The lysate was centrifuged at 12 000 × g for 10 min at 4 °C, after which the supernatant was withdrawn and stored at − 20 °C until use. The methanol extract was evaporated to dryness, and the dried extract dissolved in selleck chemicals llc an aliquot of filtered sea water. Bloom extracts, culture extracts and the medium of batch cultures (extracellular exudates) were diluted with sterilized sea water to give a dilution series of 1, 2, 3, 5, 10, 20, 50 and 100%. Sterilized sea water was used as the control. 500 μl of each

dilution was added to a 5 ml culture tube containing 25 nauplii of 48 h-hatched cysts of A. salina. The tubes were incubated at 20 °C under a continuous light flux of 90 μmol photons m− 2 s− 1. After 48 h, the percentage mortality of nauplii was calculated compared to controls. The LC50 value was determined by probit analysis ( Finney 1963). Haemolytic activity was

tested by erythrocyte lysis assay (ELA) according to Eschbach et al. (2001) and its modification by Ling & Trick (2010). ELA was carried out on bloom samples, on algal cells and on extracellular exudates of exponentially growing cultures (6 days after inoculation) of H. akashiwo. An aliquot with a known number Ibrutinib of Heterosigma cells was centrifuged (6000 × g for 10 min at 4 °C), and the supernatant containing extracellular exudates following filtration through a 0.45 μm pore size GF/C filter was collected. Algal samples were prepared following the protocols of Eschbach et al. (2001), modified Isoconazole by Ling & Trick (2010). The cells of bloom samples (10 ml) and pellets of centrifuged cultures were ruptured in ELA buffer, prepared as

described by Eschbach et al. (2001) (150 mM NaCl, 3.2 mM KCl, 1.25 mM MgSO4, 3.75 mM CaCl2 and 12.2 mM TRIS base; pH adjusted to 7.4 with HCl) by sonication for 60 s at 20 °C in a bath-type sonicator. Complete cell rupture was confirmed by microscopic observation. Ultrasonicated algal samples and supernatants were kept in the freezer until use. The dry methanol extract of H. akashiwo cells prepared for the Artemia salina assay was re-dissolved in ELA buffer before use in ELA. Blood freshly collected from a rabbit was immediately mixed with 0.1 ml 10% sodium citrate to prevent it from coagulating. For the ELA, erythrocytes were harvested from the blood by centrifugation in a 1.5 ml microcentrifuge tube at 1500 × g for 5 min at 4 °C. The pelleted erythrocytes were washed twice with ELA buffer by vortexing and centrifugation at 1500 × g for 5 min at 4 °C. Erythrocyte suspensions were adjusted to the appropriate cell density (5 × 106) in ELA buffer with a haemocytometer. The ELA method was basically that of Eschbach et al. (2001) with modifications by Ling & Trick (2010). Briefly, 0.5 ml of erythrocyte suspension and 0.5 ml of cell extract or extracellular exudates of H. akashiwo were added to 1.