Toxic Characteristics Of The Dinoflagellate Akashiwo Sanguine And Purification Of Its Toxins

Globally,blooms of harmful dinoflagellate Akashiwo sanguinea were frequent,resulting in massive mortality of aquatic organisms in coastal waters and significant economic losses.Our previous studies showed that A.sanguinea had different degrees of toxic effects on a variety of aquatic animals,and had significant allelopathic effects on a variety of phytoplankton species.At present,the toxic mechanisms and toxins composition of A.sanguinea are not clear.In this paper,the toxic characteristics of A.sanguinea were studied,the extraction and separation conditions of the toxins were optimized,and the physicochemical properties of the toxins were preliminarily discussed.The main results are as follows:(1)The crude extracts of A.sanguinea cells in different growth stages had significant hemolytic activity,and the hemolytic activity in the lag phase was significantly lower than that of other growth stages.No hemolytic toxicity was detected in the culture filtrate.The results showed that the A.sanguinea toxin was mainly stored in the algal cells,and the toxin was continuously produced and accumulated with the growth of algal cells.The hemolytic activity of the crude extracts of the algae cells of A.sanguinea JX13 and JX14 on rabbit erythrocytes and the inhibitory effect on Rhodomonas salina increased synchronously with the increase of the nutrient concentration.Existance of A.sanguinea affected the community structure of phytoplankton in natural seawater,and its toxic effects were species-specific.(2)Acetone,ethyl acetate and Methanol extracts from A.sanguinea cells were all hemolytic toxic to rabbit erythrocytes with decreasing hemolytic activity.The result showed that acetone was an effective solvent for the extraction of A.sanguinea toxins.Among the 5components obtained after SPE separation,60% and 80% of the acetone-eluted components showed strong hemolytic toxicity to rabbit erythrocytes,while the hemolytic activity of the other 3 components was very low or undetected.These results indicated that the hemolytic toxins of A.sanguinea were mainly low polar compounds.In addition,all the five fractions eluted by SPE had inhibitory effects on R.salina,but the inhibitory effects of 60% and 80%fractions on R.salina were significantly higher than that of 20% and 40% fractions.These results indicated that allelopathic compouds of A.sanguinea are compounds with different polarity and the main allelopathic compouds may be low polarity.(3)The extraction method and toxicity detection method of A.sanguinea toxin were optimized.It was confirmed that nitrogen drying did not significantly affect the hemolytic activity of A.sanguinea toxins.Compared with dimethyl sulfoxide(DMSO),ethanol was a better resoluble reagent.It was found that high irradiance may destroy the structure of A.sanguinea toxins.(4)The toxins of A.sanguinea were thermally unstable.Hemolytic activity of the crude extract was under the detection limit after being treated in 100 °C water bath for 3 h,but only decreased by 14% after being stored at-20 °C for 120 h.Dark and frozen(-20 °C)preserve of crude extracts for 30 days resulted in 50% loss in hemolytic activity,indicating that the hemolytic toxins of A.sanguinea were unstable.In addition,the UV-Vis spectra analysis of toxic components of A.sanguinea suggested that the hemolytic toxin of A.sanguinea might have a structure similar to porphyrin.