Guillard s f 2 medium

Animal culturing was essentially done as described earlier^{76 (link)}. Macrostomum lignano (DV1 line) was originally collected from sediments of the Adriatic Sea and obtained from the Department of Zoology and Limnology, University of Innsbruck, Austria, and then reared in petri dishes, fed with Nitszchia curvilineata and cultured in Guillard’s F/2 medium (Sigma G0154). Both animal and diatom cultures were incubated at RT (20 °C). 3–4 weeks old adults and the appropriate developmental stages were used for the current studies.

The thawed dinoflagellates were centrifuged (300× g, 5 min, 25 °C) for removing the CPA. Fresh FSW was added for resuspending the samples. The samples were then transferred into a 24-well microplate (Cosmo Biosciences Inc., CA, U.S.A.) which was sealed and stored in a culture incubator set to a 12 h light:12 h dark cycle (Kansin Instruments CO., LTD.). The culture medium was prepared by mixing 32 g of Coralife Salt Water Mix (WI, U.S.A) with 1 L of distilled water to create 32 ppt artificial seawater. After the Salt Water Mix was fully dissolved, 30 mL of Guillard’s f/2 medium and 1 mL of an antibiotic solution were added to 970 mL of the artificial seawater. Both the antibiotics (penicillin: 100 units/ml; streptomycin: 10 mg/ml) and Guillard’s f/2 medium were purchased from Sigma-Aldrich (St. Louis, MO., U.S.A.). The solution was filtered using 0.45 μm filter (Thermo Scientific Inc., Massa., U.S.A.) after it had evenly dissolved. The culture was replenished with fresh medium every two days. During the medium exchange, 10 μL aliquot of the culture was retrieved for cell counts.

Leaves of Posidonia oceanica were collected in April 2017 by scuba divers, at five meters depth, in a meadow off Lacco Ameno d’Ischia (Bay of Napoli, Italy: 40°44′56″ N, 13°53′13″ E). At the collection site, the temperature was about 19 °C, pH 8.12, salinity 38 psu, at an irradiance of about 400 µE m⁻². After their arrival in the laboratory, they were reared in aerated closed-cycle tanks containing sterilized water. An inspection under a stereomicroscope permitted the detection of small portions of various epiphytes, which were promptly collected by forceps and moved into multi-well plates with Guillard’s f/2 medium (Sigma Aldrich, Milan, Italy). Their development was recorded while they were kept in a thermostatic chamber at 18 °C, with an irradiance of 200 µE m⁻², and a 12:12 hour dark/light cycle. When axenic thalli were obtained, they were further transferred every two to three days in multi-wells containing fresh f/2 medium, up to a complete isolation. The first identification of the strains was performed under a light microscope, and was based on the filament and cell shape and size, the shape of the terminal cell, the presence of calyptra, sheaths, and the type of reproduction.