Molecular Characterization And Functional Study Of The Key Genes Involved In The Chitin Synthesis In Agrotis Ipsilon(Hufnagel)

Agrotis ipsilon(Hufnagel)is an underground pest that belongs to the Lepidoptera family(Noctuidae).It is a highly concerning pest that is spreading rapidly and has become increasingly serious due to global warming.Unfortunately,many areas have developed resistance to common insecticides,making it imperative to develop new control methods as soon as possible.Chitin is an excellent target for preventing and controlling pests due to its crucial role in insect tissues,including the epidermis,midgut,and trachea.Additionally,its cyclic synthesis and degradation are critical in insect growth,development,and metamorphosis.Research on key genes of the chitin synthesis pathway is of great importance for the development of new environmentally friendly green control methods.This study aimed to characterise 4 key genes involved in the chitin synthesis pathway: AiCHS1,AiCHS2,AiUAP,and AiGNA.The research investigated their sequence and spatio-temporal expression,biological function,and the impact of chitin synthesis inhibitors on growth,development,and gene expression in A.ipsilon.The main findings of the study are as follows:1.Cloning and sequence analysis of 4 key genes of the chitin synthesis pathway in Agrotis ipsilonBased on transcriptome data and PCR product sequencing,the cDNA sequence lengths of the 4 key genes,AiCHS1,AiCHS2,AiUAP,and AiGNA in A.ipsilon chitin synthesis pathway,were 4701 bp,4629 bp,1488 bp,and 453 bp,respectively,AiCHS1 had 2 alternative splice variants AiCHS1 A and AiCHS1 B,with a differential nucleic acid sequence of 177 bp;the protein sequences encoded by 4 genes were analysed by biological software and all had multiple glycosylation sites and phosphorylation sites;AiCHS1 and AiCHS2 had multiple transmembrane structures,while AiUAP and AiGNA had no transmembrane regions;none of the proteins encoded by 4 genes had signal peptide sequences;the protein secondary and tertiary structures were composed of α-helices,β-folds,extended chains and irregular coils;by homology comparison and phylogenetic analysis,4 genes have high sequence identity with their Lepidoptera homologs and are most closely related to the Noctuidae.2.Expression analysis of 4 key genes in chitin synthesis pathway in Agrotis ipsilonIn this paper,we conducted a systematic analysis of the expression patterns of AiCHS1(AiCHS1 A and AiCHS1B),AiCHS2,AiUAP,and AiGNA using fluorescence quantitative PCR(RT-q PCR)in various tissues and at different ages.Our findings revealed that the expression of AiCHS1 peaked during the pupal stage,while it was lowest in the 1st instar larvae and adult stage;the expression of AiCHS1 A,which is an alternative splicing variant of AiCHS1,was found to be highest during the 6th instar larvae and pupal stages,while it was lowest during the 1st instar larvae and adult stages;the expression level of AiCHS1 B peaked during the pupal stage and remained relatively constant throughout the other instars;AiCHS2 was expressed highest in 6th instar larvae and pupal stage and lowest in 1st instar larvae and adult stage;AiUAP transcript levels were greatest in 5th and 6th instar larvae and lowest in egg and 1st instar larvae;AiGNA expression level was highest in 5th-6th instar larvae and adult stage and lowest in pupal and 1st instar larvae.The expression patterns of different tissues showed that AiCHS1 was most abundant in the head capsule,more abundant in the epidermis and the expression levels of two alternative splice variants were consistent with AiCHS1;AiCHS2 expression level was very high in foregut and midgut with no significant difference,followed in hindgut;AiUAP was most abundant in head capsule and epidermis,followed in fat body,foregut and midgut;AiGNA expression was highest in fat body,followed by epidermis and head capsule.3.Functional studies of four key genes for chitin synthesis in Agrotis ipsilonThe biological functions of AiCHS1,AiCHS2,AiUAP,and AiGNA in the growth and development of A.ipsilon were analyzed by injecting specific dsRNA fragments into 6th instar tiger larvae.The results showed that after interfering with AiCHS1,37.6% of pupae were deformed and 16.6% of adult wings were deformed,while the expression of AiCHS1(AiCHS1 A and AiCHS1B),AiUAP and AiGNA were significantly downregulated;after interfering with AiCHS2,its transcript level was significantly reduced,the gut of larvae was shortened and 25.1% of test insects died,while surviving larvae pupated and fledged normally;after interference with AiUAP,the epidermis of 56.3% insects tested turned black,failed to molt and died,while the 4 genes’ expression levels were significantly reduced.After interference with AiGNA,36.4% of the pupae showed deformities,but the pupae were basically separated from the old epidermis,which did not affect the normal emergence of the pupae.Silencing the four genes resulted in different phenotypes,suggesting different functions.AiCHS1,AiUAP and AiGNA are involved in epidermal chitin formation,while AiCHS2 is involved in midgut chitin synthesis.Absence of AiCHS1 and AiUAP can lead to molting failure in 6th instar,pupal malformation and adult wing malformation after emergence.Interference with AiGNA resulted in pupal malformation but did not affect survival.The loss of AiCHS2 disrupted feeding and the larvae eventually died from malnutrition.4.The effects of chitin synthesis inhibitors on growth and development and expression of 4 key genes in Agrotis ipsilonFor the chitin synthesis-inhibiting pesticides diflubenzuron and lufenuron,the LC50 was 0.98 μg/m L and 0.57 μg/m L for 4th instar larvae and 1.30 μg/m L and 0.73 μg/m L for 5th instar larvae,respectively.After treatment with diflubenzuron and lufenuron,the larvae showed half-molting or blackening and deformed pupation.Diflubenzuron and lufenuron differentially affected the expression of 4 genes in the chitin synthesis pathway.The expression levels of AiCHS1,splice variant AiCHS1A and AiUAP were significantly upregulated after diflubenzuron treatment,the expression levels of other genes weren’t significantly different from control;the expression of all 4 genes was significantly upregulated after lufenuron treatment.These results indicated that diflubenzuron had a major effect on chitin formation in epidermis,while lufenuron had a great effect on chitin synthesis in epidermis and midgut.In conclusion,the results of this study enriched the biological information base of the black cutworm,revealed the expression characteristics of four genes,AiCHS1,AiCHS2,AiUAP and AiGNA,in different developmental stages and tissues,elucidated the effects of silencing the four genes and the chitin synthesis inhibitors on the growth and development of the black cutworm,and provided a theoretical basis for the study of the chitin synthesis pathway,as well as a theoretical basis for the development of new green pesticides targeting genes involved in chitin synthesis.