Molecular Mechanisms Of Insulin-like Signaling In Regulating The Reproductive Development Of Tobacco Beetle

The insulin-like signaling pathway extensively participate in regulation of material metabolism,growth and development,aging,and lifespan,its mechanism of action has always been paid more attention in insects.The cigarette beetle,Lasioderma serricorne(Fabricius),is a worldwide distributed stored pests,and the larvae cause severe economic loss to many kindsof storage materials,including cereal,tobacco,tea,and traditional Chinese medicine.The long-term unreasonable use of chemical fumigants,in together with small body size,short developmental period,and high fecundity of L.serricorne,has led to reduced agricultural product quality,increased environmental pollution,and enhanced resistance.Therefore,it is urgent to explore new control strategies and find novel targets for management of L.serricorne.Reproduction is a crucial factor for the maintenance and continuation of insect populations.Disrupting or blocking the normal reproductive development of insects is the hot research field currently.Screening the key factors and clarifying the regulation mechanism of insect reproduction,which provides new ideas and targets for pest control.This study will be focusing on the molecular regulation of insulin-like signaling in the growth and development of L.serricorne.A series of molecular techniques including the comparative transcriptome analysis,quantitative real-time PCR(qPCR),RNA interference(RNAi),and dual luciferase reporting system are utilized to annotation of insulin-like signaling pathway genes;to determine the effects of this pathway on L.serricorne reproductive development.The molecular mechanism of miR-9c-5p-Akt-Vg in regulating ovarian development is initially proposed.The main results are listed as follows:1 Transcriptomic and differentially expressed genes analysis of pupae-adult(female)in L.serricorneUsing high-throughput sequencing technology,nine samples of three critical time points for ovarian development of female L.serricorne,including 3-day-old pupae,1-day-old female adults,and 5-day-old male adults,to conduct the genes identification and comparative transcriptional analysis.A total of 11,291 expressed genes were detected,of which 10,369 were known genes and 922 were new predicted genes.A total of 2,524 significantly differentially expressed genes were identified,including 1,024 up-regulated and 1,500 down-regulated.According to GO functional enrichment analysis,the functional categories of differentially expressed genes were significantly annotated into two categories,molecular functions and biological processes,including cuticle structural components,chitin metabolism,and glucosamine compound metabolism.KEGG enrichment was conducted for co-expressed gene clusters showed that the pathways involved in reproductive system development included progesterone-mediated oocyte maturation,PI3K-Akt signaling pathway,estrogen signaling pathway,and insect hormone biosynthesis pathway.Furthermore,two candidate genes related to ovarian development,vitellogenin(LsVg)and vitellogenin receptor(LsVgR),were selected and investigated to the phenotype of ovarian development obstruction,yolk deposition decreased,oviposition amount and hatching rate decline via the inhibition of vitellogenin content by RNAi.2 Gene annotation and expression patterns of insulin-like signaling pathway genes in L.serricorneThe full-length ORF sequences of seven insulin-like signaling pathway genes(LsInR1,LsInR2,LsIRS,LsPI3K21 B,LsPI3K92 E,LsPDK,and LsAkt)were cloned using RT-PCR,with lengths of 2,943,3,696,2,967,2,271,3,181,1,419,and 1,614 bp,coding 980,1231,988,756,1060,472,and 537 amino acids,respectively.Conserved domain analysis indicated that LsInR1,LsInR2,LsPI3K21 B,LsPI3K92 E,LsPDK,and LsAkt are members of the protein kinase C family,while LsIRS is a member of the PH family.Phylogenetic analysis showed that the insulin-like signaling pathway genes of L.serricorne were clustered with genes of their respective families,and all of them had the closest relationship with Coleopteran insects.Developmental expression analysis showed that LsInR2 and LsPI3K21 B were mainly expressed in the adult stage,while LsInR1,LsIRS,LsPI3K92 E,LsPDK,and LsAkt were predominantly expressed in the pupal stage.Tissue-specific expression showed that LsInR1,LsInR2,LsIRS,LsPI3K92 E,and LsPI3K21 B were highly expressed in the midgut,LsAkt was mainly expressed in the elytra and ovary,LsPDK was primarily expressed in the head.Hormones induction results show that methoprene and bovine insulin could promote the ovarian development of L.serricorne,and induced the expression of most insulin signaling pathway genes.In addition,ovarian development was significantly delayed after 20-hydroxyecdysone(20E)treatment in L.serricorne pupae.The expression of LsAkt was significantly decreased by 20 E,whereas the expression of other genes was induced to varying degrees.3 Insulin-like signaling pathway regulates the growth and development of L.serricorneRNAi was used to clarify the biological functions of seven insulin-like signaling pathway genes in L.serricorne.The dsRNAs of LsInR1,LsInR2,LsIRS,LsPI3K92 E,LsPI3K21 B,LsPDK,and LsAkt were injected into 3-day-old female pupae,respectively.The expression levels of above these genes were significantly decreased compared with the dsGFP injection group.After silencing of LsInR2,LsPI3K92 E,LsPDK,and LsAkt,the ovarian development was severely blocked,the egg laying period was shorten,and the length of the ovarian tube was reduced,resulting in a significant decrease in the amount of eggs and the hatching rate,as well as affecting the expression of genes involved in protein synthesis,sugar uptake and metabolism,and juvenile hormone signaling pathway.After the expressions of LsInR1,LsIRS,and LsPI3K21 B was inhibited,the molting development of the pupae was blocked and deformed wings was formed,resulting in a significant reduction in the survival rate of L.serricorne,and the expression of genes related to ecdysone synthesis and signal transduction,chitin metabolism,and wing development are substantially decreased.It was speculated that the insulin-like signaling pathway may be involved in many processes such as protein storage,sugar uptake,lipid synthesis,chitin metabolism,and wing development,thereby regulating insect molting and reproductive development.4 Molecular mechanism of miR-9c-5p targeting LsAkt in regulating ovarian development of L.serricorneSilencing of LsAkt reduced the amounts of glucose,glycogen,and trehalose in female adults,and altered carbohydrate metabolism.The juvenile hormone titers and vitellogenin content of LsAkt-depletion beetles were significantly decreased.To further clarify the expression regulation mechanism of LsAkt,a micro RNA(designated miR-9c-5p)was predicted to target LsAkt by using four kindsof classic miRNA-target prediction softwares.The expression patterns analysis at the stages of pupae-adult showed that miR-9c-5p was expressed in a reverse manner compared to LsAkt.In addition,by injection of miR-9c-5p mimic into the 3-day-old female pupae of L.serricorne,the ovarian growth of the 5-day-old female adults was retarded,the fallopian tubes were shortened,and the lateral fallopian tubes were enlarged.The regulation relationship between mir-9c-5p and LsAkt gene was further verified by dual luciferase assay,and the results showed that the dual luciferase reporter vector constructed with the LsAkt gene and miR-9c-5p mimic was co-transfected and could significantly reduce the luciferase activity,indicating that LsAkt is a potential target gene of miR-9c-5p,and miR-9c-5p could inhibit the expression of LsAkt in vivo.Therefore,we conclude that miR-9c-5p is involved in regulating ovarian development and reproduction by targeting the LsAkt gene in L.serricorne.