The Molecular Mechanism Research Of Sex Determination And Differentiation In Bactrocera Dorsalis

The oriental fruit fly,Bactrocera dorsalis(Hendel),is an important agricultural pest and causes severe economic loss to over 250 kinds of fruits and vegetables.Insects have evolved diverse mechanisms for sex determination.Different species including those that are closely related show variability in the mechanism of sex determination,which using different genes and regulatory mechanisms to determine the sex differentiation.The primary chromosome signals vary in insect sex determination,and among them the typically Y chromosome-linked male determining factor(M factor)has been found to be required for male development.However,the putative M factor remains unknown in several insect species including B.dorsalis.By using RNAi,CRISPR/Cas9 editing system and high throughput sequencing technique in this study,we revealed the role of the transformer gene in sex determination in B.dorsalis,and identified one miRNA,miR-1-3p,regulates the male sex determination in early embryogenesis as the male-determining factor.We identified several sex-biased miRNAs and studied the role of these miRNAs in reproduction and lifespan in two sexes.Furthermore,we verified that the conserved miRNA Bdo-Let-7 control larval-pupal de velopment in B.dorsalis.Studying the molecular mechanism of sex determination in B.dorsalis,is not only used as a source of reference to explore homologous gene functions and new genes in sex determination but also to provide theoretical and practical foundation in insects genetic operation.This research helps developing new strategies for sterile insect technique(SIT)without radiation sterilization in insects.1.Transformer(tra)is a switch gene in the sex-determination hierarchy that regulates sexual dimorphism based on m RNA splicing in many insects.To illuminate the function of key genes in regulating sex differentiation and female fecundity,we isolated the transformer and transformer-2 genes and characterized their sex-specific transcription.Two male-specific and one female-specific isoforms of B.dorsalis transformer(Bdtra)were identifed.The presence of multiple TRA/TRA-2 binding sites in Bdtra suggests that the TRA/TRA-2 proteins are splicing regulators promoting and maintaining,epigenetically,female sex determination by a tra positive feedback loop in XX individuals during de velopment.The expression patterns of female-specific Bdtra transcripts during early embryogenesis shows that a peak appears at 15 h after egg laying.Using ds RNA to knock-down Bdtra expression in the embryo and adult stages,we showed that sexual formation is determined early in the embryo stage and that parental RNAi does not lead to the production of all male progeny.RNAi results from adult abdominal ds RNA injections show that Bdtra has a positive influence on female yolk protein gene(Bdyp1)expression and fecundity.These results may find use in improving oriental fruit fly control,such as the sterile insect technique,by using in a transgenic genetic sexing strain for male-only field releases,and improving competitive mating of sterile male flies.2.MicroRNAs(miRNAs)are a class of endogenous small non-coding RNAs that regulate various biological processes including sexual dimorphism.However,no miRNAs have been identified from the separate sex and gonads to elucidate sex gonad differentiation in B.dorsalis.In this study,we constructed four small RNA libraries from whole body of females,males and ovaries,testes of B.dorsalis for deep sequencing.The data analysis revealed 183 known and 120 novel miRNAs from these libraries.18 female-biased and 16 male-biased miRNAs that may be involved in sexual differentiation were found by comparing the miRNA expression profiles in the four libraries.Using a bioinformatic approach,we predicted doublesex(dsx)as a target gene of the female-biased miR-989-3p which is considered as the key switch gene in the sex determination of tephritid insects.By feeding the female with miR-989-3p,miR-994-5p,miR-309-3p and miR-6-3p inhibitor,it promoted the increase of spawning in females.By feeding the male with miR-8-3p,miR-34-5p,miR-1-3p and miR-12-5p inhibitor,it decreased the mating competitiveness in males.By feeding the female and male with sex-biased miRNA inhibitor,it reduced the adult lifespan.These results showed that the sex-biased miRNAs play important roles in reproduction and lifespan in two sexes.This study reveals the first miRNA profile related to the sex differentiation and gives a first insight into sex differences in miRNA expression of B.dorsalis which could facilitate studies of the reproductive organ specific roles of miRNAs.3.In some tephritids,a Y chromosome-linked male determining factor(M factor)is suggested to initiates the primary signal in the sex-determination pathway.Howe ver,the M factor and its molecular mechanism of sex-determination in B.dorsalis remain unkno wn.In this study,we identified the key stage for specifying male sexual development is from 5,6 and 7 hour post-oviposition(hpo)and three small RNA libraries were prepared from the embryos at 5,6 and 7 hpo,and 65 miRNAs were found to be differentially expressed in this embryonic sex determination stage.Among them,eight miRNAs were found to have the base-pairing binding sequences were complementary to sites in the 3? UTR(3? untranslated region)of the Bdtra,with the smallest free energy value was observed for miR-1-3p.A dual luciferase reporter assay in HEK293 T cells confirmed that miR-1-3p suppresses the expression of Bdtra,and in in-vivo experiments,the injection of a miR-1-3p mimics and inhibitor in embryos down-regulated 62% and up-regulated 109% of the Bdtra expression respectively,suggesting that the authentic target gene of miR-1-3p is Bdtra.Furthermore,injection of an miR-1-3p mimic in early embryos led to the masculinization of progeny(up to 92% phenotypic male progeny),while knock-down of miR-1-3p by inhibitor injection led to the feminization of progeny(up to 77% phenotypic female progeny).A complete sex reversal of mutant males into phenotypic females was induced by CRISPR/Cas9-mediated knockout of miR-1-3p,which resulted in the expression of female-specific splice variants of Bdtra and doublesex(Bddsx).These results suggest that miR-1-3p is required for male sex determination in early embryogenesis in B.dorsalis,possibly functioning as an intermediate male determiner that may be activated by a Y-linked primary signal.4.MicroRNAs(miRNAs)regulate various biological processes during insect development;however,their role in larval-pupal de velopment in B.dorsalis remains unkno wn.In current study,using dual luciferase reporter assay in HEK293 T cells we show that Bdo-Let-7 miRNA interacts with the 3? UTR of Bd E75 gene and suppresses its expression.The Bdo-Let-7 and Bd E75 are co-expressed in the larval-pupal stages and in different tissues of B.dorsalis.In in-vivo experiments,the injections of Bdo-Let-7 mimics and inhibitor in third instar larvae do wn-regulated 62% and up-regulated 94% of the Bd E75 expression respectively.After injection of exogenous 20-Hydroxyecdysone(20E)in 5th day of larvae,the expression levels of Bdo-Let-7 were significantly elevated 82%,91%,110% and 144% at 3,12,24 and 48 h respectively.Moreover,the abnormal pupation and eclosion was observed after larval Bdo-Let-7 antagomir injection.Based on these results,we show that Bdo-Let-7 regulates the ecdysone signaling pathway through the exact dose of Bd E75 gene,and is indispensible for normal larval-pupal development in B.dorsalis.