Screening of target genes for RNAi in Tetranychus urticae and RNAi toxicity enhancement by chimeric genes

• A dsRNA delivery method using multi-unit chambers was established.
• RNAi toxicity of 42 genes were determined based on MUC method.
• dsRNA of COPI subunit genes exhibited relatively higher toxicity.
• Recombinant dsRNAs enhanced RNA toxicity.

Due to its rapid development of resistance to nearly all arrays of acaricide, Tetranychus urticae is extremely hard to control using conventional acaricides. As an alternative control measure of acaricide-resistant mites, RNA interference (RNAi)-based method has recently been suggested. A double-stranded RNA (dsRNA) delivery method using multi-unit chambers was established and employed to screen the RNAi toxicity of 42 T. urticae genes. Among them, the dsRNA treatment of coatomer I (COPI) genes, such as coatomer subunit epsilon (COPE) and beta 2 (COPB2), resulted in high mortality [median lethal time (LT50) = 89.7 and 120.3 h, respectively]. The transcript level of the COPE gene was significantly (F3,9 = 16.2, P = 0.001) reduced by up to 24% following dsRNA treatment, suggesting that the toxicity was likely mediated by the RNAi of the target gene. As a toxicity enhancement strategy, the recombinant dsRNA was generated by reciprocally recombining half-divided fragments of COPE and COPB2. The two recombinant dsRNAs exhibited higher toxicity than the respective single dsRNA treatments as determined by LT50 values (79.2 and 81.5 h, respectively). This finding indicates that the recombination of different genes can enhance RNAi toxicity and be utilized to generate synthetic dsRNA with improved RNAi efficacy.

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