Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis

Abstract

BACKGROUND Targeting insect‐specific genes through post‐transcriptional gene silencing with RNA interference (RNAi) is a new strategy for insect pest management. However, lepidopterans are recalcitrant to RNAi, which prevents application of novel RNAi technology to many notorious pests, including Ostrinia nubilalis (ECB). Strategies for enhancing RNAi efficiency, including large doses of double‐stranded RNA (dsRNA), nuclease inhibitors, transfection reagents, and nanoparticles, have proved useful in other insects exhibiting substantial dsRNA degradation, a major mechanism limiting RNAi efficacy. To determine if similar strategies can enhance RNAi efficiency in ECB, various reagents were tested for their ability to enhance dsRNA stability in ECB tissues, then compared for their effectiveness in whole ECB. RESULTS Ex vivo incubation experiments revealed that Meta dsRNA lipoplexes, EDTA, chitosan‐based dsRNA nanoparticles, and Zn2+ enhanced dsRNA stability in ECB hemolymph and gut content extracts, compared with uncoated dsRNA. Despite these positive results, the reagents used in this study were ineffective at enhancing RNAi efficiency in ECB in vivo. To reduce assay time and required dsRNA, midguts were dissected and incubated in tissue culture medium containing dsRNA with and without reagents. These experiments showed that RNAi efficiency varied between target genes, and nuclease inhibitors improved RNAi efficiency for only a portion of the refractory target genes investigated ex vivo. CONCLUSION These results indicate that enhancing dsRNA stability is insufficient to improve RNAi efficiency in ECB and suggests the existence of additional, complex mechanisms contributing to low RNAi efficiency in ECB.

Strategies for enhancing the efficiency of RNA interference in insects

“…Similarly, lipofectamine was used in striped rice stem borer ( Chilo suppressalis ) experiments to enhance dsRNA uptake, distribution, and larval mortality 35 . In contrast, METAFECTINE© PRO did not affect RNAi efficiency in European corn borer ( Ostrinia nubilalis ) when fed to neonate larvae 33 . Whereas these experiments have shown promise in enhancing RNAi efficiency in some pest insects, the transfection reagents themselves are both expensive and potentially toxic to insects 33 and nontarget species 36 .…”

“…Since chitosan polymers are positively charged, the chitosan is electrostatically attracted to negatively charged dsRNA molecules, forming nanoparticle complexes. Chitosan‐dsRNA nanoparticles were also used in the lepidopteran pests, S. frugiperda 38 and O. nubilalis 33 . The chitosan nanoparticles effectively enhanced dsRNA stability in the larval gut in both insect species.…”

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults

“…Based on work in the red flour beetle, Tribolium castaneum (Herbest; Coleoptera: Tenebrionidae), the lethal giant larvae ( Lgl ) gene appears to be an ideal reporter gene for RNAi‐mediated gene silencing because it fits all these criteria and results in molting defects and mortality (Xiao et al, 2014). However, when this gene was suppressed with RNAi in the European corn borer, Ostrinia nubilalis (Hubner; Lepidoptera: Crambidae) no phenotypes were observed (Cooper, Song, Yu, et al, 2021), leading to questions about the suitability of Lgl as a target gene for RNAi in insects.…”

“…Coleopterans, like D. v. virgifera , are well known for being highly amenable to RNAi, whereas lepidopterans typically are not (Cooper et al, 2019; Terenius et al, 2011; Zhu & Palli, 2020). Therefore, it is hard to know if the lack of phenotype recently observed after the suppression of Lgl in multiple developmental stages of O. nubialis is due to variation in the susceptibility of this gene to RNAi in insects (Cooper, Song, Yu, et al, 2021), due to logistical differences in experimental design (Cooper et al, 2019; Terenius et al, 2011), or due to another mechanism of low RNAi efficiency in O. nubialis (Cooper et al, 2019, Cooper, Song, Shi et al, 2020, Cooper, Yu, Biondi et al, 2020; Cooper, Song, Shi, et al, 2021; Cooper, Song, Yu, et al, 2021). Therefore, identification and characterization of Lgl in D. v. virgifera may clarify the suitability of Lgl as a target or reporter gene for RNAi in insects.…”

“…We characterized gene expression profiles to determine in which developmental stages and tissues DvLgl can be targeted for comparison of expression of this gene in other insects. Also, we performed RNAi of DvLgl in D. v. virgifera for comparison with RNAi bioassays targeting TcLgl from T. castaneum (Xiao et al, 2014) and OnLgl from O. nubilalis (Cooper, Song, Yu, et al, 2021). The results confirm that Lgl is suitable for use as a target gene for RNAi, as it is a single copy gene that is expressed in all developmental stages and tissues investigated.…”