Mechanisms influencing the efficiency of RNA interference in the European corn borer

The European corn borer (ECB), Ostrinia nubilalis, is an agriculturally important lepidopteran pest that exhibits low RNA interference (RNAi) efficiency. We investigated some of the mechanisms influencing RNAi efficiency in ECB and tested commonly used reagents for improving RNAi efficiency. Ex vivo incubation experiments showed that double-stranded RNA (dsRNA) was degraded in ECB gut contents and hemolymph under physiologically relevant pH conditions. Further analysis revealed transcripts for four dsRNA-degrading endonuclease (OndsRNase) genes and one RNAi efficiency-related nuclease (OnREase) gene. Expression analysis indicated that OnREase, OndsRNase2, and OndsRNase4 might impact RNAi efficiency by dsRNA ingestion, whereas OnREase and OndsRNase1 may impact RNAi efficiency by dsRNA injection. Sequencing also revealed a single transcript for Dicer 2 (OnDcr2), R2D2 (OnR2D2), and Argonaute 2 (OnAgo2). Expression analysis indicated that these genes were expressed in all ECB developmental stages and tissues investigated. Ex vivo incubation experiments showed that Metafectene Pro, chitosan-based nanoparticles, EDTA, and Zn²⁺ enhanced dsRNA stability in ECB gut contents and hemolymph, but Lipofectamine RNAiMax, Co²⁺, and Mn²⁺ did not. Surprisingly, improving dsRNA stability did not increase RNAi efficiency in ECB in vivo. Ingestion-, injection-, and tissue culture-based RNAi assays indicated that most target genes and all ECB developmental stages investigated were highly refractory to RNAi. These findings suggest that multiple mechanisms likely contribute to low RNAi efficiency in ECB. This investigation is expected to help improve RNAi efficiency in lepidopteran insects, so effective RNAi-based biopesticides can be developed.