Using RNAi To Silence Bactrocera Dorsalis (Diptera: Tetriphitidae) Reproduction Behavior Related Genes

Bactrocera dorsalis is a destructive pest which harms fruits and vegetables mainly through sneaking into the internal by larvae and affects their yield and quality. Currently, the control research of B. dorsalis is mainly focused on lure research and other traditional control methods, but the molecular biology research is still in the blank. RNAi is a technology using dsRNA to induce post transcriptional silence of target gene. RNAi through feeding dsRNA has been successfully conducted in many insect species, but an effective feeding RNAi platform hasn't been established in Diptera species.We have selected the reproduction-related genes noa and rabll as target genes and studied gene expression, number of eggs and mortality using construction of dsRNA-expressing vector and Real-time PCR methods. Our research have explored the feasibility of feeding dsRNA to silence target gene and provided a functional tool to study function of genes and offered a broader perspective for adoption of RNAi in agricultural pest control.We have cloned cDNA of noa and rabll, covering 51% and 92% each. Analysis using BLAST indicated that the homology of each gene fragment is 82% and 85% between B. dorsalis and Drosophila melanogaster. Analysis of corresponding amino acid sequence using CLUSTAL X2.0 indicated that the homology of noa and rab11 between B. dorsalis and D. melanogaster is 94% and 98%.Escherichia coli strain HT115 was genetically engineered to express dsRNA targeting noa and rab11. qRT-PCR showed that mRNA level of two target genes was reduced compared to ds-egfp control by feeding either engineered bacteria or dsRNAs. The maximum down-regulation of noa and rab11 is 71.3% to 66.7%. Egg production was affected through feeding ds-noa and ds-rab11 compared to ds-egfp group. Silencing of rab11 through ingestion of dsRNA killed 20% of adult flies. Adult flies were continuously fed with dsRNA and bacteria expressing dsRNA for 14 days and up-regulations of target genes were observed during this process. The transcripts of noa showed up-regulation compared to ds-egfp control group on day 2 after continuous feeding engineered bacteria. The maximum over-expression is 2.55 times compared to ds-egfp control group. The transcripts of rabll showed up-regulation compared to ds-egfp control group on day 7 after continuous feeding ds-rab11. The maximum over-expression is 14.77 times compared to ds-egfp control group.Our results suggested that it is feasible to silence genes by feeding dsRNA and bacteria expressing dsRNA in B. dorsalis. Additionally the over-expression of the target gene after continuously feeding dsRNA or bacteria was observed.