Insights into systemic RNAi: The role of extracellular vesicles in Western corn rootworm

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism induced by small, noncoding RNA molecules. It is widely used for studying gene function and in pest management. However, aspects of the RNAi mechanism, such as its systemic spread, remain less understood. Recent findings suggest that extracellular vesicles (EVs), nanosized structures involved in intercellular communication, may facilitate the systemic spread of RNAi signals. EVs are formed through different biogenesis pathways, including the ESCRT-dependent pathway, which involves endosomal sorting complex required for transport (ESCRT), and the ESCRT-independent lipid-mediated pathway. This study explored the role of EVs in the systemic spread of RNAi in the western corn rootworm (WCR) using parental RNAi (pRNAi). To examine the role of EVs in RNAi signal spread, critical steps in both EV biogenesis pathways were disrupted using specific pharmacological inhibitors, and hunchback (hb) was used as a marker gene. We hypothesized that inhibition of crucial steps of EV biogenesis in adult females would interfere with EV production and consequently reduce the occurrence and intensity of hb knockdown phenotype in offspring. Adult WCR females were fed diet treated with pharmacological inhibitors and hb dsRNA alongside positive and negative controls. The effect of the treatments on the hb knockdown phenotype was evaluated by quantifying transcript levels of hb in adult females using RT-qPCR, and by recording the hatching rate and intensity of hb phenotype. Our findings suggest that EVs contribute to the systemic spread of the RNAi signal in WCR, providing new insights into RNAi mechanisms in insects.