Extracellular vesicle-mediated RNAi spread in Western corn rootworm

Extracellular vesicles (EVs) are nanosized lipid-bound structures that play a key role in intercellular communication by transporting different cargoes, such as RNA. Recent findings in cell lines suggest that EVs may be involved in intercellular transmission of RNA interference (RNAi) signals. EVs can be formed through different biogenesis pathways, including the ESCRT-dependent pathway, which involves endosomal sorting complex required for transport (ESCRT), and the ESCRT-independent pathway, which relies on lipid-mediated mechanisms. This study investigated the role of EVs in the systemic spread of RNAi in the western corn rootworm (WCR) using parental RNAi (pRNAi). To investigate the role of EVs in RNAi signal spread, critical steps of both ESCRT-dependent and ESCRT-independent biogenesis pathways were disrupted using specific pharmacological inhibitors, and hunchback (hb) was used as a marker gene. We hypothesized that inhibition of crucial parts of EV biogenesis in adult females would interfere with EV production and consequently reduce the occurrence and intensity of hb knockdown phenotype in the offspring. Adult WCR females were fed diet treated with pharmacological inhibitors and hb dsRNA with the respective 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, in addition to recording the hatching rate and intensity of hb phenotype. Our results suggest that EVs are involved in the systemic spread of the RNAi signal in WCR, contributing to a better understanding of RNAi mechanisms in insects.