The Formation,Survival And Germination Of Temporary Cysts Of Scrippsiella Trochoidea And Its Response To Darlmess And 5-azacytidine

The formation of temporary cysts of Scrippsiella trochoidea,an important algal bloom species,was induced by darkness at different temperatures,and the survival and germination of temporary cysts under different conditions were studied.The purpose of this study is to understand the formation,survival and germination of temporary cysts of S.trochoidea.The responses of antioxidant systems of S.trochoidea to darkness stimulation were studied in order to discuss the mechanism of darkness stimulation on S.trochoidea.In addition,the effects of 5-azacytidine,an inhibitor of DNA methyltransferase,on the growth and antioxidant system of algal cells and the germination of temporary cysts were studied.The results of this study revealed the role of temporary cyst in the life cycle and population dynamics of S.trochoidea,elucidated the physiological and biochemical responses of the algae cells to darkness,and preliminarily revealed effects of 5-azacytidine on the growth,physio-biochemical responses,and the life cycle of S.trochoidea.The results of this study will provide scientific basis for understanding the mechanism and control of blooms of S.trochoidea.The main results of this study are as follows:?1?Temporary cysts began to form on the first day after darkness treatment and the formation rates reached the highest at the third to the fifth day,while the peaks of cysts formation delayed at normal temperature?20?,25??darkness treatments compared to the cold darkness treatment?8??.The final accumulative cyst formation rates were 15.9%,20.1%and 9.9%at 8?,20?and25?,respectively.The formation of temporary cysts did not require the fusion of gametes,and could be considered as the asexual production.The sizes of temporary cysts were similar to the vegetative cells,with smooth cyst wall without calcareous spines.?2?Temporary cysts could not survive for a long time,and cyst death occurred just after one day of formation.All cysts died after 8?16 days storage at darkness.The survival time was longer at low temperature,and the half-lethal time were 5.9 d,2.9 d and 2.7 d at 8?,20?and 25?,respectively.There was no mandatory resting period for temporary cysts,and germination occurred within 24 hours after incubation.The final maximum accumulative germination rates of43.3%?65.6%were obtained after 3?6 days incubation.The germination rate was higher for cysts formed at low temperature.The results of this study indicated that S.trochoidea was easy to form temporary cysts,which germinated rapidly under suitable environmental conditions.This study highlighted the important roles of temporary cysts in the population dynamic,and bloom occurrence and collapse of S.trochoidea.?3?The growth of S.trochoidea was significantly inhibited in darkness.Meanwhile,the contents of soluble proteins increased significantly.The content of soluble protein increased more significantly under the dark treatment at 20?,which was 2.18 times of that before treated just after 24 hours treatment,while only 1.51 times of that before treated under the dark treatment at8?.The significant increase of soluble proteins provided the material basis for algal cells to resist the darkness stresses.The activities of superoxide dismutase?SOD?in algal cells only increased a bit under darkness at 8?,which could not reduce the damage of membrane lipid,and resulted in the persistent increase of malondialdehyde?MDA?content.However,the SOD activity increased rapidly and persistently under darkness at 20?,with the highest value up to 2.39 times of that before treated.The high SOD activity protected algal cells from the damage of reactive oxygen,and thus MDA contents decreased.The contents of glutathione?GSH?increased rapidly and peaked at 48 h under darkness at 8?and 20?,which were 2.38 and 1.90 times of those before treated,respectively.Generally,GSH contents increased more significantly under darkness at 8?.The results suggested that GSH might not only help algal cells to resist the darkness stress,but also play an important role in resistance to cold stress.?4?5-azacytidine showed significant inhibition on the growth of S.trochoidea,and displayed an evident dose-response relationship.The 96h EC₅₀ value of 5-azacytidine to the growth of S.trochoidea was 146.77?mol/L?35.84 mg/L?,which belonged to the moderate toxicity level.5-azacytidine had adverse effects on the germination of the temporary cysts of S.trochoidea.The EC₅₀ value on the cyst germination was 8.08 mmol/L?1.97 g/L?,which belonged to the low toxicity level.Low concentrations of 5-azacytidine?10?100?mol/L?promoted the germination of temporary cysts at the early stage of incubation,however,the final accumulative germination rates in all exposure groups were lower than that of control group.?5?The antioxidative systems of S.trochoidea responded to 5-azacytidine rapidly and significantly.The contents of soluble proteins,GSH,MDA,and SOD activities increased significantly after exposed at different concentrations of 5-azacytidine,and showed clear dose-response relationships.The highest values of these parameters were 2?3 times of those in the control group and those before exposure.The soluble proteins and SOD activities were more sensitive to 5-azacytidine,and significant increases occurred under concentration of 10?mol/L after 24 hours exposure.While,significant increases of GSH and MDA were recorded at concentrations over 100?mol/L.The results suggested that the enhancement of antioxidant systems under the low concentrations of 5-azacytidine and during the late exposure would help the algal cells to extinguish the reactive oxygen species?ROS?,and thus reduced the contents of MDA.Furthermore,5-azacytidine can reduce the DNA methylation level of algal cells,and enhance the expression of related genes to synthesize more proteins and enzymes,therefore increase the activities and contents of various antioxidant compounds.