Polymorphisms in Anopheles gambiae immune genes associated with natural resistance to Plasmodium falciparum

Abstract

Many genes involved in the immune response of Anopheles gambiae, the main malaria vector in Africa, have been identified, but whether naturally occurring polymorphisms in these genes underlie variation in resistance to the human malaria parasite, Plasmodium falciparum, is currently unknown. Here we carried out a candidate gene association study to identify single nucleotide polymorphisms (SNPs) associated with natural resistance to P. falciparum. A. gambiae M form mosquitoes from Cameroon were experimentally challenged with three local wild P. falciparum isolates. Statistical associations were assessed between 157 SNPs selected from a set of 67 A. gambiae immune-related genes and the level of infection. Isolate-specific associations were accounted for by including the effect of the isolate in the analysis. Five SNPs were significantly associated to the infection phenotype, located within or upstream of AgMDL1, CEC1, Sp PPO activate, Sp SNAKElike, and TOLL6. Low overall and local linkage disequilibrium indicated high specificity in the loci found. Association between infection phenotype and two SNPs was isolate-specific, providing the first evidence of vector genotype by parasite isolate interactions at the molecular level. Four SNPs were associated to either oocyst presence or load, indicating that the genetic basis of infection prevalence and intensity may differ. The validity of the approach was verified by confirming the functional role of Sp SNAKElike in gene silencing assays. These results strongly support the role of genetic variation within or near these five A. gambiae immune genes, in concert with other genes, in natural resistance to P. falciparum. They emphasize the need to distinguish between infection prevalence and intensity and to account for the genetic specificity of vector-parasite interactions in dissecting the genetic basis of Anopheles resistance to human malaria.

Figure 1. Association between AgMDL1-40910564 genotype and P. falciparum infection intensity.

The square root transformed mean number of oocysts in infected females and their standard errors are shown for each genotype. For clarity, plotted values are corrected for the additive isolate effect so that visual differences only reflect the genetic contribution to phenotypic variation (see Methods). Sample sizes are indicated above the bars.

Figure 2. Association between CEC1-12441661 and SpPPOact-58805968 genotypes and P. falciparum infection prevalence.

A) CEC1-12441661 and B) SpPPOact-58805968. The proportion of infected females is shown for each genotype. For clarity, plotted values are corrected for the additive isolate effect so that visual differences only reflect the genetic contribution to phenotypic variation (see Methods). Vertical bars represent the confidence intervals of the standardized proportions. Sample sizes are indicated above the bars.

Figure 3. Association between SpSNAKElike-40693950 genotype and infection phenotype as a function of P. falciparum isolates.

A) Percentages of infected females (infection prevalence) and their confidence intervals. B) Numbers of oocysts in infected females (infection intensity) and their standard errors. Sample sizes are indicated next to each data point. Blue squares: Isolate 1; green triangles: Isolate 2; red circles: Isolate 3.

Figure 4. Association between TOLL6-41490803 genotype and infection prevalence as a function of P. falciparum isolates.

The percentages of infected females and their confidence intervals are shown by isolate for each genotype. Sample sizes are indicated next to each data point. Blue squares: Isolate 1; green triangles: Isolate 2; red circles: Isolate 3.

Figure 5. Effect of A. gambiae Sp SNAKElike (SNL) silencing on P. falciparum infection.

The number of oocysts that developed for mosquitoes injected either with dsSp SNAKElike or dsLacZ in five independent gene knockdown assays using different P. falciparum isolates are shown. The red bars indicate the mean numbers of oocysts and their standard error. Sample sizes are indicated. *P<0.05; **P<0.01; ***P<0.001.