Transcriptomic Analysis Of Small RNAs In The Testis And Ovary Of Helicoverpa Armigera And Functional Characterization Of Differentially Expressed MicroRNAs

The cotton bollworm,Helicoverpa armigera(Hübner),is a polyphagous crop pest of the Lepidoptera Noctuidae.Owing to its broad range of host plants and strong fertility,H.armigera causes huge economic losses to agricultural production of our country.MicroRNA(miRNA),a class of endogenous non-coding single-stranded smallRNAs(sRNAs)of ~22 nucleotides(nucleotide,nt)with in length can bind to a 3’UTR fragment of its target mRNA,leading to degradation of the target mRNA or translation suppression of the target mRNA.Acting as one type of post-transcriptional regulatory factors,miRNAs participates in various biological processes of insect growth and development,including sex determination,gonadal development and reproductive regulation.This study uses H.armigera as a research object to study the expressional differences and functions of miRNAs in the testis and ovary of this destructive crop pest.The main findings are as follows:1.Morphological analysis showed that H.armigera male testis matured in the newly emerged adults whereas its female ovary did not mature until 48 h post emergence.Male testis was readily visible in the larval stage with a microscope whereas female ovary developed relatively slowly and more concentrated in the later stages(pupae and adults)of development.2.A total of 7,592,150 and 8,815,237 clean reads were obtained by constructing sRNA libraries of testis and ovary,respectively.Length distribution analysis of these sequences showed that the main types of sRNAs in the testis and ovary were different.The sRNA sequences were divided into several sRNA families and a total of 173 novel miRNAs and 74 known miRNAs were identified from the two libraries.Through the analysis of the 74 known mature miRNAs,a total of 60 miRNAs were identified showing significant differential expression(|log2FC| ≥ 1),of which 50 miRNAs were significantly up-regulated in the testis and 10 were significantly up-regulated in the ovary.q RT-PCR proved that miR-252a-5p,miR-2c,miR-998,miR-2763,and miR-2765 had a higher expression level in the testis whereas miR-263b-5p,miR-989 a and miR-34 displayed a higher expression level in the ovary.These 8 miRNAs also showed different expression patterns in the other body parts/tissues including head,thorax,abdomen,feet,wings and fat body.3.The 8 differentially expressed miRNAs together had a total of 30,172 putative target genes(transcripts).GO functional enrichment analysis and KEGG pathway enrichment analysis indicated that they had target genes involved in reproduction process.At the cellular level,dual-luciferase reporter gene assay verified that miR-252a-5p targets on OVOL while miR-263b-5p targets EGFR and FOXO.In live insects,injection the artificially synthesized analogues(agomir)of miRNAs down regulated the corresponding target genes,while injection the inhibitors(antagomir)of miR-252a-5p or miR-263b-5p up-regulated the corresponding target genes.These results confirmed that miR-252a-5p and miR-263b-5p negatively regulated the expression of OVOL and EGFR or FOXO in H.armigera.4.Single-paired mating experiment involving a normal male/female with a female/male injected with miR-252a-5p or miR-263b-5p agomir/antagomir found that the fecundity and hatching rate of the experimental groups were significantly reduced.The same phenomenons were observed when one of the single-paired mating parents was injected with the siRNA of corresponding target gene to knock down the expression level of OVOL,EGFR or FOXO.In addition,significant ovarian defect phenotypes were observed when mothers were injected with the siRNA of OVOL,EGFR or FOXO.The results suggested that miR-252a-5p and miR-263b-5p balance the expressions of OVOL and EGFR or FOXO,respectively,which in turn regulate the development of ovaries or fertilized eggs,and thus affect fecundity and hatching rate.In conclusion,this study constructed two smallRNA libraries of H.armigera testis and ovary to reveal testis/ovary-differntially expressed miRNAs.Among them,miR-252a-5p and miR-263b-5p were confirmed to post-transcriptionally regulate OVOL and EGFR or FOXO,respectively,and thus control gonadal development and reproduction of H.armigera.