Accumulation Of Paralytic Shellfish Toxins In Ostrea Rivularis Gould By Stable Isotope ¹⁵N Tracer Technology

Paralytic shellfish toxins?PSTs?are a common natural micro-algae neurotoxins with the abundant source,a worldwide distribution,lots of analogs and highly toxic.Due to incomplete statistics,the occurrence of food poisoning caused by PSTs are about 2000and the death rate is 15%every year.Therefore,many countries and relevant international organizations have strengthen the management and control of shellfish aquatic products strict,established the regulatory limit of PSTs in shellfish.For the intensive study on PSTs,this study used stable isotope ¹⁵N tracer technology to domestication Alexandrium minutum?A.minutum?species producing high abundance ¹⁵N-PSTs.It described purification procedures for¹⁵N-PSTs isolated from domesticated algae.The uptake,distribution and transformation of ¹⁵N-PSTs in Ostrea rivularis Gould exposed to toxic dinoflagellates A.minutum were researched primarily.In order to study metabolic mechanism of PSTs in aquatic products and paralytic shellfish poison-binding protein?PSPBP?intensively,as well as being medicine in clinical application,this paper provides theoretical basis and new ideas.The main contents and conclusions of the experiments are as follow:?1?Using ¹⁵N-Na NO₃instead of nitrogen source Na NO₃in f/2 nutrient solution,A.minutum producting high abundance of ¹⁵N-PSTs were acclimated by ¹⁵N tracer labeling method.The effects of¹⁵N/P on the growth and toxin production of A.minutum,and ¹⁵N abundance changes in ¹⁵N-PSTs extracts,were analysized under five different ¹⁵N/P concentrations of batch culture.When ¹⁵N/P concentration is 1.5 times of N/P content in f/2nutrient solution,miroalgae cells'physiological condition is better.After three succession cultivation,the final results show that when the ¹⁵N/P concentration is equal to the N/P content in f/2 nutrient solution,which is suitable for acclimating toxic algae cell contained high abundance¹⁵N-PSTs.By stepwise enrichment culture,the PSTs extracted from the N generation A.minutum cells,which ¹⁵N marker abundance reached to 82.36%.Using¹⁵N-PSTs as a tracer,it will provide theoretical basis for the further study on the PSTs metabolism of shellfish.?2?Mass culture the A.minutum,which produced high abundance of ¹⁵N-PSTs,aim to gain enough high abundance ¹⁵N-PSTs crude.the crude extract were purified by polyacrylamide gel Bio-Gel P-2 and carboxylic acid type ion exchange resin Bio-Rex70,and the quantitative and qualitative analysis were analyzed by MBA,HLPC-FLD,UPLC-MS/MS and fluorescence method.The results show that GTX-1,-2,-3 and-4 are the only toxins that occur in A.minutum,GTX1/4 and GTX2/3 can be separated under the optimized conditions of the isolation and purification.There are 2?6¹⁵N atoms are introduced into the ¹⁵N-PSTs,and the 4 ¹⁵N atoms are most likely to be introduced,by Mass spectrometry identification.?3?Simulated the natural environment that oyster contaminated PSTs,¹⁵N-PSTs crude extract?5 mg/L?+Isochrysis zhanjiangensis sp.nov?5.0×10⁵cells/m L?and toxic dinoflagellates A.minutum?0.5×10⁴cells/m L?producting high abundance of ¹⁵N-PSTs were feed Ostrea rivularis Gould respectively.Each tank received a continuous flow of 10L day^-1of each microalgal treatment,and the exposure period continued for two days.¹⁵N abundance,toxicity,toxin content and composition in different oyster tissues were analyzed by stable isotope analyzer,MBA and HPLC-FLD,and the accumulation,distribution and transformation of ¹⁵N-PSTs in Ostrea rivularis Gould were studied.The results show that the ¹⁵N-PSTs toxicity?in the reference of viscera?in oyster exposed to toxic dinoflagellates was significantly higher than that oyster exposed to¹⁵N-PSTs crude extract,were 430.0 MU/100g and 342.5 MU/100g respectively.The ¹⁵N-PSTs in adductor muscle,mantle,gills and visceral mass?digestive gland and pancreas?of the oyster were detected,and the main accumulation tissue was visceral mass,followed by gills,mantle and adductor muscle.Biotransformation in different oyster tissues are different,mainly occurred that the?type isomeride are converted into?type isomeride,and GTX2/3converted into GTX1/4.In addition,there is a certain positive correlation between the¹⁵N marker abundance and PSTs toxicity,which can provide a new idea for the study of the metabolism of PSTs in shellfish.