| Background:Aedes-borne diseases such as dengue fever,Zika and Chikungunya are becoming major global health emergencies,and old threats such as yellow fever are re-emerging.As an important vector,Aedes albopictus has spread to other continents except Antarctica,showing a grim situation of global spread and has become one of the most dangerous 100 invasive species.Due to the lack of safe and effective vaccines and special effects drugs,mosquito vector control has long been a key part of the management of mosquito-borne diseases.The control of mosquitoes mainly includes population reduction and population replacement.The main methods of population reduction is to reduce mosquito populations by applying chemical pesticides or to prevent mosquito breeding by removing larval habitats.However,with the large-scale use of chemical pesticides,the insecticides resistance of mosquitoes has increased.In recent years,the infection rate of mosquito-borne diseases has increased year by year,and environmental pollution has also appeared.The development of mosquito densovirus(MDV)as a new mosquito bio-insecticide will provide a new idea for the biological control of mosquito-borne diseases.MDV specifically infects mosquitoes and their life history,making it a potential for efficient and safe bio-mosquitoicides.Because wild-type MDV has the characteristics of long acting time and weak toxicity,it is a feasible method to carry out genetic recombination and enhance its pathogenicity to mosquitoes.However,in previous studies,exogenous nucleic acids were introduced in MDV,but with the introduction of foreign genes,the virus also lost its ability to replicate autonomously.Since mosquito-borne viruses transmit through bites of female mosquitoes,controlling the population of female mosquitoes is the mainly control methods.At present,genetic strategies for controlling mosquito-borne diseases such as dengue fever based on the release of sterile mosquitoes to replace wild-type mosquitoes have been tried and achieved success.Studies have confirmed the male specificity of AalNix,and it is necessary for the normal development of male phenotype,but the effect of AalNix on the internal genitalia of Aedes albopictus remains to be further studied.C6/36 is derived from male Aedes albopictus,but whether C6/36 cells can be used as a model for sex determination mechanism remains unknown.Objective:1.Using the expression strategy of intronic small RNA(sRNA),construct the recombinent MDV(rMDV)vector expressing endogenous miRNAs,miRNA sponges and artificial miRNAs(amiRNAs).rMDV specificly infects and enters mosquito cells,inducing overexpression and silencing of endogenous miRNAs,and inducing endogenous gene silencing,respectively.2.Compare the knockout efficiency of AalNix between C6/36 and embryos to determine whether C6/36 cells can be used to test the sgRNA efficiency in Aedes albopictus;explore AalNix whether participate in the regulation of sex determination pathways in C6/36;investigate whether AalNix knocked-out causes malformation and femaleization of male genitalia,further elucidating the male determination function of AalNix in Aedes albopictus.Methods:1.rMDV vectors that expressing miRNA,miRNA sponge and amiRNA were constructed by intronic small RNA expression strategy.The splicing efficiency of the artificial intron in the rMDV was evaluated in mosquito cells,and the overexpression and silencing effect of the rMDV on the endogenous miRNA was verified in vitro and in vivo,respectively.Transmission electron microscopy was used to detect the integrity of rMDV.PCR and DNA sequencing were used to detect the genetic stability of rMDV and its vector in their host C6/36 cells and E.coli.qPCR was used to detect the proliferation of rMDV in C6/36 cells and the rearing water of larvae.2.Design sgRNA for AalNix exon 1,construct vector which could express the corresponding sgRNA and Cas9 and transfect it into C6/36 cells to test the gene editing efficiency of each sgRNA.The RFP and Puromycin resistance genes were knocked into AalNix,AalNix-deficient cells were enriched by puromycin screening and flow cytometry,and the expression of aaldsxF and aalfruF was detected by RT-qPCR.sgRNA synthesized in vitro and Cas9 protein were co-injected into Ae.albopictus embryos,and the genital phenotype was observed.HRMA and sequencing confirmed the knockout efficiency of AalNix.The expression level of doublesex and fruitless female and male specific splicing isoform were detected respectively.Results:1.non-defective MDV vectors and fluorescent defective rMDV vectors were constructed.After the rMDV vectors were transfected into C6/36,the artificial intron can be efficiently splicing.Following the mosquito larvae been infected,the virus can spread to whole body.Intronic miRNA expression strategy of rMDV promoted over-expression of miR-210 and let-7 in vivo and in vivo,respectively.Intronic miRNA sponge expression strategy of rMDV induced silencing of miR-210 and let-7 in vivo and in vitro,respectively.Intronic amiRNA expression strategy of rMDV induces the target V-ATpase gene silencing in vivo and in invito.The intact rMDV was detected by transmission electron microscopy.The proliferation ability in C6/36 cells and larvae rearing water were not signifigant difference with wild-type MDV.Moreover,rMDV and its vectors are genetically stable.2.Five sgRNA expression vectors were constructed,in which sgRNA 1 and sgRNA2 have gene editing activity for AalNix,the mutation rate of sgRNA1 is 10.00%(4/40),and the mutation rate of sgRNA2 is 17.50%(7/40).After C6/36 knocked out AalNixthe expression levels of aaldsxF and aalfruF increased.sgRNA 1,sgRNA2 and Cas9 proteins were co-injected into Ae.albopictus embryos.36 malformed and feminazied mosquitoes were observed.Most of these individuals also had deformity and femizied genitalia,and 20 of them were determined mutations with HRMA.The melt curve of one sgRNA1 site and 15 sgRNA2 sites were different with wild-type individuals.All 9 sequenced individual were detected sgRNA2 site mutations,while only 3 indivdual of them were found sgRNAl site mutations.The knockout efficiency of sgRNA2 was higher than that of sgRNAl,which is consistent with C6/36 cells.Following AalNix knocked-out,both aaldsx and aalfru showed a bias to female isoforms.Conclusion:1.rMDV can induce over-expression and silencing of endogenous miRNAs,as well as induce efficient gene silencing in vivo by expressing amiRNAs.rMDV has wild MDV-like proliferation and secondary transmission capabilities.This study is the first to construct the non-defective rMDV miRNA delivery system,which can be used as a tool for mosquito gene function analysis,laying the foundation for the use of viral transgenic virions(viral paratransgenesis)for mosquito control.2.C6/36 cells can be used to test the knockout efficiency of sgRNA and apply it to embryos in Ae.albopictus.C6/36 cells can be used to study the molecular machanism of AalNix in sex-determining.AalNix is critical for male genital development,further confirming that AalNix is required for male development.Despite-70 MY of divergence,Nix functions as a conserved male-determining factor in the two most important arboviral vectors,namely Ae.aegypti and Ae.albopictus. |
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