RNA interference (RNAi) has become a widely used technique of knocking down a gene's expression in insects, but its efficacy in some species is limited by a reduced ability of the cells to take in and disperse the double-stranded RNA (dsRNA) throughout the cytoplasm. While RNA transport proteins such as SID-1 and its orthologues can facilitate dsRNA uptake in some invertebrate species, dsRNA uptake in many insects examined to date appears to be facilitated by clathrin-mediated endocytosis (CME). In this study, we used pharmacological inhibitors and RNAi-mediated knockdown of endocytic genes to provide evidence that CME is the primary means of dsRNA uptake in the mosquito Aedes aegypti. Inhibition of clathrin-mediated endocytosis was sufficient to supress uptake of short (21 nt) interfering RNAs (siRNAs), short (23 nt) hairpin RNAs (shRNAs), and long (>200 nt) dsRNA molecules in Aedes aegypti cultured cells and larvae. In contrast, we observed that short (23 nt) “paperclip” RNAs (pcRNAs), with partially closed ends, efficiently enter cells via a clathrin-independent pathway and effectively facilitate transcript knockdown. This alternative dsRNA structure may prove useful in insects generally considered recalcitrant to RNAi and in insect populations where resistance to RNAi-insecticides may arise through changes in dsRNA uptake mechanisms.

RNA干扰（RNAi）已成为一种广泛使用的敲除昆虫中基因表达的技术，但是其在某些物种中的功效受到细胞吸收和分散双链RNA（dsRNA）遍及整个昆虫的能力的限制。细胞质。尽管RNA转运蛋白（例如SID-1及其直向同源物）可以促进某些无脊椎动物物种吸收dsRNA，但迄今为止，网格蛋白介导的内吞作用（CME）促进了许多昆虫中dsRNA的吸收。在这项研究中，我们使用了药理学抑制剂和RNAi介导的内吞基因敲低研究，以提供证据表明CME是埃及伊蚊中dsRNA摄取的主要手段。。抑制网格蛋白介导的内吞作用足以抑制埃及伊蚊培养的细胞和幼虫中短（21 nt）干扰RNA（siRNA），短（23 nt）发夹RNA（shRNA）和长（> 200 nt）dsRNA分子的摄取。。相反，我们观察到，短的（23 nt）“回形针” RNA（pcRNA）具有部分封闭的末端，可通过网格蛋白非依赖性途径有效进入细胞，并有效促进转录本的敲低。这种可替代的dsRNA结构可能在通常被认为对RNAi不利的昆虫中以及在通过dsRNA摄取机制的变化可能产生对RNAi杀虫剂的抗性的昆虫种群中有用。