Molecular mechanisms of dsRNA uptake and intracellular trafficking in the fat body of Locusta migratoria

Abstract

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism initiated by exogenous double-stranded RNA (dsRNA), which leads to the degradation of target mRNA within the cell. Since its discovery, RNAi has become a powerful tool for functional genomics and a promising strategy for pest control. However, the cellular uptake and intracellular trafficking of dsRNA, especially in specific insect tissues, remain poorly understood. In this study, we systematically investigated the uptake, transport, and endosomal escape mechanisms of dsRNA in the fat body of Locusta migratoria. Our results reveal that L. migratoria apolipophorin III (LmApoLp-III), a lipid-binding protein, functions as a key carrier that facilitates the initial transport of dsRNA from the hemolymph to the fat body cell membrane. There, dsRNA is recognized and bound by a set of membrane receptors, including scavenger receptors (LmSRA, LmSRC) and lipoprotein receptors (LmLPR, LmLRP1, LmLRP2, and LmLRP3), forming receptor complexes that mediate cellular internalization via clathrin-mediated endocytosis and macropinocytosis. Intracellular trafficking of dsRNA is orchestrated by several L. migratoria small Rab GTPases (LmRab4, LmRab7, and LmRab9), which are critical for vesicular transport. Furthermore, vacuolar-type H⁺-ATPase (V-ATPase) facilitate the escape of dsRNA from endosomes, allowing its entry into the RNAi machinery to exert its gene-silencing function. Together, these findings provide the first comprehensive characterization of the dsRNA uptake pathway in insect fat body tissue, offering valuable insights into RNAi mechanisms and informing the development of more effective RNAi-based pest control strategies.