| Paralytic shellfish poisoning(PSTs)is one of the most widely distributed and most harmful shellfish toxins in the world.Alexandrium sp as the main producer of PSTs,are widely distributed and existed in the natural sea water,which has brought serious risk to food safety and ecological security.At present,in terms of food safety research,PSTs research is focused on the environment and distribution of toxic algae in aquatic organisms PSTs accumulation,distribution and elimination of the rare of this important process of PSTs endogenous metabolism profile;in the aspect of ecological safety,focused on the inhibitory effect of PSTs on the biological individual intake of exogenous energy the interference mechanism of biological cells and no level of energy utilization research.Therefore,this study considered toxic dinoflagellates as part of the natural environment Scallop food foundation,to assess the impact of single cell algae existing in water to Scallop physiological activity from nutrition level angle;At the same time by Scallop feed with toxic algae,the conversion process of toxins and growth changed,to understand the influence of toxic algae as food foundation on the growth of Scallop.The purpose of this study is to investigate the toxic dinoflagellate as a basis composition food of Scallop,that has influenced on the safety(PSTs risk)and quality(growth traits)of Scallop,and that could elaborate ‘Causes and mechanisms of biomass degradation by shellfish toxins at environmental level'.(1)By collecting phytoplankton in the four seasons of the north-yellow-sea,to study the community structure and nutrient contents,and then compared the characteristics of phytoplankton nutrients in different latitude.At the same time,through the determination of the common microalgae nutrition which cultured in laboratory,compare the differencesthe nutritional components between of common microalgae and phytoplanktonof north-yellow-sea,aiming to evaluate the nutritional status ofphytoplanktonof north-yellow-sea.The experimental results show that the composition of community structure in northern the Yellow Sea is complex and changeable,mainly composed of diatom and dinoflagellate,and the producing algae is the basal bait of scallop.The phytoplankton in the northern yellow sea is rich,the protein dry weight percentage is between 21.26 % ~ 45.46 %;The content of amino acid in summer is low,and the amino acid content in other seasons can meet the growth needs of shellfish.The DHA content in position 19 was the highest in four seasons and was abundant;In laboratory,the nutrient content of producing poisonous algae is rich,and it can provide essential nutrition for scallop growth.(2)A comprehensive method for simultaneous identification and detection of 13 paralytic shellfish toxins(PSTs)in bivalve molluscs was developed using liquid chromatography coupled with quadrupole/linear ion trap tandem mass spectrometry(LC-QTrap/MS).Sample was extracted with acetic acid aqueous solution,cleaned-up by ENVI-Carb solid-phase extraction column.The separation of 13 PSTs were performed on a HILIC column using gradient elution of 95% acetonitrile and water containing 2mmol/L ammonium formate,50mmol/L formic acid as mobile phase.A scheduled multiple reaction monitoring(MRM)in negative mode as survey scan and an enhanced product ion(EPI)scan as dependent scan in an information-dependent acquisition(IDA)experiment was adopted in mass spectrometry acquisition.The calibration curves were linear well with correlation coefficient over 0.99.The detection limit of the method: GTX5,NEO,and STX,and dcSTX,and dcNEO 10 ?g/kg,C2,GTX3,GTX4,dcGTX3 4 ?g/kg,C1 13 ?g/kg,dcGTX2 14 ?g/kg,GTX1 and GTX2 12 ?g/kg,a total of 62?g STX eq./kg after conversion by toxic equivalency factors.The average spiked recoveries were 70.3%~105.2%,with relative standard derivations(RSDs)for 3.45%~13.8%.This method effectively reduce shellfish matrix suppression effect,prior to simplify processes and reduce the dilution,increased sensitivity,isomers meet baseline separation.The proposed method can be used to identify and detect the 13 PSTs in bivalve molluscs.(3)In this paper,we exposed scallop directly to toxins algae,in order to characterization of harmful process caused by PSTs in Chlamys farreri.Furthermore,PSTs accumulation,and metabolic biotransformation in different organ of scallop were followed using liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS).The result showed that the AT5-3strain of Alexandrium tamarense which separated from South China Sea were stable both in growth and toxins production.Its main toxin production were Gonyautoxins-1&-4,with a cell toxin-production of 7.95 fmol/cell.A rapid PSTs accumulation was observed,while total accumulation efficiency reached 84.4%.The highest accumulation concentration was 1903?g STX eq./kg.Accumulation ability of different tissues were in order with viscera>gonad>pallium>muscle.Moreover,the PSTs metabolic ability of visceral was the strongest,and visceral was the target organ.In addition,the proportions among PSTs in bivalves differ from the profile of the dinoflagellate that produces the toxins.Chlamys farreri has performed a strong biological transformation ability,resulting in the content of Neosaxitoxin(NEO)which have high toxicity increased significantly.The main transformation way were N-Sulfocarbamoylgonyautoxin-2(C1)? Gonyautxins-2(GTX2)/ Decarbamoylgonyautoxins-2(dcGTX2),and N-Sulfocarbamoylgonyautoxin-3(C2)? Gonyautxins-3(GTX3).In this experiment,Chlamys farreri showed the characteristic of rapid PSTs-accumulation and slow PSTs-metabolism,meanwhile,the increase of high toxin component ratio,resulting in the PSTs toxicity and the risk of Chlamys farreri increased significantly.This study is helpful to the scientific evaluation of the formation mechanism of PSTs,to provide scientific support for the monitoring of PSTs in china.(4)Through exposure experiments,the PSTs toxicological effects of the ultrastructure of visceral and gill tissues of Chlamys farreri were studied,and the changes of redox enzymes in antioxidant defense system in the visceral and gill tissues were detected,inorder to study the toxicological stress of visceral and gill tissues of Penaeus monodon caused by PSTs.The experimental results showed that the ultrastructure of visceral and gill in Chlamys farreri under the action of PSTs showed pathological changes,the intestinal epithelial cells vacuolization,the nuclear atrophy,and the cell necrosis was severe;In the early stage of exposure,the mitochondria and lysosomes in the midline of the gill epithelial cells increased and concentrated,the epithelial cells began to rupture and rupture,mucus cells released a large number of mucus particles.In the early stage of exposure experiment,the redox enzymes in the visceral and gill were increased,lipid peroxidation,the corresponding GSH px and pod increased,the ACP activity and NADPH content decreased,some NADPH may participate in the biological transformation of toxins.The damage of the ultrastructure of visceral and gill tissue of Chlamys farreri and the activation of redox enzymes in tissues can provide theoretical basis for the establishment of tissue toxicological index of shellfish under PSTs stress. |
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