Study On Susceptibility Of Armigeres Subalbatus To Zika Virus And Dengue Virus And Sex Determination Mechanism

Background:Zika virus(ZIKV)and dengue virus(DENV)are closely related flaviviruses primarily transmitted by Aedes mosquitoes.ZIKV can cause Zika virus disease which was pandemic in 2015,and over twenty cases were imported to China in 2016.DENV is the pathogen of Dengue fever which is epidemic over 128 countries and regions in the world.Armigeres subalbatus is an emerging and widely distributed mosquito,and ZIKV has been detected and isolated from it.However,it is not clear whether Ar.subalbatus could be a vector for ZIKV and DENV or not.At present,due to the lack of effective vaccines for most mosquito-borne infectious diseases,the inhibition of mosquito population is still the most effective measure.The method of inhibiting the external environmental population quantity by releasing sterile male mosquitoes has been verified and applied.However,the molecular mechanism of sex determination of Ar.subalbatus is still unknown.Objective:1.To explore the susceptibility and transmissibility of Ar.subalbatus to ZIKV and DENV-2.To investigate the possibility of the larvae infect ZIKV and DENV-2 from the artificial urine,and the route of virus invades larvae.2.Screening and identification of male determinant of Ar.sulbalbatus.To explore whether the knockout of AsuMf can cause malformation or feminization phenotype of male mosquitoes,and whether effect splicing patterns of doublesex and fruitless.To clarify the male-determining mechanism of Ar.sulbalbatus.Methods:1.A line of Ar.subalbatus isolated from Guangzhou,China,and further identified by the mitochondrial cytochrome oxidase subunit I(COI)gene.The adults of Ar.subalbatus were fed with blood meal containing ZIKV or DENV-2.At 4,7,10,14,and 21 days post-inoculation(dpi),ZIKV or DENV-2 was detected and quantified in the midgut,ovaries and salivary glands by RT-qPCR and RT-qPCR.To assess transmissibility,suckling mice were bitten by ZIKV-infected mosquitoes,and ZIKV was detected in brain tissue by RT-qPCR and plaque assays.Furthermore,the larvae of Ar.subalbatus were reared in artificial urine containing ZIKV or DENV-2.The infection rates and viral titers of larvae and adults were detected by RT-PCR and RT-qPCR,and the viral distribution in the larvae tissues were observed by immunohistochemistry.2.Based on the results of genome and transcriptome sequencing of Ar.subalbatus,the male-specific gene(AsuMf)was successfully screened by chromosome quotient(CQ)method.The full length sequence of AsuMf gene was obtained by cDNA end amplification,and the structure and function of the gene were analyzed.Preparing the chromosome karyotype specimen of Ar.subalbatus and analysing the position of AsuMf gene on the chromosome by fluorescent in situ hybridization.The sgRNA was designed in the second exon of AsuMf gene,and the gene was knockout using CRISPR/Cas9 technology.Internal phenotype and external phenotype of male individuals were observed and recorded.The expression of dsxF,dsxM,fruF,and fruM in individuals with AsuMf gene knockouted were detected by RT-PCR and RT-qPCRResults:1.Following oral inoculation,ZIKV but not DENV-2 was detected in Ar.subalbatus midguts(18.0%)at 4 dpi,ovaries(8.3%)at 7 dpi and salivary glands(4.2%)at 10 dpi.Eight days after bitten by ZIKV-positive mosquitoes,ZIKV was detected in the brain tissues of three of four suckling mice.ZIKV can be detected in the larvae reared in artificial urine contained ZIKV at a high concentration of 105 pfu/mL and various tissues of grow-up adults with a low infection rate(0.93%).ZIKV can be observed in anal papillae and midgut of larvae at 4 dpi under laboratory conditions.DENV-2 was not detected in the larvae reared in artificial urine.2.Based on sequencing data and laboratory results,we successfully selected one male-specific gene(AsuMf)from 15 candidate genes.RT-PCR and RT-qPCR results showed that the AsuMf gene was consistent with the characteristics of male determinant fator,including early embryo(E4-8h)and male-specific expression.The results of fluorescence in situ hybridization confirmed that Ar.subalbatus have homomorphic chromosomes and the AsuMf gene was located on chromosome 3.SgRNA designed in the coding region could successfully knock out the AsuMf gene with an average knock-out rate of 59.5%in the three replicates.The external and internal phenotypes of the knockout G0 individuals were malformed and feminized,and the alternative splicing of the downstream dsx and fru genes also showed a trend to female.Conclusion:1.Ar.subalbatus is a potential vector for ZIKV,and larvae can also serve as a route of viral infection and transmission.2.The M factor(AsuMf)located on chromosome 3 in Ar.subalbatus is male determinant.The deletion of the AsuMf gene may directly or indirectly regulate the formation of alternative splicing of the downstream dsx and fru genes.