Regulation Of SHSP20.8 On Pupal Development And Reproduction Of Agasicles Hygrophila (Coleoptera:Chrysomelidae)

Agasicles hygrophila is a natural enemy of Alternanthera philoxeroides,which has been successfully controlled this weed worldwide.Small heat shock protein(sHSP)genes play an important role in insect growth,development and resistance to environmental stress.Our previous study indicates that sHSP genes are closely related to the growth and development of A.hygrophila,which is highly expressed in the pupal stage.In order to reveal the function of these genes in the growth and development of A.hygrophila,we used sHSP20.8,which is highly expressed in the pupal stage,as the research object,and analyzed its expression level in all developmental stages and at different times during pupal stage of A.hygrophila by q PCR.Furthermore,the effects of silencing sHSP20.8 in pupae on the pupal development duration,eclosion rate,life span,oviposition duration,fecundity and hatching rate of offspring eggs were studied by RNAi.The main results are as follows:1)Cloning and identification of sHSP20.8.The complete coding sequence of sHSP20.8 was 543 bp,which encoded 180 amino acids with a molecular weight of 20.8 k Da and the isoelectric point of 6.45.The protein,containing typical conserved domains and characteristic motifs of small heat shock proteins,was located in the nucleus.Phylogenetic analysis further showed that sHSP20.8 belonged to the small heat shock protein family and clustered with Coleoptera insects.2)The spatiotemporal expression pattern of sHSP20.8.q PCR was used to analyze the expression pattern of sHSP20.8 in different developmental stages and at different times during pupal stages.Results showed that the expression level of sHSP20.8 was higher in egg stage and pupal stage,and gradually increased after pupation.The relative expression level of sHSP20.8 reached the highest in 1-day-old pupa,and sharply decreased and remained stable in 2-day-old pupa.It is suggested that sHSP20.8 may be involved in the regulation of metamorphosis from pupal stage to adult stage.3)Function verification based on RNAi technology.The expression of sHSP20.8 at pupal stage was reduced by RNAi.RNAi-treated pupae had significantly shortened developmental duration of pupae,and the number of eggs laid by adults at the later stage reduced significantly when compared with the control group.In addition,the hatching rate of eggs laid by adults in the first 8 days was significantly lower than that of the control group,especially the hatching of eggs in the first 2 days was 4% and 9%,significantly lower than the control group of 89% and 86%.In order to further explore the underlying causes for sHSP20.8 affecting the fecundity,we conducted a cross pairing test using wild type female or male mating with RNAi-treated male or female.The results showed that RNAi-treated female is the main reason for the decline of fecundity.In conclusion,the sHSP gene is closely related to the growth,development and reproduction of A.hygrophila,and plays an important regulatory role in reproduction,but the underlying mechanism needs to further study.This study expands our traditional understanding of heat shock protein genes,especially sHSP genes,and provides an important reference for revealing the new functions of heat shock protein genes.