Detrimental Impacts And Mechanisms Of The Dinoflagellate Karenia Mikimotoi In Fujian Coastal Waters On Typical Marine Organisms

In recent decades,harmful algal blooms have occurred frequently in coastal waters of China,and they have caused serious problems with significant economic,social,and human health consequences.Dinoflagellate Karenia mikimotoi is a typical HABs species.It has bloomed in large scales almost every year since 1998,which caused massive mortalities of cultured fish and shellfish,and led to huge economic losses.Blooms of K.mikimotoi were responsible for massive mortalities of abalones in Fujian coastal in 2012,with an economic loss of more than 2 billion yuan.However,little is known about the effects and mechanisms of these blooms on marine organisms.In this thesis,the toxic effects and the possible mechanisms of toxicity of K.mikimotoi from Fujian coastal waters on typical marine organisms at different trophic levels,including zooplankton?Brachionus plicatilis,Artemia salina,Neomysis awatschensis,and Calanus sinicus?and aquaculture species?Penaeus vannamei and Scophthalmus maximus?were investigated.The results of acute toxicity test showed that at a bloom density of 3×10⁴ cells/m L,the Fujian strain of K.mikimotoi significantly affected the tested organisms,which had mortality rates at 96 h of 100,23,20,97,33,and 53%,respectively.Rotifer B.plicatilis was the most sensitive species of the six test organisms to K.mikimotoi,for the survival rate of rotifer was inhibited to 57%by K.mikimotoi at a density of 30 cells/mL,and 0%at a density of 3200 cells/m L in 24 h.Those results indicate that HABs of K.mikimotoi may have negative impacts on marine organisms from different trophic levels,including wildlife and culture organisms.K.mikimotoi also affected biological behavior and body structure of marine organisms.Under the microscopic observation,the rotifers B.plicatilis had an immediate avoidance response to K.mikimotoi,and gradually lose their swimming ability in several minutes.After a 1 h treatment,we found that the cilia of rotifers stopped swing,and the body of rotifers dehydrated and shrank significantly.Several vesicles and ulcerations were also observed.Scanning electron microscopy observations also found out that rotifers affected by K.mikimotoi showed significant body shrinkage,and their cilia lose significantly compared to the control.The enzymes activities were significantly affected by K.mikimotoi.Results showed that algae significantly inhibited the activities of esterase,acetylcholinesterase,total ATPase,and Na⁺-K⁺-ATPase at densities of 30,30,300,1000 cells/m L,respectively.After 3 h K.mikimotoi treatment at a density of 1000 cells/mL,their activities,compared to the controls,decreased 19.1,34.0,17.7,31.4%,respectively.Results indicate that K.mikimotoi can affect energy metabolism and substance transport in organisms by inhibiting their enzymes activities.In terms of the toxic mechanism of K.mikimotoi,it was found that the toxicity of K.mikimotoi to turbot S.maximus increased under non-aeration conditions,but we also found that dissolved oxygen in the water remained at high levels?5.53,6.41 ppm in algae treatment at densities of 1×10⁴,3×10⁴ cells/m L?.Combined that turbot was able to tolerate 2.15 ppm of hypoxic conditions in blank,we believe that the toxicity of the algae is the main reason for fish mortalities,although hypoxia might contribute to it.In this study,the Fujian strain of K.mikimotoi was tested for its ability to generate reactive oxygen species,including·O₂^-and H₂O₂,and their concentrations were 0.014±0.004 OD/?10⁴ cells?and 3.00±0.00 nmol/?10⁴ cells?,respectively.However,reactive oxygen species at that concentrations cannot harm rotifers,and there were no significant changes in the mortality rate of rotifers when reactive oxygen species were eliminated by enzymes.These results suggest that ROS are not the main mechanism of toxicity of K.mikimotoi.Lipophilic extracts,with hemolytic toxicity and cytotoxicity,from K.mikimotoi was tested as nontoxic to marine organisms at a bloom density of 3×10⁴ cells/mL.It is found that lipophilic extracts only affect the survival of rotifers at much higher densities than the bloom density of K.mikimotoi.Those results indicate that the toxins produced by K.mikimotoi may not directly affect marine organisms.We also extracted algae in different ways with different mediums,and found that K.mikimotoi may produce more toxins,which had high activities and decomposed easily during extracting.Further investigation on the toxicity mechanism of K.mikimotoi had revealed that the toxicity of this species was associated with intact living algal cells,and it also associated the close contact of algal cells.The cell-free culture supernatant showed nontoxic to all test organisms,and toxicity also disappeared when algal cells were ruptured or separated with organisms by semipermeable membrane.We also found toxicity of the intact cell suspension significantly decreased compared with unfiltered cell culture,but gradually recovered over time.This result suggests that the toxic effect occurs on or near the algal cell membrane.This Fujian strain of K.mikimotoi was proved to be toxic to all test species,including zooplankton and aquaculture species varying in size and feeding habit at or below the bloom density of 3×10⁴ cells/m L,these results indicate that HABs caused by the Fujian strain of K.mikimotoi can have detrimental effects on local ecology and lead to huge finical losses to the marine aquaculture?shrimp,fish and shellfish?industry along the Fujian coast.K.mikimotoi may affect marine organisms by contact,and was associated with intact living algal cells,while hypoxia might contribute to organisms'mortalities,especially in still water.Neither reactive oxygen species nor the hemolytic lipophilic extract had a lethal effect on the test organisms at a bloom density.K.mikimotoi may produce more toxins with high activities,and toxic effect may occur on or near the algal cell membrane.