| Anopheles sinensis is widely distributed in China and is an important vector for malaria and other mosquito-borne diseases,and vector control has been one of the key strategies for global mosquito-borne disease prevention and control.However,with the long-term and massive use,it has brought about the general resistance of mosquitoes to pyrethriods.The process of insecticide exposure involves multiple metabolic pathways in mosquitoes,but the metabolic mechanisms of detoxification of pyrethroid insecticides such as deltamethrin by Anopheles sinensis are currently unknown.In this study,we used liquid chromatography tandem mass spectrometry(LC-MS/MS)-based metabolomics and transcriptomics techniques to analyze the metabolites and differentially expressed genes in sensitive and resistant Anopheles sinensis after exposure to deltamethrin.The expression of these genes in different tissues and developmental stages of resistant and sensitive Anopheles sinensis was verified by realtime fluorescence PCR.The homologous genes were also validated in the Aedes albopictus C6/36 cell line.The results provide a scientific basis and theoretical reference for the in vivo metabolism and resistance studies of pyrethroid insecticides in Anopheles sinensis.This study is divided into three parts:Ⅰ.Metabolomic analysis of deltamethrin in resistant adult Anopheles sinensis mosquitoes after exposure to deltamethrinIn the early stage,the types and contents of metabolites in sensitive and resistant larvae and sensitive adult of Anopheles sinensis exposed to deltamethrin were compared,and the biochemical function and KEGG metabolic pathway enrichment of mosquitoes after exposure were analyzed.On this basis,the metabolic changes of deltamethrin exposed to deltamethrin in adult Anopheles sinensis captured in the field were further analyzed by LC experiment MS/MS method.The results compared with the sensitive adult Anopheles sinensis exposed to deltamethrin,there were 168 unique differential metabolites of resistant Anopheles sinensis.The biochemical functional classification was mainly concentrated in the families of organic oxygen compounds,carboxylic acids and their derivatives,glycerol phospholipids and purine nucleotides,and the metabolic pathways were mainly concentrated in glycerol metabolism,amino acid metabolism and ABC transporters.Compared with the metabonomics of deltamethrin resistant larvae studied in our group,it was found that the common differential metabolites of Anopheles sinensis resistant larvae and adult mosquitoes belonged to glycerolipids,purine nucleotides and organic oxygen compounds,respectively.the changes of common metabolites suggest that these three compounds may play an important role in deltamethrin metabolism and detoxification of Anopheles sinensis.Ⅱ.Transcriptomic analysis of sensitive and resistant Anopheles sinensis larvae after deltamethrin exposureTranscriptome analysis was used to further study the difference of specific gene expression levels between sensitive and resistant populations of Anopheles sinensis.As the number of resistant adult mosquitoes in the field could not meet the requirements of the experiment,the larvae of sensitive and resistant Anopheles sinensis were studied by bioassay after exposure to deltamethrin.The differentially expressed genes of sensitive and resistant Anopheles sinensis were screened,and the differential gene expression levels were analyzed by cluster analysis,GO enrichment analysis and KEGG enrichment analysis.The results showed that there were 423 differentially expressed genes between deltamethrin treatment group and anhydrous ethanol control group,including 170 up-regulated genes and 253 down-regulated genes between deltamethrin-treated group and anhydrous ethanol control group,and 1651 differentially expressed genes between deltamethrin-resistant Anopheles sinensis group and ethanol control group,including 580 up-regulated genes and 1071 down-regulated genes.Cluster analysis showed that lipid metabolism was the main metabolic pathway,which was consistent with the results of the first part.Further comparative analysis showed that there were 20 co-expressed genes between resistant and sensitive strains,of which 5 genes related to lipid metabolism were GLB1,D11 DS,FASN,mogat2-an and PLA2G3.This part of the study revealed the functional classification and metabolic pathway of differentially expressed genes in Anopheles sinensis larvae after insecticide treatment,so as to provide scientific basis and reference for further development of the biological function of Anopheles sinensis related genes.Ⅲ.In vitro and vivo expression characterization of differential genes related to lipid metabolismCombining the results of the previous two parts,it was found that lipid metabolism played an important role in deltamethrin resistance of Anopheles sinensis,and differentially expressed genes related to lipid metabolism of Anopheles sinensis were screened.In this part,q RT-PCR method was used to analyze the expression of lipid metabolism related genes GLB1,D11 DS,FASN,mogat2 and PLA2G3 in different tissues such as midgut and ovary of Anopheles sinensis as well as larvae,pupae and adult mosquitoes.In addition,in order to further verify that differentially expressed genes related to lipid metabolism are involved in insecticide resistance,the above lipid metabolism homologous genes of Anopheles sinensis were compared and analyzed in vitro before and after deltamethrin treatment in Aedes albopictus C6/36 cell line.The results showed that GLB1 expression was significantly down-regulated in both resistant Anopheles sinensis and Aedes albopictus C6/36 cells after exposure to deltamethrin,suggesting that GLB1 may be involved in the lipid metabolism pathway of Anopheles sinensis and thus play an important role in causing insecticide resistance.The results of this part of the study further validate the role of lipid metabolism in the development of insecticide resistance in Anopheles sinensis,and provide theoretical guidance and basis for the subsequent development of resistance management. |
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