Hemolytic and cytotoxic activity from cultures of Aureococcus anophagefferens-a causative species of brown tides in the north-western Bohai Sea, China

Brown tides were first observed in 2009 in the north-western Bohai Sea (Qinhuangdao sea area), China, and blooms have occurred at different scales in late spring every year since then. Although the detrimental effects on marine organisms of the causative phytoplankton species Aureococcus anophagefferens have been extensively studied, the mechanism remains poorly understood. We used erythrocytes and adrenal gland chromaffin tumor cells (PC12) to explore the hemolytic activity and cytotoxicity, respectively, of chloroform and methanol extracts of cultured A. anophagefferens isolated from the north-western Bohai Sea area. The methanol extracts showed no hemolytic or cytotoxic activity. Chloroform extracts had a potent hemolytic effect on rabbit erythrocytes; thin layer chromatography (TLC) indicated that the hemolysin was a kind of glycolipid compound. Erythrocyte lysis assay showed that erythrocytes of sea bream were sensitive to the hemolysin, whereas those of human and chicken erythrocytes were insensitive. The hemolytic effects were elevated as temperatures rose from 4 °C to 37 °C. Hemolytic blocking experiments showed that sphingomyelin and d-xylose can inhibit hemolysis significantly, while osmotic protectants with different hydrated molecular diameters had no inhibition, and the hemolysins had no obvious phospholipase activity. The chloroform extracts of A. anophagefferens had significant inhibitory effects on the viability of PC12 cells, and can induce efflux of lactic dehydrogenase (LDH) of PC12 cells and lead to their necrosis.