Involvement of clathrin-dependent endocytosis in cellular dsRNA uptake in aphids

RNAi is an essential technology for studying gene function in eukaryotes, and is also considered to be a potential strategy for pest control. However, the mechanism behind the cellular uptake of dsRNA in aphids, a group of important agricultural sucking pests, remains unknown. Here, using the pea aphid Acyrthosiphon pisum as model for aphids, we identified two core genes of clathrin-dependent endocytosis (CDE), Apchc and Apvha16. We confirmed that expression of Apchc, Apvha16 and RNAi core component genes (ApAgo2, ApDcr2 and ApR2d2) were simultaneously induced at 12 h after feeding dsRNA. By using an RNAi-of-RNAi approach, we demonstrated that suppression of Apchc and Apvha16 transcripts by RNAi significantly impaired RNAi efficiency of selected reporter genes (RGs), including ApGNBP1, Apmts and Aphb, suggesting the involvement of CDE in cellular dsRNA uptake in aphids. Further confirmation was also provided using two inhibitors, chlorpromazine (CPZ) and bafilomycin A1 (BafA1). Administration of CPZ and of BafA1 both led to an impaired silencing efficiency of the RGs in the pea aphid. Finally, these RNAi-of-RNAi results were reconfirmed in the peach aphid Myzus persicae. Taking these findings together, we conclude that CDE is involved in cellular dsRNA uptake in aphids.