Transcriptomic And Metabolomic Analysis Of Diapause In Exorista Civilis

Exorista civilis(Diptera: Tachinidae),a kind of forage dominant parasitic natural enemy,is a parasitic fly of a variety of lepidopteran pests.The main problems faced by this natural enemy insect in application were the short storage period of artificial expansion and the serious drawbacks of traditional low-temperature refrigeration technology,making the natural enemy insects vulnerable to low-temperature damage and affecting the quality of products,so improving the resistance of the natural enemy insects is the inherent root of solving the above problems.Diapause is an important behavior to help insects survive in the unfavorable environment,maintain individual survival and ensure population reproduction.Thus,artificially regulating diapause of Exorista civilis can greatly extend the shelf life of the product and is of great significance to achieve release accurately.In this study,we performed transcriptome sequencing,quantitative real-time PCR and metabolomic assay on diapause and non-diapause pupae,and screened out differentially expressed genes and metabolites related to diapause,so as to lay a theoretical foundation for elucidating the molecular mechanism of dipause regulation in pupae of Exorista civilis.1.The transcriptomic analysis of diapause and non-diapause pupaeThe number of differentially expressed genes was determined to be 7513,of which 4355 were up-regulated and 3158 were down-regulated in diapause and non-diapause pupae.The successfully annotated 3257 differentially expressed genes were enriched by GO database,and they were mainly classified into three categories: biological process,cellular component,and molecular function,with the most enriched pathways in biological process.896 differentially expressed genes were mapped to 223 pathways by KEGG pathway enrichment analysis,mainly involving in signal transduction,carbohydrate metabolism,and endocrine system.In order to confirm the accuracy of transcriptomic data of diapause and non-diapause pupae,five diapause-associated genes were selected for quantitative real-time PCR validation,namely cytochrome C oxidase subunit 6a,actin β/γ1,ring-box protein 1,gamma-aminobutyric acid receptor subunit alpha and glucose-6-phosphate isomerase;the internal reference gene was set as glyceraldehyde-3-phosphate dehydrogenase.The results revealed that the selected differentially expressed genes showed consistent expression trends in quantitative real-time PCR and transcriptomic sequencing,indicating that the transcriptomic sequencing results were reliable.2.The metabolomic analysis of diapause and non-diapause pupaeIn the negative ion mode,169 differential metabolites were screened,of which 99 were up-regulated and 70 were down-regulated.In the positive ion mode,332 differential metabolites were screened,of which 268 were up-regulated and 64 were down-regulated.KEGG enrichment analysis of differential metabolites revealed that in the negative ion mode,differential metabolites were enriched in38 pathways,except for human diseases,only 7 pathways were significantly enriched,including amino acid biosynthesis,protein digestion and absorption,histidine metabolism,arginine and proline metabolism,in which L-proline,L-phenylalanine,L-histidine were all up-regulated;in the positive ion mode,differential metabolites were enriched in 46 metabolic pathways,of which only 3 pathways were significantly enriched,namely histidine metabolism,aminyl-t RNA biosynthesis,and amino acid biosynthesis,and the main differential metabolites involved were L-histidine,L-phenylalanine,L-tyrosine,and 1-methylhistidine,all of which were up-regulated in expression.The pathways of the histidine metabolism and amino acid biosynthesis were significantly enriched in both positive and negative ion modes,but the substances enriched in the two ion modes were different.3.The combined transcriptomic and metabolomic analysisThe combined transcriptomic and metabolomic analysis of diapause and non-diapause pupae in Exorista civilis by KEGG Pathway showed that the differentially expressed genes and differentially expressed metabolites were enriched in 31 pathways in the negative ion mode and 39 pathways in the positive ion mode.The c AMP signaling pathway was involved in the regulation of calcium signaling pathway,insulin signaling pathway,FOXO signaling pathway,and gluconeogenesis by adenosine-5’-monophosphate,further affecting the expression of calmodulin,antioxidant enzymes,and PEPCK.The expression of phenylalanine was up-regulated in the biosynthetic pathway of aminyl-t RNA,which may be related to the formation of melanin in diapausing pupae of Exorista civilis.