Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene

Abstract

Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector-parasite interactions. Here, we developed a simple in vivo double-stranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and reproducible silencing of Defensin. Analysis of the knockdown phenotype revealed that this peptide is required for the mosquito antimicrobial defense against Gram-positive bacteria. In contrast, in mosquitoes infected by Plasmodium berghei, no loss of mosquito viability and no significant effect on the development and morphology of the parasite midgut stages were observed in the absence of Defensin. We conclude that this peptide is not a major antiparasitic factor in A. gambiae in vivo. Our results open new perspectives for the study of mosquito gene function in vivo and provide a basis for genome-scale systematic functional screens by targeted gene silencing.

Fig. 1. RNA analysis of the Defensin gene knockout by dsRNA. RNA blots demonstrate that, over a period from 1 to 8 days (numbers above the images), E. coli challenge stably induces the expression of Defensin (DEF) mRNA (arrowheads) above the uninfected control level (C) in wild-type (WT) (A and B) and in dsGFP-treated, but not in dsDEF-treated, mosquitoes (B and C). In (D), the input dsRNAs match in size the signals (DEF and GFP) that are detected in dsRNA-injected mosquitoes (asterisks). (A), (C) and (D) were run on 1.2% agarose gels and (B) on an 1.4% agarose gel. The ribosomal protein S7 transcript was used as a loading control. GFP, green fluorescent protein. (E) RT–PCR analysis of the DEF (19 cycles) gene expression in dsGFP- and dsDEF-treated mosquitoes before (C) and after (days 1, 2 and 4) bacterial challenge. The expression of the ribosomal protein gene S7 (19 cycles) served as control.

Fig. 2. MALDI-TOF MS analysis of the Defensin gene knockout by dsRNA. (A) Mosquitoes were challenged with a mixture of E. coli and S. aureus, and 36 h later the presence of Defensin peptide was detected in the hemolymph of control dsGFP, but not of dsDEF, mosquitoes. (B) Defensin is present in the anterior midgut 24 h after infectious bloodmeal in control dsGFP, but not in dsDEF, mosquitoes. The peaks corresponding to Defensin, and their molecular weights are indicated by arrows.

Fig. 3. Lethality of control dsGFP-treated mosquitoes after infection with different doses of bacteria. Survival rates (%) are presented for mosquitoes infected with (B) the Gram-negative bacterium E. coli or the Gram-positive bacteria (A) B. subtilis, (C) S. aureus and (D) M. luteus. The bacterial concentrations are expressed as optical densities (OD) of suspensions at 600 nm: OD₆₀₀ = 0.0005 (open circles), OD₆₀₀ = 0.005 (open triangles), OD₆₀₀ = 0.05 (open squares), OD₆₀₀ = 0.4 (crosses) and OD₆₀₀ = 5 (asterisks). Each experiment was performed with 50 mosquitoes for each bacterial species, and the results shown are representative of three independent experiments. Standard errors are indicated by bars.

Fig. 4. Defensin knockdown mosquitoes are susceptible to Gram-positive, but not to Gram-negative, bacteria. The survival rates (%) of (A) dsGFP-treated mosquitoes and (B) dsDEF-treated mosquitoes after injection of PBS (open squares), E. coli (OD₆₀₀ = 0.005, open triangles), M. luteus (OD₆₀₀ = 0.4, open circles) or S. aureus (OD₆₀₀ = 0.4, asterisks) are shown. Each experiment was performed with 50 mosquitoes for each bacterial species, and the results shown are representative of three independent experiments. Standard errors are indicated by bars.