The A. ceratii parasite strain AT5.2 (19) was isolated from Alexandrium cells sampled from the Gulf of Maine (USA) and was used to infect the A. catenella strain Alex5; RCC3037 (formerly described as Alexandrium tamarense) isolated from the North Sea coast of Scotland. A. ceratii was cultured by infecting A. catenella (Alex5; RCC3037) and then transferred to a fresh host culture every 3 to 4 days (18). Cultures were grown at 15°C in K medium, with cool-white fluorescent lamps providing photon irradiation of 150 μmol m−2 s−1 on a 14-hour/10-hour light-dark cycle.

To obtain axenic cultures, the host A. catenella strain (Alex5; RCC3037) was grown in 500 ml of K medium in four Erlenmeyer flasks supplied with a mixture of antibiotics [ampicillin (165 μg/ml), gentamicin (33.3 μg/ml), streptomycin (100 μg/ml), chloramphenicol (1 μg/ml), and ciprofloxacin (10 μg/ml)] 1 week before the experiment. The upper phase (approximately 400 ml) was transferred to a new Erlenmeyer flask, and new K medium was added, with a final volume of 500 ml. Before infecting the experimental cultures, 1 ml of each stock culture was stained with acridine orange and checked for bacterial contamination. Infection of the host culture was performed following the procedures described by Coats and Park (18). Parasite dinospores (5 μm) were harvested from infected host cultures (A. catenella > 20 μm) on day 4 by gravity filtration through a 10-μm pore size mesh. The harvested dinospores were examined microscopically to ensure the absence of host cell contamination and additionally incubated for 24 hours to allow lysis of eventual host cells as well as degradation of host environmental DNA. Dinospores were collected via gentle centrifugation at 4°C for 10 min. The supernatant was decanted, and the resulting cell pellet was ground to a fine powder with liquid nitrogen in a precooled mortar and pestle. The ground tissue was transferred immediately to a 2-ml screw-cap tube with 2 ml of Buffer G2 [with ribonuclease A (RNase A)] and 0.1 ml of the Proteinase K (Sigma-Aldrich, Germany) stock solution, according to the manufacturer’s protocol (Qiagen Genomic DNA Handbook). DNA extraction was performed using the Blood & Cell Culture DNA Kits (Qiagen, Hilden, Germany) and Qiagen Genomic-tips. DNA quality and quantity were determined using a NanoDrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany) and a DNA Nano Chip assay on a 2100 Bioanalyzer device (Agilent Technologies, Böblingen, Germany).

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