The raw data of the figures and tables, mass spectrometry and RNA-sequencing data

RNA interference (RNAi), a naturally occurring gene silencing mechanism found in almost all eukaryotic organisms, has proven to be an adaptable and powerful tool in therapeutics, bioengineering, and agriculture. Differential responses to RNAi, however, are a key limiting factor, in which cellular uptake of exogenous dsRNA in target organisms remains poorly understood. Here, to fill this knowledge gap, we integrated omics tools with phenotypic assays to characterize dsRNA uptake mechanisms across tissues in the migratory locust, Locusta migratoria (Orthoptera). Our findings clearly demonstrate that cellular uptakeof dsRNAis tissue-dependent, involving multiple cell membrane receptors and pathways. In hemocytes, uptake is rapid and mediated by clathrin-mediated endocytosis and macropinocytosis. Epidermal cells utilize clathrin- and caveolin-mediated endocytosis, while midgut cells employ caveolin-mediated endocytosis and Sid-like channel transport. Comparatively, clathrin-mediated endocytosis appears to be the most conserved mechanism across insects, including the red flour beetle, Tribolium castaneum (Coleoptera), and the Asian corn borer, Ostrinia furnacalis (Lepidoptera). Takentogether, dsRNA enters the cells of different tissue types through diverse pathways. This systematic and comprehensive study not only advances our understanding of the cellular uptake of extracellulardsRNA and the resultant differential sensitivity to RNAi in insects, but also facilitates the ongoing integration of this species-specific biotechnology into sustainable Integrated Pest Management practices.