RNAi Efficiency through dsRNA Injection Is Enhanced by Knockdown of dsRNA Nucleases in the Fall Webworm, Hyphantria cunea (Lepidoptera: Arctiidae)

Abstract

RNA interference (RNAi) technology is a promising approach used in pest control. The efficiency of RNAi varies considerably among different insect species, and growing evidence suggests that degradation of double-stranded RNA (dsRNA) prior to uptake is an important factor that limits RNAi efficiency in insects. Our recent work on fall webworm (Hyphantria cunea), an important invasive pest in China, showed a relatively low silencing efficiency of RNAi through dsRNA injection, which is considered the most feasible dsRNA delivery method for inducing RNAi, and the factors involved in the mechanism remain unknown. Herein, we first detected the dsRNA-degrading activity in the hemolymph and gut content of H. cunea in ex vivo assays and observed rapid degradation of dsRNA, especially in the hemolymph, which was complete within only 10 min. To determine whether dsRNA degradation could contribute to the low effectiveness of RNAi in H. cunea, four dsRNA nuclease (dsRNase) genes, HcdsRNase1, HcdsRNase2, HcdsRNase3, and HcdsRNase4, were identified by homology searching against the H. cunea transcriptome database, and their transcript levels were subsequently investigated in different tissues, developmental stages, and after dsRNA injection. Our results show that HcdsRNases are highly expressed mainly in gut tissues and hemolymph, and the expression of HcdsRNase3 and HcdsRNase4 were significantly upregulated by dsGFP induction. RNAi-of-RNAi studies, using HcCht5 as a reporter gene, demonstrated that silencing HcdsRNase3 and HcdsRNase4 significantly increases RNAi efficacy via dsHcCht5 injection, and co-silencing these two HcdsRNase genes results in a more significant improvement in efficacy. These results confirm that the RNAi efficacy in H. cunea through dsRNA injection is certainly impaired by dsRNase activity, and that blocking HcdsRNases could potentially improve RNAi, providing a reference for related studies on insects where RNAi has low efficiency.

Gene cloning, protein expression, and enzymatic characterization of a double‐stranded RNA degrading enzyme in Apolygus lucorum

“…Recent studies have found 4 dsRNases in H. cunea , HcdsRNase1 , HcdsRNase2 , HcdsRNase3 , and HcdsRNase4 (Zhang et al. , 2022). There are 3 different dsRNases in Cylas puncticollis and 5 dsRNases in the tobacco cutworm Spodoptera litura (Peng et al.…”

“…In contrast, SgdsRNase1, SgdsRNase3, and SgdsRNase4, which are also highly expressed in the midgut, do not affect RNAi efficiency (Wynant et al, 2014). Recent studies have found 4 dsRNases in H. cunea, HcdsRNase1, HcdsRNase2, HcdsRNase3, and HcdsRNase4 (Zhang et al, 2022). There are 3 different dsRNases in Cylas puncticollis and 5 dsRNases in the tobacco cutworm Spodoptera litura (Peng et al, 2020).…”

“…In Lepidoptera insects, RNAi is often inefficient (Liu et al, 2012), one of the reasons is that dsRNAs are rapidly degraded by dsRNAdegrading nuclease (dsRNase) (Wang et al, 2016). Recent work on the fall webworm (Hyphantria cunea), a lepidopteran insect, has proved that HcdsRNase3 and HcdsRNase4 cause the low efficiency of RNAi of H. cunea in vivo and the rapid degradation of dsRNA in gut fluids and hemolymph (Zhang et al, 2022). DsR-Nase belongs to Mg 2+ -dependent nuclease capable of degrading single-stranded RNA (ssRNA) or DNA (ss-DNA), double-stranded DNA (dsDNA), RNA/DNA hybrid, but mainly dsRNA (Siwecka, 1997;Meiss et al, 1999).…”

Influence of diverse storage conditions of double‐stranded RNAin vitro on the RNA interference efficiency in vivo insect Tribolium castaneum

“…31 Among these factors, the stability of dsRNA in vivo is considered to be the critical element for the variable RNAi efficiency in insects. 17,[32][33][34] Many recent studies have focused on the factors affecting dsRNA stability in living insects, mainly referring to the dsRNA degradation attributed to the exocellular nuclease in the gut, 26,27,35 hemolymph, 28,30,36,37 and salivary secretions. 38,39 However, little attention has been paid to the stability of dsRNA in vitro prior to being administered into insects.…”

Silenciamento gênico de >i<Diatraea saccharalis>/i< utilizando a técnica de RNA de interferência (RNAi)

“…Em Hyphantria cunea (Lepidoptera), usando o gene chitinase 5 (Cht5) como genealvo, demonstrou-se que silenciar genes que codificam dsRNases (especialmente HcdsRNase3 e HcdsRNase4) aumenta significativamente a eficácia de RNAi via injeção de dsHcCht5, e co-silenciar esses dois genes HcdsRNases resultam em uma melhora ainda mais significativa na eficácia (Zhang et al, 2022). Também, em Cnaphalocrocis medinalis (Lepidoptera), o cosilenciamento de CmCHS (quitina sintase de C. medinalis) e CmdsRNase afetou significativamente o crescimento e desenvolvimento do inseto e, assim, melhorou a eficácia do RNAi .…”