Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

Abstract

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Figure 1:. f-Agdsx and fkh knockdown in adult An. gambiae reduces fkh mRNA levels in the SGs and affects the female ability to blood-feed.

(A) Representative image of the plasmid design utilized for dsRNA production in this methodology. The second T7 promoter sequence is added to the plasmid by including it in the 3’ primer used to amplify the insert to be cloned into the pGEMT plasmid. The plasmid is then transformed into E. coli HT115 (DE3) bacteria and a feeding solution is made of a suspension of induced heat-killed bacteria in 10% sugar water. (B) Animals fed with a dsRNA feeding solution for either f-Agdsx or fkh, showed significantly lower levels of fkh transcripts (one-way ANOVA with multiple comparisons; n=15). However, only the group fed with fkh dsRNA (C) showed a significant difference in the number of biting attempts needed to acquire a blood meal. Mosquitoes in this group needed, on average, five times the number of probing attempts to obtain a successful blood meal than needed by the control or the dsx-dsRNA fed groups (one-way ANOVA with multiple comparisons; n=15). Error bars indicate the Standard Error of the Mean (SEM). Each experiment was conducted in three separate biological replicates. Please click here to view a larger version of this figure.

Figure 2:. fkh knockdown in adult An. gambiae salivary glands reduces SG transcription factor levels.

Shown are representative images from day 13 adult female An. gambiae SGs after 8 days (days 5–13) of oral exposure to either (A) non-related dsRNA control (ant) or (B) dsRNA targeting the SG TF fork head (fkh, AGAP001671) in 10% sucrose stained with the dyes DAPI (DNA; red), labeled wheat germ agglutinin (WGA, chitin/ O-GlcNAcylation; blue), antisera against the SG TFs Sage (green) and CrebA (purple). Scale bar lengths shown are microns. SGs (i) are outlined with white dashes. Yellow lines in zoomed lobe images (of the regions enclosed by yellow boxes, and labeled “inset”) indicate where the line scans of signal intensity were conducted. Green and purple channel intensities corresponding to line scans for each zoomed lobe are plotted (always from left to right in the SG) in the graphs below the images; X-axis = distance (in pixels) and Y-axis = gray unit (pixel intensity). The pixel intensity’s dynamic range is delimited by red (maximum) and blue (minimum) dotted lines and the corresponding values are shown on each graph. MIP = maximum intensity 3D projection through the entire SG depth. DL: distal lateral lobe; M: medial lobe; PL: proximal lateral lobe; SD: salivary duct. Please click here to view a larger version of this figure.

Figure 3:. fkh knockdown in adult An. gambiae salivary glands reduces SG secreted protein levels.

Shown are representative images from day 13 adult female An. gambiae SGs after 8 days (days 5–13) of oral exposure to either (A) non-related dsRNA control (ant), or (B) dsRNA targeting the SG TF fork head (fkh,AGAP001671) in 10% sucrose stained with the dyes DAPI (DNA; red), labeled wheat germ agglutinin (WGA, chitin/ O-GlcNAcylation; blue), and the saliva proteins AAPP (green) and Mucin (MUC2, purple). Scale bar lengths shown are microns. SGs (i) are outlined with white dashes. Yellow lines in zoomed lobe images (of the regions enclosed by yellow boxes) indicate where the line scans of signal intensity were conducted. Green and purple channel intensities corresponding to line scans for each lobe are plotted (always from left to right in the SG) in the graphs below the images; X-axis = distance (in pixels) and Y-axis = gray unit (pixel intensity). The pixel intensity’s dynamic range is delimited by red (maximum) and blue (minimum) dashed lines and the corresponding values are shown on each graph. MIP = maximum intensity 3D projection through the entire SG depth. DL: distal lateral lobe; M: medial lobe; PL: proximal lateral lobe; SD: salivary duct. Italic “DL” labels (Bi) indicate two visible regions of the same DL lobe. Please click here to view a larger version of this figure.

Figure 4:. fkh knockdown in adult An. gambiae salivary glands reduces SG secretion markers.

Shown are representative images from day 13 adult female An. gambiae SGs after 8 days (days 5–13) of oral exposure to either (A) non-related dsRNA control (ant), or (B) dsRNA targeting the SG TF fork head (fkh, AGAP001671) in 10% sucrose stained with the dyes DAPI (DNA; red), labeled wheat germ agglutinin (WGA, chitin/ O-GlcNAcylation; blue), Nile Red (lipids; purple), and antisera against the recycling endosome vesicle marker Rab11 (green). Scale bar lengths shown are microns. SGs (i) are outlined with white dashes. Yellow lines in zoomed lobe images (of the regions enclosed by yellow boxes) indicate where the line scans of signal intensity were conducted. Green and purple channel intensities corresponding to line scans for each lobe are plotted (always left to right in the SG) in the graphs below the images; X-axis = distance (in pixels) and Y-axis = gray unit (pixel intensity). The pixel intensity’s dynamic range is delimited by red (maximum) and blue (minimum) dashed lines and the corresponding values are shown on each graph. MIP = maximum intensity 3D projection through the entire SG depth. DL: distal lateral lobe; M: medial lobe; PL: proximal lateral lobe; SD: salivary duct. Please click here to view a larger version of this figure.