Molecular Mechanism Of MiR-9b-ABCG4-Insulin Regulated Wing Formation In Aphis Citricidus

The brown citrus aphid,Aphis citricidus,is one of the most important pests of citrus which is the largest production fruits around the world.In together with the directly sucking on citrus' stem and leaf,it is also the main vector of Citrus triseza virus(CTV).Similar with other aphids,the brown citrus aphid present wing dimorphism,while the frequency of alate morph in A.citricidus is relatively higher in comparison with other aphid species.Being an important ecological strategy of aphids,wing dimorphism play crucial role in the outbreak disaster of this pest.Therefore,it is of great theoretical and practical significance in determining the molecular mechanism of wing dimorphism and development for the sustainable control of the aphids.In the current study: based on the transcriptome,the different gene expression profiling and small RNA sequencing of alate and apterous adults of A.citricidus datasets,the key gene involved in the development of alate morphs—ABCG4 were selected;next evidences from the feeding mi RNAs mimics and inhibitors,dual-glo luciferase report system and RNA-binding protein immunoprecipitation pointed out that mi R-9b target on ABCG4;RNAi technology was used to analyze the insulin signing pathway and its upstream mi R-9b-ABCG4 in wing development,which revealed the mi R-9b-ABCG4-insulin model in regulation of the wing development of A.citricidus.The main results in this study are as follow:1 Exploration of the differentially expressed genes and mi RNAs between alate and apterous adults of A.citricidus.A library covered different developmental stages of A.citricidus was constructed and further assembled into 44,199 unigenes.Digital gene expression profiling and small RNA analysis were performed to obtain a global view of the aphid transcriptome between differences alate and apterous adults.In comparsion with apterous adults,a total of 5,749 genes and 6 mi RNAs were significantly differential expressed,including 279 genes and 4 mi RNAs were highly expressed.Among the highly expressed genes in alate morphs,an ABC transport gene,ABCG4,was hilighted.The full c DNA sequence of ABCG4 was cloned by RACE PCR.Five mi RNA-m RNA predictive softwares were used to predict the potential mi RNAs which could regulate the expression of ABCG4.The results indicated that mi R-9b,mi R-10 and mi R-236 could be the candidated mi RNAs in regulatin the expression of ABCG4.2 Regulation of the expression of ABCG4 by mi R-9b.The results indicated that feeding miR-9b mimics decreased the relative expression of ABCG4 and feeding mi R-9b inhibitor increased the relative expression level of ABCG4.Feeding mi R-10 or mi R-236 mimics/inhibitor did not effect on ABCG4.The results suggested that mi R-9b maybe regulate ABCG4.In vitro dual-glo luciferase report system conbined the site-directed mutagenesis of ABCG4 was used to elucidate that mi R-9b regulate ABCG4 expression through binding to predicted ORF region.In vivo RNA-binding protein immunoprecipitation(RIP)assay showed that overexpression of mi R-9b,Ago-1 antibody enriched more ABCG4 m RNA.Comprehensive analysis of the results of feeding mi RNAs mimics/inhibitor,dual-glo luciferase assay and RIP assay showed that mi R-9b directly regulate the expression of ABCG4.3 Involvment of miR-9b and ABCG4 in wing developmentThe relative expression of mi R-9b and ABCG4 in different developmetal stages and tissues were tested by q RT-PCR.The results indicated that mi R-9b and ABCG4 present the opposite expression pattern among developmental stages,specifically there was a sharp increase of the expression level of ABCG4 between fourth instar winged nymphs to alate adults transition while a sharp decrease of the expression leve of mi R-9b,wherease,there were no difference among each instar nymphs in apterous morphs.mi R-9b and ABCG4 were highly expressed in fatbody as well as muscle and wing in alate morphs.These results suggested that mi R-9b and ABCG4 may be involved during fourth instar winged nymphs to alate adults transition.RNAi was used to explore the effects of ds ABCG4 on wing development of A.citricidus.The results demonstrated that ABCG4 regulate the wing development process of A.citricidus and adults with malformed or undeveloped wing.The similar feeding assays were taken in feeding mi R-9b mimics and inhibitor.Overexpression of mi R-9b resulted the similar phenotype with feeding ds ABCG4 while there were no phenotypes observed upon feeding mi R-9b inhibitor.The similar phenotypes were also observed in A.pisum that ingestion of mi R-9b mimics or ds ABCG4 disrupted the wing development indicating that the role of mi R-9b and ABCG4 were conserved in aphid species.According to the results of temporal and spatial expression patterns of ABCG4 and mi R-9b,feeding mi R-9b mimics/inhibitor and ds ABCG4 in brown citrus aphid and peaaphid,it is evident that mi R-9b regulates ABCG4 and influences wing development of aphids.4 Mechanism of mi R-9b-ABCG4 in regulation of wing developmentSilencing of two insulin receptor genes(Ac In R1 and Ac In R2)resulted in aphids with deformed wings.q RT-PCR was used to examined expression patterns of three insulin related peptide genes(IRP1,IRP3 and IRP5)in different developmental stages and tissues.The results indicated that IRP1 was highly expressed in CNS,IRP3 and IRP5 were highly expressed in fatbody.Silencing of IRP3 resulted the adults with malformed wings.Silencing of ABCG4 decreased the expression of IRP3 and silencing of IRP3 increased the expression of ABCG4 implying afeedback regulation between ABCG4 and IRP3.The relative expression of IIS pathway genes were tested after feeding mi R-9b mimics/inhibitor and ds ABCG4.The results showed that silencing of ABCG4 or overexpression of mi R-9b decreased the expression level of IIS pathway genes while silencing of mi R-9b increased the expression level of IIS pathway genes.Feeding bovine insulin assay was used to rescue the deformed wings that resulted by ds ABCG4.The results indicated that the ingestion of bovine insulin increased the expression of IRP3 but decreased the expression of ABCG4,which again implying the feedback regulation between ABCG4 and IRP3.Feeding bovine insulin and ds ABCG4 together showed no abnormal phenotypes in wing.In conclusion,we can propose a new wing development mechanism in aphids: mi R-9b negatively regulates the expression of ABCG4,and ABCG4 controls the secration of IRP3 thereby modulating the IIS pathway activity,which further regulate the wing development of A.citricidus.