RESUMEN
Colorado potato beetle (CPB) is an agricultural pest of solanaceous crops, notorious for its rapid resistance development to chemical pesticides. Foliar spraying of dsRNA formulations is a promising innovative technology providing highly specific and environmentally acceptable option for CPB management. We designed dsRNA to silence CPB mesh gene (dsMESH) and performed laboratory feeding trials to assess impacts on beetle survival and development. We compared the effectiveness of in vivo and in vitro produced dsRNA in a series of laboratory experiments. We additionally performed a field trial in which the efficacy of dsRNA sprayed onto potato foliage was compared to a spinosad-based insecticide. We showed that dsMESH ingestion consistently and significantly impaired larval growth and decreased larval survival in laboratory feeding experiments. In vivo produced dsRNA performed similarly as in vitro synthesized dsRNA in laboratory settings. In the field trial, dsMESH was as effective in controlling CPB larvae as a commercial spinosad insecticide, its activity was however slower. We discuss limitations and benefits of a potential dsMESH-based CPB management strategy and list some important RNAi based CPB research topics, which will have to be addressed in future.
RESUMEN
RNA interference (RNAi) has become an efficient tool for inducing resistance to viruses in many organisms. In this study, Escherichia coli cells were engineered to produce stable double-stranded RNA (dsRNA) against the nucleopolyhedrosis virus to elicit RNAi in silkworms. The immediate-early-1 (ie-1) and late expression factor-1 (lef-1) genes of the Bombyx mori nucleopolyhedrovirus (BmNPV) involved in viral DNA multiplication were cloned in the plasmid L4440 under the influence of the double T7 promoter and transformed to E. coli HT115 DE3 host cells. On induction with isopropyl ß-D-thiogalactopyranoside, these cells efficiently produced dsRNA of the cloned genes. The B. mori larvae were fed with 50 µL of E. coli cells expressing ie-1 and lef-1 dsRNAs (each approximately 25 µg) to elicit RNAi. The semi-quantitative and quantitative PCR analysis of RNA from the midgut of the dsRNA-fed larvae revealed a significant reduction in the expression of the target genes involved in BmNPV multiplication, which restricted virus copy numbers to 100 compared with 1.9 × 105 in the infected controls. Furthermore, the dsRNA-fed infected larvae showed > 50% increased survivability compared with the infected controls. The study revealed the successful use of bacteria as vectors for efficiently delivering dsRNA to elicit RNAi against BmNPV in silkworms.