom Thalassiosira wessiflogii (CCMP 1051) and the cyanobacterium Synechococcus elongates_cf (CCMP 1379) were obtained from the National Center for Culture of Marine Algae and Microbiota (NCMA). Replicated (n = 3) Batch cultures were grown in artificial seawater (Berges et al. 2001) containing nitrogen, phosphorus and silicon at 400 µM (as NaNO3), 25 µM (NaH2PO4), and 400 µM (Na2SiO3), respectively. Culture temperatures were maintained at 20 ± 1 °C. Photon flux density on the surface of the culture bottles was 40 to 45 µmol m-2 s-1 on a 14 hour light: 10 hour dark cycle. During exponential growth, each culture was split into three treatments in which oxidative stress was induced by the addition of hydrogen peroxide at final concentrations of 0 (control), 10 and 100 µM H2O2. The treatments were sampled once a day over the next three days.

Measures of phytoplankton abundance and biomass
Counts of 400 cells from each culture were made using hemocytometers (Guillard and Sieracki 2005) from samples preserved in Lugol’s iodine (Parsons et al. 1984) using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Turbidity of the cultures, used as an indicator of growth, was measured by absorbance at 750 nm in a 1 cm path cuvette using a UV-Mini 1240 spectrophotometer (Shimadzu Corporation).

Chlorophyll a concentration 90% acetone extractions from biomass retained on GF/C (Whatman) were measured using a Turner Designs 700 fluorometer, which was calibrated using chlorophyll a standards (Sigma) (Arar and Collins 1997). The extract was diluted with 90% acetone if the chl a concentration were too high.

Cell permeability
Uptake and staining with the membrane-impermeable SYTOX Green (Invitrogen) was used to determine what proportion of the diatom population had permeable cell membranes (Veldhuis et al. 2001, Franklin et al. 2012). Four hundred cells were examined using an epifluorescence microscope (Axioplan 2, Carl Zeiss MicroImaging) and the number of cells that stained with SYTOX Green was enumerated.

TEP staining and analysis
Transparent exopolymer particles (TEP) were sampled according to Alldredge et al. (1993) and TEP abundance was enumerated by image analysis (Logan et al. 1994, Engel 2009). Ten photomicrographs were taken of each slide using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Images were analyzed using ImageJ software (National Institutes of Health) based on the method of Engel (2009). Thresholding during image processing was done using the triangle method (Zack et al. 1977).

CSP staining and analysis
Coomassie staining particles (CSP) were sampled according to Long and Azam et al. (1996) and CSP abundance was enumerated by image analysis (Logan et al. 1994, Engel 2009). Ten photomicrographs were taken of each slide using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Images were analyzed using ImageJ software (National Institutes of Health) based on the method of Engel (2009). Thresholding during image processing was done using the triangle method (Zack et al. 1977).

Quantum yield of photosystem II
The quantum yield of photosystem II was used as an indicator of phytoplankton health and measured using the saturating pulse method (Genty et al. 1989) using a pulse amplitude modulated fluorometer (PAM-210, Heinz Walz GmbH) folowing a protocol based on Marwood et al. (1999).

Caspase-like activity
Caspase-like activity was measured based on the method of Bouchard & Purdie (2011). Phytoplankton were collected by centrifugation, then lysed in a buffer, and the caspase-3 like activity was measured in the extracted proteins using a Enzcheck Caspase-3 Assay Kit #1 (Invitrogen inc.). The fluorescent product was measured by fluorescence using a microplate reader (SPECTRAmax GeminiEM, Molecular Devices).

References cited
Alldredge, A. L., Passow, U. & Logan B. E. 1993. The abundance and significance of a class of large, transparent organic particles in the ocean. Deep-Sea Res. Oceanogr., I. 40: 1131-1140. doi:10.1016/0967-0637(93)90129-Q

Arar, E. J. & Collins, G. B. 1997. Method 445.0. In Vitro Determination of Chlorophyll a and Pheophytin a in Marine and Freshwater Algae by Fluorescence U.S. Environmental Protection Agency, Cincinnati, Ohio.

Berges, J. A., Franklin D. J. & Harrison, P. J. 2001. Evolution of an artificial seawater medium: Improvements in enriched seawater, artificial water over the last two decades. J. Phycol. 37:1138-1145. doi:10.1046/j.1529-8817.2001.01052.x

Bouchard, J. N., Purdie, D. A. 2011. Effect of elevated temperature, darkness, and hydrogen peroxide treatment on oxidative stress and cell death in the bloom-forming toxic cyanobacterium Microcystis aeruginosa. J. Phycol., 47(6), 1316-1325. doi:10.1111/j.1529-8817.2011.01074.x

Engel, A. 2009. Determination of Marine Gel Particles. In Wurl, O. [Ed.] Practical Guidelines for the Analysis of Seawater. CRC Press, Taylor & Francis Group, Boca Raton, Florida, pp.125-142.

Franklin, D. J., Airs, R. L., Fernandes, M., Bell, T. G., Bongaerts, R. J., Berges, J. A. & Malin, G. 2012. Identification of senescence and death in Emiliania huxleyi and Thalassiosira pseudonana: Cell staining, chlorophyll alterations, and dimethylsulfoniopropionate (DMSP) metabolism. Limnol. Oceanogr. 57: 305–317. doi:10.4319/lo.2012.57.1.0305

Genty, B., Briantais, J. M., Baker N. R. 1989. The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence, Biochimica et Biophysica Acta, 990(1), 87-92. doi:10.1016/S0304-4165(89)80016-9

Guillard, R. R. L. & Sieracki, M. S. 2005. Counting cells in cultures with the light microscope. In Andersen R. A. [Ed.] Algal Culturing Techniques. Elsevier Academic Press, Burlington, MA, pp. 239-252.

Logan, B. E., Grossart, H. P. & Simon, M. 1994. Direct observation of phytoplankton, TEP and aggregates on polycarbonate filters using brightfield microscopy. J. Plankton Res.16: 1811-1815.doi:10.1093/plankt/16.12.1811

Marwood, C. A., Smith, R. E. H., Soloman, K. R., Charlton, M. N., Greenberg, B. M. 1999. Intact and photomodified polycyclic aromatic hydrocarbons inhibit photosynthesis in natural assemblages of Lake Erie phytoplankton exposed to solar radiation. Ecotox Environ Safe 44:322-327. doi:10.1006/eesa.1999.1840

Parsons, T. R., Maita, Y. & Lalli, C. M. 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, Oxford, UK.

Passow, U. & Alldredge, A. L. 1995. A dye-binding assay for the spectrophotometric measurement of transparent exopolymer particles (TEP). Limnol. Oceanogr. 40: 1326-1335. doi:10.4319/lo.1995.40.7.1326

Veldhuis, M. J. W., Kraay, G. W. & Timmermans, K. R. 2001. Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth. Eur. J. Phycol. 36: 167–177. doi:10.1080/09670260110001735318

Zack, G. W., Rogers, W.E., Latt S. A. 1977. Automatic-measurement of sister chromatid exchange frequency, J. Histochem. Cytochem., 25(7), 741-753. doi:10.1177/25.7.70454