Study On Toxigenic Physiology And Toxin Preparation Of Diarrhetic Shellfish Toxins Produced By Harmful Benthic Dinoflagellate Prorocentrum Lima

Diarrhetic Shellfish Toxins?DSTs?belonged to marine shellfish toxins are widely distributed in coastal areas around the world,including okadaic acid?OA?,dinophysistoxins?DTXs?and other form toxins.Large amounts of these toxins are mainly produced by dinoflagellates from the genera Prorocentrum and Dinophysis.OA and DTXs are responsible for intoxication episodes breakout,leading to disorder syndrome?vomiting,diarrhea,and abdominal pains?in human consumers of contaminated shellfish.Marine ecological environment and seafood products security are confronted with potential hazardous under the influence of other toxins released to marine environment.The DSTs-producing dinoflagellate Prorocentrum lima are widespread all over the world.They have an effected on marine ecological environment and seafood products security in China.In this study,P.lima located from Wenchang,Hainan,China?N 19°37?57??,E110°58?32???was taken as research object.A high resolution mass spectrum?HRMS?method was successfully established for screening and identifying OA,DTX-1 and their derivatives by optimizing pretreatment method and mass spectrometer parameters.Diversified parameters of P.lima in different regions were collected by combining HRMS with high-performance liquid chromatography,molecular biology methods and molecular network technology.Furthermore,the effects of temperature and nitrogen and phosphorus limitation on the toxicity and transformation characteristics of free form toxins and esterified toxins in P.lima were investigated.In addition,preparation method were determined for extraction,separation and purification of OA,DTX-1 and 35S DTX-1.The mainly content of paper are as follows.?1?A high resolution mass spectrum?HRMS?method was successfully established for screening and identifying free form and esterified toxins of DSTs by optimizing pretreatment method and mass spectrometer parameters.DSTs extracted by 80%methanol in P.lima were diluted 2.50 folds before purifying by strata^TM-X solid-phase extraction,and they were detected on the condition of full-MS/dd-MS² positive ion scan mode,step collision energy?10/30 e V?.The obtained results revealed that the linear relationship of OA and DTX-1 was good,the correlation coefficient was?0.999.And the recovery rate of single spiked OA was between 74.0%and 147%.Three free toxin?OA,DTX-1 and 35S DTX-1?and four diol esters?OA-D9b,OA-D8a,DTX-1-D6 and DTX-1 D9b?were discovered by applying the described HRMS method.The standard deviation of mass accuracy of seven toxins were all less than 2.92 ppm.OA per cell concentrations varied from 5.73 to 32.5 pg/cell,the toxin content of DTX-1 and 35S DTX-1 less than1.00 pg/cell at different growth phases.Moreover,OA-D9b was the major diol-ester toxin,accounting for 68.3%on total toxin content.Therefore,the proposed HRMS method has excellent practicality and availability for simultaneous screening and identifying OA,DTX-1 and derivatives,and it can provide a technical basis for comprehensively and accurately evaluate potential risks of DSTs in P.lima.?2?The fingerprint database of three P.lima in different regions were established.DSTs and characteristic compounds of P.lima were screening and identifying by using HRMS and molecular network technology,and pigments composition and gene sequence were also collected by assistanting LC-MS with molecular biology methods.The results showed that a total of 519 characteristic compounds were screened in our study.It was verified that 32 main characteristic compounds and 15 differentially esterified toxins.The m/z 958 compound can be directly used as a characteristic esterified toxin that distinguishes 2WZD12 from other P.lima.The method was a preliminary exploration based on the need of identification between toxic algae and bivalve shellfish in the complex toxin contaminated area and it can provide data basis for establishing a DSTs fingerprint database.?3?P.lima?HN45?was cultured in laboratory under different temperatures?20?,25?,30??,nitrogen and phosphorous limitation conditions?nitrogen-to-phosphorus ratio,N/P:4.08,8.17,16.3,24.5,73.5,147?in this experiment.The physiological characteristics,such as cell density,growth rate,pigments,protein and soluble sugar,as well as the production characteristics of DSTs were investigated and analyzed.Based on ANOVA,the results showed that temperature and phosphorus-limited levels had significant effect on production ability for single cells of P.lima.DSTs toxin contentd reached the highest value?11.3 pg/cell?at the temperature of 25?and phosphorus concentration of 6?M.Phosphorus deficiency reduced the contents of cells density,specific growth rate,chlorophyll a and soluble sugar,but significantly increased per cell content of OA.Moreover,The results further confirmed that chlorophyll a concentration was negatively correlated with DSTs.In addition,esterified toxin accounted for 3.00%to14.0%of total toxins in P.lima,which was much lower than free form toxins.Further study for potential risk assessment of the toxicity of esterified toxins should be valued.?4?DSTs preparation methods were preliminary established with souce of P.lima.P.lima cells in stationary phase were collected in our experiment,and OA,DTX-1 and35S DTX-1 were extracted,purified and separated by methanol extraction,ethyl acetate extraction,Sephadex^TM LH-20 purification and HITACHI primaide separation.The collected DSTs components were analyzed by HRMS.The results showed that OA,DTX-1 and 35S DTX-1 were effectively separated and prepared,and OA?1.80 mg?,DTX-1?0.44 mg?and 35S DTX-1?9.85?g?were acquired based on 20.0 g collected algal cell.The developed DSTs preparation methods laid the foundation of prepared technical performance and declared reference materials.