Exceptional diversity, maintenance of polymorphism, and recent directional selection on the APL1 malaria resistance genes of Anopheles gambiae.

The three-gene APL1 locus encodes essential components of the mosquito immune defense against malaria parasites. APL1 was originally identified because it lies within a mapped QTL conferring the vector mosquito Anopheles gambiae natural resistance to the human malaria parasite, Plasmodium falciparum, and APL1 genes have subsequently been shown to be involved in defense against several species of Plasmodium. Here, we examine molecular population genetic variation at the APL1 gene cluster in spatially and temporally diverse West African collections of A. gambiae. The locus is extremely polymorphic, showing evidence of adaptive evolutionary maintenance of genetic variation. We hypothesize that this variability aids in defense against genetically diverse pathogens, including Plasmodium. Variation at APL1 is highly structured across geographic and temporal subpopulations. In particular, diversity is exceptionally high during the rainy season, when malaria transmission rates are at their peak. Much less allelic diversity is observed during the dry season when mosquito population sizes and malaria transmission rates are low. APL1 diversity is weakly stratified by the polymorphic 2La chromosomal inversion but is very strongly subdivided between the M and S "molecular forms." We find evidence that a recent selective sweep has occurred at the APL1 locus in M form mosquitoes only. The independently reported observation of a similar M-form restricted sweep at the Tep1 locus, whose product physically interacts with APL1C, suggests that epistatic selection may act on these two loci causing them to sweep coordinately.

Wild Anopheles funestus mosquito genotypes are permissive for infection with the rodent malaria parasite, Plasmodium berghei.

BACKGROUND: Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development. METHODS: An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae. RESULTS: P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 18-20 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections. CONCLUSIONS: The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the awaited genome sequence of A. funestus.

No evidence for positive selection at two potential targets for malaria transmission-blocking vaccines in Anopheles gambiae s.s.

Human malaria causes nearly a million deaths in sub-Saharan Africa each year. The evolution of drug-resistance in the parasite and insecticide resistance in the mosquito vector has complicated control measures and made the need for new control strategies more urgent. Anopheles gambiae s.s. is one of the primary vectors of human malaria in Africa, and parasite-transmission-blocking vaccines targeting Anopheles proteins have been proposed as a possible strategy to control the spread of the disease. However, the success of these hypothetical technologies would depend on the successful ability to broadly target mosquito populations that may be genetically heterogeneous. Understanding the evolutionary pressures shaping genetic variation among candidate target molecules offers a first step towards evaluating the prospects of successfully deploying such technologies. We studied the population genetics of genes encoding two candidate target proteins, the salivary gland protein saglin and the basal lamina structural protein laminin, in wild populations of the M and S molecular forms of A. gambiae in Mali. Through analysis of intraspecific genetic variation and interspecific comparisons, we found no evidence of positive natural selection at the genes encoding these proteins. On the contrary, we found evidence for particularly strong purifying selection at the laminin gene. These results provide insight into the patterns of genetic diversity of saglin and laminin, and we discuss these findings in relation to the potential development of these molecules as vaccine targets.

Anopheles gambiae APL1 is a family of variable LRR proteins required for Rel1-mediated protection from the malaria parasite, Plasmodium berghei.

BACKGROUND: We previously identified by genetic mapping an Anopheles gambiae chromosome region with strong influence over the outcome of malaria parasite infection in nature. Candidate gene studies in the genetic interval, including functional tests using the rodent malaria parasite Plasmodium berghei, identified a novel leucine-rich repeat gene, APL1, with functional activity against P. berghei. PRINCIPAL FINDINGS: Manual reannotation now reveals APL1 to be a family of at least 3 independently transcribed genes, APL1A, APL1B, and APL1C. Functional dissection indicates that among the three known APL1 family members, APL1C alone is responsible for host defense against P. berghei. APL1C functions within the Rel1-Cactus immune signaling pathway, which regulates APL1C transcript and protein abundance. Gene silencing of APL1C completely abolishes Rel1-mediated host protection against P. berghei, and thus the presence of APL1C is required for this protection. Further highlighting the influence of this chromosome region, allelic haplotypes at the APL1 locus are genetically associated with and have high explanatory power for the success or failure of P. berghei parasite infection. CONCLUSIONS: APL1C functions as a required transducer of Rel1-dependent immune signal(s) to efficiently protect mosquitoes from P. berghei infection, and allelic genetic haplotypes of the APL1 locus display distinct levels of susceptibility and resistance to P. berghei.