Enhanced dsRNA Production via a Three-Terminator Vector and Transcriptomic Correlates of RNAi Exposure in Thrips

RNA interference (RNAi) represents a promising alternative to chemical insecticides, but its efficacy depends on efficient double-stranded RNA (dsRNA) uptake, a process poorly characterized in thrips. To enable sufficient dsRNA production for functional studies, we first optimized an E. coli expression system by constructing a vector containing three tandem terminators, which substantially enhanced dsRNA yield by approximately 11-fold. Using this optimized production system, this study identified a conserved muscle actin fragment for dsRNA synthesis and evaluated RNAi responses in Megalurothrips usitatus and Frankliniella occidentalis. Insect mortality, target-gene suppression, and transcriptomic responses were evaluated via RT-qPCR and RNA-seq analyses using artificial diets supplemented with muscle actin dsRNA. The designed dsactin shared > 97% sequence identity between the two species. Oral ingestion of 1500 ng µL^-1 dsRNA caused concentration-dependent mortality (72% in M. usitatus, 48% in F. occidentalis) and significant down-regulation of muscle actin mRNA within 72 h. Transcriptomic analysis in M. usitatus revealed upregulation of genes associated with clathrin-mediated endocytosis and SID-1-like transmembrane transport, suggesting a potential dual-pathway model for dsRNA uptake. These findings provide correlative insights into RNAi efficiency in thrips.