A Comparative Analysis of RNAi Trigger Uptake and Distribution in Mosquito Vectors of Disease

Abstract

In mosquitoes, the utilization of RNAi for functional genetics is widespread, usually mediated through introduced double-stranded RNAs (dsRNAs) with sequence identity to a gene of interest. However, RNAi in mosquitoes is often hampered by inconsistencies in target gene knockdown between experimental setups. While the core RNAi pathway is known to function in most mosquito strains, the uptake and biodistribution of dsRNAs across different mosquito species and life stages have yet to be extensively explored as a source of variation in RNAi experiments. To better understand mosquito-RNAi dynamics, the biodistribution of a dsRNA to a heterologous gene, LacZ (iLacZ), was tracked following various routes of exposure in the larval and adult stages of Aedes aegypti, Anopheles gambiae, and Culex pipiens. iLacZ was largely limited to the gut lumen when exposed per os, or to the cuticle when topically applied, but spread through the hemocoel when injected. Uptake of dsRNA was noted in a subset of cells including: hemocytes, pericardial cells of the dorsal vessel, ovarian follicles, and ganglia of the ventral nerve cord. These cell types are all known to undergo phagocytosis, pinocytosis, or both, and as such may actively take up RNAi triggers. In Ae. aegypti, iLacZ was detected for up to one week post exposure by Northern blotting, but uptake and degradation drastically differed across tissues. The results presented here reveal that the uptake of RNAi triggers is distinct and specific to the cell type in vivo.

Keywords: Aedes; Anopheles; Culex; RNA interference; biodistribution; dsRNA; per os.

Figure 1

Multiple cell types uptake iLacZ in Ae. aegypti ovaries. Confocal microscopy of ovaries dissected 24 h post intrathoracic injection of iLacZ vs. no treatment control. Cell types shown include primary follicles composed of: follicular epithelia (FE), nurse cells (NC), and oocytes (OO), as well as secondary follicles (SF), epithelial cells of the ovarian sheath (SH), and atretic follicles (AT). Scale bar = 100 µm.

Figure 2

Tracking iLacZ by Northern blot in Ae. aegypti adult females. Overlaid gel electrophoresis and Northern blot runs following peritoneal exposure of iLacZ. (A) iLacZ in tissue extracts including the head (HE), thorax (TH), abdomen (AB), midgut (MG), and ovary (OV) at 4 and 24 h post exposure. (B) Time-course of iLacZ clearance in whole-body RNA extracts from 0.25 to 168 h post exposure. (C) Whole body extracts with iLacZ at 168 h post exposure with (+) and without (-) DNase treatment, single replicate performed. (D) Degradation time-course of a 200 bp, 400 bp, and 600 bp iLacZ products at 24, 72, and 168 h post exposure. L = 100 bp ladder, rRNA = ribosomal RNA loading controls imaged immediately prior to Northern membrane transfer, iLacZ = iLacZ 377 bp Northern blot probe, (+) ctrl = iLacZ stock solution, (-) untreated Ae. aegypti whole body RNA.