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Double-stranded RNA (dsRNA) has been applied to control insect pests due to its induction of RNA interference (RNAi) of a specific target gene expression. However, developing dsRNA-based insecticidal agent has been a great challenge especially against lepidopteran insect pests due to variations in RNAi efficiency. The objective of this study was to screen genes of chymotrypsins (SeCHYs) essential for the survival of the beet armyworm,  Spodoptera exigua , to construct insecticidal dsRNA. In addition, an optimal oral delivery method was developed using recombinant bacteria. At least 7 SeCHY genes were predicted from  S .  exigua  transcriptomes. Subsequent analyses indicated that  SeCHY2  was widely expressed in different developmental stages and larval tissues by RT-PCR and its expression knockdown by RNAi caused high mortality along with immunosuppression. However, a large amount of dsRNA was required to efficiently kill late instars of  S .  exigua  because of high RNase activity in their midgut lumen. To minimize dsRNA degradation, bacterial expression and formulation of dsRNA were performed in HT115  Escherichia coli  using L4440 expression vector. dsRNA (300 bp) specific to  SeCHY2  overexpressed in  E .  coli  was toxic to  S .  exigua  larvae after oral administration. To enhance dsRNA release from  E .  coli , bacterial cells were sonicated before oral administration. RNAi efficiency of sonicated bacteria was significantly increased, causing higher larval mortality at oral administration. Moreover, targeting young larvae possessing weak RNase activity in the midgut lumen significantly enhanced RNAi efficiency and subsequent insecticidal activity against  S .  exigua .

Optimization of recombinant bacteria expressing dsRNA to enhance insecticidal activity against a lepidopteran insect, Spodoptera exigua