Developing novel RNAi-based biopesticides targeting the central nervous system of Spodoptera littoralis

Abstract

The Egyptian cotton leafworm, Spodoptera littoralis, is a highly polyphagous lepidopteran capable of damaging > 80 crop species. Most commercial insecticides targeting S. littoralis threaten non-target organisms and the environment. RNA interference (RNAi), a natural immune defence mechanism that regulates endogenous gene expression, can be triggered by double-stranded RNA (dsRNA), causing insect mortality. Novel sprayable biopesticides incorporating dsRNA or genetically engineered plants expressing dsRNA offer highly specific pest control methods. This project explored the feasibility of controlling S. littoralis via an RNAi approach, with the end goal of developing a dsRNA-based biopesticide targeting this insect. Three genes essential to central nervous system functionality, acetylcholinesterase 1 (ace-1), nicotinic acetylcholine receptor (nAChR) and voltage-gated sodium channel (para) were targeted; NADPH cytochrome P450 reductase (NADPHcytP450r) was targeted to elucidate its role in insecticide detoxification. Oral dsRNA delivery to 4th instar larvae via artificial diet and to 5th instar via gavage feeding did not significantly reduce (P > 0.05) gene expression, survival, pupation or adult emergence likely because dsRNA is rapidly degraded by nucleases in S. littoralis midgut juice, as revealed by ex vivo dsRNA incubation. Continuous feeding of dsRNA to neonates significantly reduced larval weight (P < 0.05), suggesting reduced nuclease activity in earlier instars, supported by the lack of significant effect on gene expression and survival upon direct dsRNA haemolymph injection into 4th instar larvae compared to significant developmental delays upon 3rd instar injection. Soaking eggs in ace- 1, nAChR and NADPHcytP450r dsRNA significantly reduced (P < 0.05) hatching, indicating their non-cholinergic roles. Formulation with chitosan, a nanoparticle capable of protecting dsRNA and increasing cellular uptake, did not enhance dsRNA stability in pure midgut juice nor significantly reduce (P > 0.05) survival of 4th instar or neonate larvae. Pre-exposure to NADPHcytP450r dsRNA reduced the LC50 of deltamethrin against S. littoralis suggesting its role in detoxification. Bioinformatic analyses suggested the safety of these molecules against non-target organisms, confirmed by bioassay against Bombus terrestris. This work highlights the efficacy of targeting these genes whilst also indicating the need for optimised delivery techniques.