Paperclip RNA induces lethal gene silencing in fall armyworm (Spodoptera frugiperda)

The fall armyworm (FAW, Spodoptera frugiperda) is a major lepidopteran pest threatening global agriculture. RNA interference (RNAi) has emerged as a promising strategy for sustainable pest management, yet it remains ineffective against FAW due to physiological barriers. In this study, we present the first report evaluating mortality and silencing efficacy of paperclip-structured RNAs (pcRNAs) in FAW larvae. Incubation with gut fluids from FAW larvae demonstrated higher stability of pcRNA compared to linear dsRNA. Distinct pcRNAs targeting the Juvenile Hormone Esterase (JHE), Chitin Synthase 2 (CHS), Acetylcholinesterase (ACE), Ecdysteroid Receptor (ECR), and MESH genes were synthesized and delivered orally on the surface of artificial diet to FAW neonates. All pcRNAs induced specific gene silencing when applied individually. While mortality increased for all pcRNAs compared to negative controls (pcGFP and water), this increase was only significantly different in the case of MESH pcRNA. Furthermore, MESH pcRNA induced higher mortality than the linear MESH dsRNA, reinforcing the enhanced stability and efficacy of the pcRNA structure. Moreover, the simultaneous silencing of multiple genes using pcRNA cocktails resulted in the highest levels of larval mortality. In contrast, a viroid-structured dsRNA vector pyramiding ACE/JHE/CHS genes did not induce significant mortality, suggesting the superior performance of the paperclip design for multiplex gene targeting. These results highlight the importance of gene target combinations and structural RNA design for efficient RNAi in FAW. The pcRNA format demonstrated superior silencing efficiency and mortality induction over traditional dsRNA formats, supporting its potential for use in next-generation RNAi-based biopesticides for FAW control.