Designing and Validating T. castaneum dsRNA

We designed the T. castaneum cactus primer sequences for dsRNA synthesis from the iBeetle Database [30 (link)] (Table I in S1 Tables). We then verified the primer pair for the absence of secondary effects by comparing phenotypes with a non-overlapping dsRNA fragment [30 (link)] and by blasting the primer pair against the new T. castaneum genome using NCBI’s Primer-BLAST [85 (link)]. Next, we generated T7 promoter sequence-tagged DNA from T. castaneum cDNA via PCR using the Platinum Green Hot Start kit (Invitrogen). We then purified the PCR product using the QIAquick PCR Purification kit (Qiagen). Using the Megascript T7 kit (Invitrogen), we synthesized dsRNA overnight [76 (link)]. As a control for the induction of beetle RNAi, we used E. coli DNA as a template to produce dsRNA against a maltose binding protein E (malE) sequence [58 (link)]. Finally, we quantified dsRNA concentration using the Qubit microRNA Assay Kit (Invitrogen).