Study On The Toxin Production And Environmental Response Characteristics Of Gymnodinium Catenatum In The Coastal Waters Of Fujian,China

The chain-forming dinoflagellate Gymnodinium catenatum is the only known gymnodinioid dinoflagellate that produces paralytic shellfish toxins which can restrain nerve conduction by blocking Na⁺conductance and cause poisoning,even death.Dense blooms caused by the dinoflagellate have been frequently reported in coastal waters of China,and G.catenatum has become a common red tide organism which poses a great threat to coastal ecological health and food safety.The growth and toxicity characteristics of G.catenatum showed higher intraspecific variability（strain differences）,so as to its response to environmental factors.Limited studies have been conducted to explore the physiological responses of this strain to major environmental factors.Thus,the growth,enzyme activity,and toxin production were investigated in batch cultures of G.catenatum under different environmental conditions in the present study.The molecular mechanism of toxin production was furtherly explored with RNA-seq.The main results are listed as follows:（1）The G.catenatum（strain TIO523）isolated from coastal waters of Fujian shared similar LSU and ITS sequences with other strains and derived from different geographical regions clustered into a clade in the phylogenetic tree.The 5^th base in the5.8S rDNA gene of the strain was cytosine,indicating it belonged to the C-gene ribotype.The equivalent cellular toxicity of this strain was 12.26±0.10 pg STX eq/cell.The toxin profile of G.catenatum was consisted of C1,C2,dcneoSTX,and GTX3-5,with C1/2 as the dominant derivatives.Toxicological experiments showed that G.catenatum exhibited acute toxicity to Penaeus vannamei.The survival rate of P.vannamei decreased with the increase of G.catenatum density,as well as the time of significant inhibition of survival rate.However,G.catenatum exhibited no acute toxicity to Brachionus plicatilis,Artemia salina,Neomysis awatschensis,Scophthalmus maximus.（2）The light intensity,temperature,and salinity showed a significant influence on the growth of G.catenatum.The maximum cell density increased with irradiance in the range of 0-70μmol/（m² s）and no growth was observed without irradiance.Low temperature inhibited the growth of G.catenatum,and the optimal temperature was 20℃.The optimal salinity range for growth was 25-35.The toxin profile remained constant in all the environmental conditions.Low temperature and low salinity promoted the production of toxins.Inappropriate temperature（over 30℃ or under 15℃）can lead to the decrease of C1 relative content and the increase of C2relative content.Similarly,low salinity（20）and high salinity（40）can also lead to a decrease of C1 relative content.Furthermore,the light intensity has no effect on the toxin production of Gymnodinium catenatum.（3）Gymnodinium catenatum grew well on multiple N substrates within appropriate ranges of concentrations.The dinoflagellate showed the greatest affinity for NH₄⁺,while high concentrations of NaNO₂,NH₄Cl and urea had toxic effects on cells.The strain was also able to grow well on multiple P substrates with the lowest capacity for utilizing ATP.Nitrate reductase was structurally expressed with low activity.Glutamine synthetase and urease were induced by their substrate ammonium and urea,respectively.The alkaline phosphatase activity was induced to hydrolyze organic phosphorus to meet its phosphorus requirements.Nitrate-repletion and phosphate-depletion conditions enhanced the cellular toxicity.The toxin profile remained constant in different nitrogen concentraitons,while the ratio of C2:C1 toxins decreased in low phosphorus concentration conditions.（4）Using transcriptome technology,we investigated the effects of different nitrogen concentrations（25,100,800μmol N/L）and temperatures（15,20,30℃）on the transcriptional expression of intracellular toxigenic related cellular processes.Compared with the 100μmol N/L nitrogen treatment group,the genes related to the photosynthetic pathway in the algae cells were significantly down-regulated,while the trend of celluar toxicity was opposite.The expression of G.catenatum also showed a similar pattern under low temperature conditions（15℃）.However,the expression of the metK involved in the catalytic synthesis of S-adenosylmethionine was significantly down-regulated（p<0.01）under high temperature conditions,indicating that high temperature can inhibit the expression of the metK gene,thereby limiting the synthesis of S-adenosylmethionine.Furthermore,high temperature also inhibited the expression of met3 gene involved in the synthesis of 3’-phosphoadenosine-5’-phosphosulfate（PAPS）,thus limiting sulfate assimilation,which together resulted in decreased toxicity.Under high nitrogen conditions,the expression of met3 and cysC genes involved in the synthesis of PAPS was upregulated,while the expression of sal genes involved in the decomposition of PAPS was downregulated,indicating that high nitrogen promotes the accumulation of PAPS in cells,leading to increased cell toxicity.The expression of toxin-producing related genes,sxt A and sxtG,remained stable in different nitrogen concentration or temperature.In this study,the growth,enzyme activity and toxin production characteristics of G.catenatum were investigated in various environmental conditions,which provided a comprehensive analysis of toxicological effects of G.catenatum in offshore China.Moreover,the molecular mechanisms related to toxin production caused was also explored.The results will provide important references for the exploration of environmental regulation mechanism in G.catenatum blooms occurrence.