Abstract
BackgroundMembers of the Anophelesgambiae species complex are primary vectors of human malaria in Africa. It is known that a large haplotype shared between An. gambiae and Anophelescoluzzii by introgression carries point mutations of the voltage-gated sodium channel gene para, including the L1014F kdr mutation associated with insensitivity to pyrethroid insecticides. Carriage of L1014F kdr is also correlated with higher susceptibility to infection with Plasmodium falciparum. However, the genetic mechanism and causative gene(s) underlying the parasite susceptibility phenotype are not known.MethodsMosquitoes from the wild Burkina Faso population were challenged by feeding on natural P. falciparum gametocytes. Oocyst infection phenotypes were determined and were tested for association with SNP genotypes. Candidate genes in the detected locus were prioritized and RNAi-mediated gene silencing was used to functionally test for gene effects on P. falciparum susceptibility.ResultsA genetic locus, Pfin6, was identified that influences infection levels of P. falciparum in mosquitoes. The locus segregates as a ~3 Mb haplotype carrying 65 predicted genes including the para gene. The haplotype carrying the kdr allele of para is linked to increased parasite infection prevalence, but many single nucleotide polymorphisms on the haplotype are also equally linked to the infection phenotype. Candidate genes in the haplotype were prioritized and functionally tested. Silencing of para did not influence P. falciparum infection, while silencing of a predicted immune gene, serine protease ClipC9, allowed development of significantly increased parasite numbers.ConclusionsGenetic variation influencing Plasmodium infection in wild Anopheles is linked to a natural ~3 megabase haplotype on chromosome 2L that carries the kdr allele of the para gene. Evidence suggests that para gene function does not directly influence parasite susceptibility, and the association of kdr with infection may be due to tight linkage of kdr with other gene(s) on the haplotype. Further work will be required to determine if ClipC9 influences the outcome of P. falciparum infection in nature, as well as to confirm the absence of a direct influence by para.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0924-8) contains supplementary material, which is available to authorized users.
Highlights
Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa
Structure of the para gene locus Wild mosquitoes were collected as larvae in Burkina Faso, were reared to adults in the insectary, and were challenged with wild P. falciparum parasites by membrane feeding on blood from gametocyte carriers, yielding 363 mosquitoes belonging to the An. gambiae species complex that were used in the study
The mosquito DNAs were genotyped with 105 single nucleotide polymorphisms (SNP) spanning a broad ~15 Mb region containing the para gene across the centromere of chromosome 2, between coordinates chromosome 2R:55 Mb to chromosome 2L:10 Mb (Additional file 1: Table S1; Fig. 1, bottom track of 105 SNPs shown as black vertical lines)
Summary
Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa. It is known that a large haplotype shared between An. gambiae and Anopheles coluzzii by introgression carries point mutations of the voltage-gated sodium channel gene para, including the L1014F kdr mutation associated with insensitivity to pyrethroid insecticides. The genetic mechanism and causative gene(s) underlying the parasite susceptibility phenotype are not known. Throughout sub-Saharan Africa, members of the Anopheles gambiae species complex are primary vectors of the human malaria parasite Plasmodium falciparum, which is responsible for extensive morbidity and mortality. Phenotypic differences for traits that influence malaria transmission, such as behaviour, Plasmodium susceptibility and insecticide resistance can be influenced by genetic variation [1,2,3,4]. Increased resistance to pyrethroid insecticides is influenced in part by a SNP of the voltagegated sodium channel gene para ( called VSG). The geographic labels are misnomers as these resistance loci are not geographically restricted, because L1014F co-occurs with L1014S in East Africa [16] and rates of L1014S have been increasing in West Africa [17, 18]
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