Stress Response And Regulation Mechanism Of Chlamys Farreri Against Azaspiracids

Nowadays,marine biotoxins are widely accumulated in shellfish,which has potential threats to aquaculture,foreign trade and food safety under the background of frequently blooming of red tide.In view of the highly toxic characteristics and the trend of global distribution of Azaspiracids(AZAs),in this study,a toxic algae of AZAs(Azadinium poporum,AZDY06)was used as baits,Chlamys farreri was taken as the stress object,metabonomics and transcriptomics techniques were comprehensively applied to study the response mechanism of C.farreri to AZAs at multiple levels.So that we can further explore the detoxification mechanism of AZAs in C.farreri,and provide scientific basis for risk assessment and establishment of limit standards.The main research contents are as follows:(1)The stress regularity was clarified by data of blood cells and eight enzyme indexes.The results showed that there were three stages of physiological stress:stage?was the stress of blood cells within one hour,which played the role of recognition,phagocytosis and decomposition.In stage?,SOD and POD played an antioxidant role within one day,and?-GCS,GST and NQO1 promoted metabolic reaction,so as to reduce peroxide damage and accelerate AZAs excretion;In stage?,Ca²⁺/Mg²⁺-ATPase and Na⁺/K⁺-ATPase were significantly stressed within three days to regulate energy homeostasis.In the whole process,AZAs damaged the villi and lumen of C.farreri,which had a dose-effect relationship.In this study,the stress regularity of C.farreri to AZAs was expounded from three aspects:metabolism,immunity and energy,which provided theoretical support for the further toxic mechanism.(2)The metabolic dynamics in different stages in C.farreri were studied by non-targeted metabonomics.The results showed that the differential metabolites were significantly enriched in steroid metabolism,retinol metabolism,synthesis and degradation of various amino acids,and 43 differential metabolites were screened,among which glutathione,taurine,choline phosphate,tryptophan,etc.were most related to toxic effects.The toxic effects were manifested as peroxide damage,destruction of cell membrane,and metabolic disorder of amino acids and nucleotides.In addition,abnormal changes of tryptophan suggested the possibility of neurotoxicity.In this study,the response of C.farreri was studied from the metabolic level,and the differential metabolites and pathways were identified to provide data support for exploring the detoxification pathway of AZAs.(3)The difference,enrichment and trend analysis of expressed genes in C.farreri were carried out by transcriptomics.The results showed that genes related to oxidative stress,energy regulation and substance metabolism were significantly expressed,and there were differences in different stages.There were two main expression trends:up-regulation and down-regulation.The former mainly enriched in apoptosis,amino acid metabolism and Toll receptor signaling,while the latter was lysosome,sphingolipid metabolism and endocytosis.In the end,411 highly expressed genes were enriched in 191 pathways,among which many genes related to energy regulation were most significantly expressed,such as ATP6 and COX3,and the expression could reach more than 104 FPKM,which constituted the basis of stress.Then,the significant expression of specific functional genes,such as ATP5F1B,ATF5 and KCNK16,drived the signal transmission of the second messenger c AMP and the balanced regulation of apoptosis and ion exchange.In this study,the stress mechanism was studied in the gene level,and the differential genes and pathway were identified to establish the foundation for exploring the molecular mechanism in C.farreri.(4)Multi-level analysis was carried out by combining biological stress,metabonomics and transcriptomics.The results showed that genes related to oxidative stress,energy regulation and substance metabolism were interrelated and regulated together.Among them,the oxidative stress module dominated the regulation of apoptosis,and the metabolic module was the most complex.It could regulate the consumption and production of energy and also closely related to signal transmission,substance synthesis and oxidative stress.Time series analysis showed that steroid hormones secreted quickly to send out stress signals in early stage and secreted adrenaline to assist in activating immune functions in medium stage.In the whole process,the body consumed energy to cause ATP hydrolysis,and excessive consumption of energy break the balance of the body and then activated the de novo synthesis of purine in later stage to remedy the imbalance.This study explored the molecular mechanism of stress from various angles,which provided theoretical support and data basis for further analyzing the detoxification mechanism of C.farreri to AZAs.(5)Through linkage analysis of metabolic enzymes,metabonomics and transcriptomics data,the key phase I and phase II metabolic pathways of AZAs in C.farreri were further locked.The phase I metabolism was mainly cytochrome P450pathway,and the regulatory factors such as CYP1A1 and ALDH2 were differentially expressed.And they also participated in the metabolism of arachidonic acid and tryptophan,and thus participated in the phase I metabolic reaction collectively.While glutathione metabolic pathway was the mainly phase II metabolism,which involved the differential expression of key regulatory factors such as GST1 and GSTT2B.In addition,this study also enriched the important products of glucuronic acid II phase metabolism.