12 well tissue culture plates

All C. reinhardtii cell lines used in this research were made from the parental line TN72 [40 (link)], which is a cw15 (i.e. cell wall deficient) mutant with an aadA spectinomycin resistance cassette replacing part of psbH in the chloroplast genome. TN72 is available from the Chlamydomonas Resource Center (http://www.chlamycollection.org) as strain CC-5168. Transformation of TN72 was performed using the glass bead vortexing method [40 (link)] and a plasmid containing an intact copy of psbH in addition to the genes of interest, resulting in markerless phototrophic transformants whose homoplasmicity was confirmed by PCR as described previously [35 (link)]. DNA sequencing (Source BioScience, Nottingham, UK) was used to confirm that the introduced genetic elements were intact. C. reinhardtii was grown in Tris-acetate phosphate (TAP) medium [60 ], with 2% agar (Fisher Bioreagents, New Hampshire, USA) added for solid plates. For selection of phototrophic transformants, high-salt minimal (HSM) medium with 2% agar was used [60 ]. Liquid cultures were grown in glass flasks or in clear plastic 12 well tissue culture plates (VWR, Pennsylvania, USA). For some experiments (see Additional file 1: Table S2), two parallel Algem photobioreactors were used (Algenuity, Stewartby, UK).

Adult N. vectensis were maintained in glass bowls containing 0.2 μm filtered 11ppt saltwater in the dark, at room temperature. Full strength saltwater was sourced from Biscayne Bay of Miami, FL, USA and diluted using reverse osmosis fresh water to bring the final concentration to 11ppt. Animals were fed 5 days per week with freshly hatched Artemia (Utah Sea). 50% water changes were done 3 times a week^{33 (link)}. In preparation for cell culture, animals were removed from bowls and rinsed three times with “anemone gentamicin medium” (AGM) which consists of sterile 11 ppt saltwater supplemented with 10 μg/ml Gentamicin Reagent Solution (Gibco by Life Technologies)^{31 (link)} (Table S1). For 3–7 days, individual anemones were isolated in AGM with daily media changes and starved. Each animal was then individually rinsed in a 2.5 μg/ml Penicillin/Streptomycin/Amphotericin B solution (PSAb) (Sigma) in 0.2 μm-filtered 11ppt saltwater and incubated at room temperature in 2.5 μg/ml PSAb for 10 min. Animals were then transferred into individual sterile 12-well tissue culture plates (VWR, Radnor, Pennsylvania) with selected media detailed below.