Preliminary Mechanism Of GPCRs In Regulating P450 Genes Mediated The Sulfoxaflor Metabolic Resistance In Aphis Gossypii Glover

The cotton aphid,Aphis gossypii Glover（Hemiptera:Aphididae）,is one of the most serious insect pests in the cotton field worldwide,which causes economic damage through direct feeding and indirectly virus transmission.So far,the control of cotton aphids in China is largely depended on chemical insecticides.Consequently,cotton aphids have evolved resistance to many types of insecticides.G protein-coupled receptors（GPCRs）are important protein receptors to exist on cell membranes and can sense various signals.They are involved in the regulation of many important physiological processes in insects,including growth,development,reproduction,metabolism,and behaviors.Studies have shown that GPCRs mediated the resistance of Culex quinquefasciatus to permethrin through regulating the expression of resistance-related P450 genes.It has been proved that cytochrome P450 mediates the main metabolic resistance mechanism of sulfoxaflor in cotton aphids.However,the upstream regulation pathway of GPCRs is unclear.In this study,a series of biological,biochemical,and molecular experiments were conducted on the susceptible strain and a sulfoxaflor-resistant strain.Therefore,A.gossypii GPCR genes with typical seven-transmembrane（7TM）domains were systematically identified,the expression pattern of A.gossypii GPCR genes were analyzed in different strains,developmental stages,tissues,and sulfoxaflor treatments,the molecular resistance mechanism of GPCRs regulating the P450 gene expression was clarified in the sulfoxaflor resistance in A.gossypii.The main results are as follows.1.Identification of A.gossypii GPCR genes with typical seven transmembrane domainsBased on previous studies,the GPCRs of Drosophila melanogaster,Tribolium castaneum,and Acyrthosiphon pisum were collected and used as references.By performing BLASTP searches in the genome database of A.gossypii,120 GPCR genes were found in candidate pools.According to the criteria,88 A.gossypii GPCR genes were identified,among which there are 66 family-in A members,12 family-in B members,7 family-in C members,and 3 family-in F members.2.Analysis of expressing pattern of A.gossypii GPCR genesThe expression of eleven GPCR genes was significantly induced by sulfoxaflor with concentrations of LC₁₅（31.03 mg/L）and LC₅₀（157.68 mg/L）.The expression levels of 57A.gossypii GPCR genes in the susceptible strain SS and sulfoxaflor resistant（Sul R）strain were performed by quantitative real-time PCR.The results showed that a total of 34 GPCR genes were up-regulated in the Sul R strain.Age-specific and tissue-specific expression analysis indicated that the expression levels of 18 GPCR genes in the adult stages were higher than those in other stages and were significantly differentially expressed in the wingless or winged aphids;twelve,five,and two GPCR genes were highly expressed in the head,epidermis,and midgut,respectively.3.Study on regulation of GPCRs to the expression of P450 gene related to A.gossypii resistance to sulfoxaflorIn order to elucidate the mechanism of GPCRs regulating the P450 gene expression,knocking down Ago GPCR49 or Ago GPCR54 by RNAi was performed,the susceptibility of A.gossypii to sulfoxaflor increased significantly and the relative activities of detoxification enzymes P450 decreased significantly.It was further found that 20 of P450genes were significantly down-regulated with Ago GPCR49 knocked down.After silencing Ago GPCR54,12 of P450 genes were significantly down-regulated.In addition,CYP6DB1,CYP6CY18,CYP301A1,CYP6CY4,CYP6CY14,CYP4CJ3,CYP6A14,CYP4CK1,and CYP306A1 were significantly decreased after feeding with ds Ago GPCR49 or ds Ago GPCR54,among which the expression level of CYP6CY14 was significantly reduced by 79%and 71%,respectively.These findings layed a theoretical foundation for preliminary mechanism of GPCRs in regulating P450 genes mediated the sulfoxaflor metabolic resistance in Aphis gossypii Glover.