Transmission blocking immunity in the malaria non-vector mosquito Anopheles quadriannulatus species A

Abstract

Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa, Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vector mosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus is largely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodium berghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits and manifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequent stages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and the thioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completely abolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatus into a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of natural refractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to control transmission of the disease.

Figure 1. Plasmodium parasite killing in An. quadriannulatus.

(A, B) Melanized ookinetes (arrows) of P. falciparum (A) and P. berghei (B) while crossing the An. quadriannulatus midgut. (C) Melanized ookinete and live oocyst density in the midguts of An. quadriannulatus and An. gambiae females infected with P. berghei. Four independent paired experiments were performed and their results were analysed by REML variance components analysis by fitting the mixed effect model. The geometric means±SD of the pooled data from the four independent experiments are shown. The melanized parasite densities (black bars) were 12.4±3.1 for An. quadriannulatus (n = 167) and 0.2±0.5 for An. gambiae (n = 118; P<0.001), and the oocyst densities were 3.6±3.3 and 12.8±2.6, respectively (P<0.001). n, number of midguts. (D) Prevalence (% of mosquitoes with at least one live parasite) of midguts at day 10 and salivary glands in corresponding infections at day 21–22 showing live P. berghei oocysts and sporozoites, respectively. The results of three independent experiments (see Table S1) were pooled and analyzed using the Chi-square goodness-of-fit test. A significant decrease in prevalence is detected in An. quadriannulatus (P<0.001), but not in An. gambiae. Bars represent standard errors.

Figure 2. An. quadriannulatus refractoriness to Plasmodium is heritable and dominant.

F1 hybrids were obtained by crossing male (square) An. quadriannulatus with female (circle) An. gambiae. F1 hybrid females were then backcrossed with An. quadriannulatus males to obtain F2 backcrossed mosquitoes. The prevalence of melanized parasites (inner pie, black) and live oocyst (outer pie, green) in the midguts of females from the parental An. quadriannulatus (n = 49) and An. gambiae (n = 53) populations, F1 (n = 66) and F2 (n = 86) are shown as concentric pie charts. Box plots depict the distribution of melanized ookinetes (black) and live oocysts (green) in each female mosquito group, with the respective median value shown in red. Two independent experiments were performed and the pooled data were analyzed using the Chi-square goodness-of-fit test for the prevalence and the Kruskal-Wallis non parametric ANOVA for the parasite densities. n refers to the number of mosquito midguts in the pooled data.

Figure 3. Silencing LRIM1, LRIM2 and TEP1 transforms An. quadriannulatus into a vector species.

(A) Representative microscopy images of midguts dissected from P. berghei-infected mosquitoes which were injected with either LacZ dsRNA (control) or dsRNAs for each of the examined genes. GFP-fluorescent oocysts are shown in the right panels whereas arrows in the bright field images indicate melanized ookinetes. No melanized ookinetes and higher oocyst densities are observed in kd mosquitoes. (B) Quantitative effects of gene kds on melanized ookinete density (black bars) and oocyst density (green bars) in mosquito midguts compared to LacZ dsRNA-treated controls. The pooled results from two independent experiments were analyzed using the REML variance component analysis by fitting the mixed effects model. Geometric means and standard deviations are shown. Experiments with LRIM1 and LRIM2 kds were performed separately from those with TEP1 kd; thus they are presented in separate graphs.