Establishment Of Large-scale Mosquito Densovirus Production Methods And Identification Of Circular RNAs In Aedes Albopictus

Background:Mosquito-borne diseases(MBDs)cause a significant proportion of the global infectious disease burden.Vector control remains the primary strategy available to reduce the transmission of MBDs.However,long-term,wide-scale and large-scale traditional chemical pesticide application has caused significant and increased negative effects on ecosystems and broader emerging insecticide resistance in vectors;therefore,the development of a novel alternative approach is urgently needed.Mosquito densoviruses(MDV)are entomopathogenic viruses that exhibit a narrow host range and multiple transmission patterns,making MDV a great potential bioinsecticide.However,the application process has been relatively stagnant over the past three decades.The major obstacle has been that viruses must be produced in mosquito cell lines;therefore,the production process is both expensive and time-consuming.Circular RNA(circRNA)is a newly discovered non-coding RNA(ncRNA).Unlike linear RN A,circRNA form covalently closed loop structure by jointing 3' and 5' ends together via exon circularization or intron circularization.Through which way it is more stable and conserved than linear RNA.It exists in a large number of eukaryotic cells,has a high degree of conservation and specificity in spatiotemporal,tissue,and disease expression,high stability,and plays a key role in various biological functions,such as the regulation of gene expression and alternative splicing.Evidence is emerging that some circRNAs might regulate microRNA(miRNA),because of circRNA containing many miRNA binding sites,which can play a role as competitive endogenous RNA(ceRNA)and as the miRNA "sponge"to remove the inhibition effect of the target genes.CircRNAs have been identified in some species,including western honeybees,drosophila and Bombyx mori.However,the understanding of mosquito circRNA is still very limited,and to date,no study on Aedes albopictus circRNA has been conducted.Here,the circRNAs in the Aedes albopictus were identified and validated.Differentiall expressed circRNAs(DEcircRNAs)were further investigated.Objective:1.MDV has huge potential in the prevention and control of mosquito-borne diseases.Explore an economical,fast,and commercial method for producing MDV.2.By studying the circRNA of Aedes albopictus,to further understand the function and mechanism of circRNA in mosquitoes,to provide a basis for the research of circRNA in other insects,and to provide new ideas for mosquito controlMethods1.Two wild-type(wt)MDVs,Aedes albopictus densovirus-3(AalDV-3)and Aedes aegypti densovirus(AaeDV),and a recombinant rAaeDV-210 were used to infect the Aag2 and C6/36 mosquito cell lines and the 1st-2nd instar and 3rd-4th-instar larvae of Ae.albopictus,Ae.aegypti and Cx.quinquefasciatus.Viral titers and yields in cells,media,larvae and rearing water and total viral yield were evaluated.Three kinds of virus displayed higher maximum virus titers in vivo than in vitro,and they displayed higher maximum viral yields in rearing water.2.High-throughput RNA sequencing(RNA-seq)was performed to assess circRNA expression profiles in Aedes albopictus,and significantly dysregulated circRNAs were validated by reverse transcription polymerase chain reaction(RT-PCR)and real-time quantitative polymerase chain reaction(qPCR).Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed to predict the potential functions of high expression circRNAs.Results:1.The three viruses displayed higher total maximum viral yields in C6/36 cells than in Aag2 cells.The three viruses displayed higher total maximum viral yields in larvae than in water body.Higher viral yields were produced by 1st-2nd-instar larvae compared to 3rd-4th-instar larvae.The recombinant viruses did not display significantly lower yields than wt viruses in nearly all samples.In summary,by using 100 1 st-2nd-instar Aedes mosquito larvae in 200 ml of rearing water,more than 1013 genome equivalents(geq)MDV yield can be obtained.2.Female and male Aedes albopictus were sequenced using RNA-seq,and a total of 1531 circRNAs were predicted using bioinformatics.These circRNAs were approximately 200-1000 nt in length and could be classified into four types:exon-exon,exon-intergenic,intergenic-exon and intergenic-intergenic.The exon-intergenic circRNAs were the most abundant.Moreover,286 DEcircRNAs were identified in the female vs male comparison group,including 103 highly expressed in famale and 183 highly expressed in male.Interestingly,10 high reads count circRNAs were in both female and male.Finally,randomly selected circRNAs were verified by RT-PCR and qPCR,confirming the reliability of our sequencing dataConclusions:1.Considering the lower production cost,this in vivo method has great potential for the large-scale production of MDV.MDV exhibits promising prospects and great potential for mosquito control in the future.2.In this study,1,531 circRNAs were identified in Aedes albopictus using the DCC software(v 0.4.4 https://github.com/dieterich-lab/DCC)algorithm.CircRNA was divided into 4 types,and the existence of circRNA was verified by RT-PCR and qPCR.The results of this study lay the foundation for further study of circRNA in mosquitoes.