Insecticidal action of the botanical insecticide wilforine on Mythimna separata (Walker) related with the changes of ryanodine receptor expression.

The detailed molecular mechanism of wilforine, a novel botanical insecticidal component, remains unclear, except for the knowledge that it affects the calcium signaling pathway. The aim of the current study was to examine the underlying molecular mechanism of wilforine in Mythimna separata (Walker) by transcriptome and RNA interference (RNAi), with chlorantraniliprole as control. RNA sequencing showed that the relative expression of genes related to the calcium signaling pathway and muscle contraction in M. separata treated with wilforine significantly changed and was further validated by qRT-PCR. Interestingly, the expression level of the ryanodine receptor (MsRyR) gene was downregulated by wilforine at relatively high concentrations and long treatment time, contrary to that observed using chlorantraniliprole. Furthermore, a putative MsRyR was cloned using a 16,258-bp contiguous sequence containing a 308-bp 5'-untranslated region and 578-bp 3'-untranslated region by RT-PCR and RACE. The results of the RNAi experiment showed that injection of dsMsRyR significantly reduced MsRyR mRNA levels, and growth and development were inhibited. Importantly, silencing of the MsRyR gene resulted in decreased susceptibility to both wilforine and chlorantraniliprole. Together with the results of our previous studies on toxic symptoms and muscle tissue lesions between wilforine and chlorantraniliprole, we propose that RyR Ca release channel dysfunction is closely related with significant lethal mechanisms of wilforine.