Molecular characterization and genetic authentication assay for Anopheles 'hemocyte-like' cell lines 4a-3A and 4a-3B.

BACKGROUND: Anopheles cell lines are used in a variety of ways to better understand the major vectors of malaria in sub-Saharan Africa. Despite this, commonly used cell lines are not well characterized, and no tools are available for cell line identification and authentication. METHODS: Utilizing whole genome sequencing, genomes of 4a-3A and 4a-3B 'hemocyte-like' cell lines were characterized for insertions and deletions (indels) and SNP variation. Genomic locations of distinguishing sequence variation and species origin of the cell lines were also examined. Unique indels were targeted to develop a PCR-based cell line authentication assay. Mitotic chromosomes were examined to survey the cytogenetic landscape for chromosome structure and copy number in the cell lines. RESULTS: The 4a-3A and 4a-3B cell lines are female in origin and primarily of Anopheles coluzzii ancestry. Cytogenetic analysis indicates that the two cell lines are essentially diploid, with some relatively minor chromosome structural rearrangements. Whole-genome sequence was generated, and analysis indicated that SNPs and indels which differentiate the cell lines are clustered on the 2R chromosome in the regions of the 2Rb, 2Rc and 2Ru chromosomal inversions. A PCR-based authentication assay was developed to fingerprint three indels unique to each cell line. The assay distinguishes between 4a-3A and 4a-3B cells and also uniquely identifies two additional An. coluzzii cell lines tested, Ag55 and Sua4.0. The assay has the specificity to distinguish four cell lines and also has the sensitivity to detect cellular contamination within a sample of cultured cells. CONCLUSIONS: Genomic characterization of the 4a-3A and 4a-3B Anopheles cell lines was used to develop a simple diagnostic assay that can distinguish these cell lines within and across research laboratories. A cytogenetic survey indicated that the 4a-3A and Sua4.0 cell lines carry essentially normal diploid chromosomes, which makes them amenable to CRISPR/Cas9 genome editing. The presented simple authentication assay, coupled with screening for mycoplasma, will allow validation of the integrity of experimental resources and will promote greater experimental reproducibility of results.

Genetics and immunity of Anopheles response to the entomopathogenic fungus Metarhizium anisopliae overlap with immunity to Plasmodium.

Entomopathogenic fungi have been explored as a potential biopesticide to counteract the insecticide resistance issue in mosquitoes. However, little is known about the possibility that genetic resistance to fungal biopesticides could evolve in mosquito populations. Here, we detected an important genetic component underlying Anopheles coluzzii survival after exposure to the entomopathogenic fungus Metarhizium anisopliae. A familiality study detected variation for survival among wild mosquito isofemale pedigrees, and genetic mapping identified two loci that significantly influence mosquito survival after fungus exposure. One locus overlaps with a previously reported locus for Anopheles susceptibility to the human malaria parasite Plasmodium falciparum. Candidate gene studies revealed that two LRR proteins encoded by APL1C and LRIM1 genes in this newly mapped locus are required for protection of female A. coluzzii from M. anisopliae, as is the complement-like factor Tep1. These results indicate that natural Anopheles populations already segregate frequent genetic variation for differential mosquito survival after fungal challenge and suggest a similarity in Anopheles protective responses against fungus and Plasmodium. However, this immune similarity raises the possibility that fungus-resistant mosquitoes could also display enhanced resistance to Plasmodium, suggesting an advantage of selecting for fungus resistance in vector populations to promote naturally diminished malaria vector competence.

Comprehensive Ecological and Geographic Characterization of Eukaryotic and Prokaryotic Microbiomes in African Anopheles.

Exposure of mosquitoes to numerous eukaryotic and prokaryotic microbes in their associated microbiomes has probably helped drive the evolution of the innate immune system. To our knowledge, a metagenomic catalog of the eukaryotic microbiome has not been reported from any insect. Here we employ a novel approach to preferentially deplete host 18S ribosomal RNA gene amplicons to reveal the composition of the eukaryotic microbial communities of Anopheles larvae sampled in Kenya, Burkina Faso and Republic of Guinea (Conakry). We identified 453 eukaryotic operational taxonomic units (OTUs) associated with Anopheles larvae in nature, but an average of 45% of the 18S rRNA sequences clustered into OTUs that lacked a taxonomic assignment in the Silva database. Thus, the Anopheles microbiome contains a striking proportion of novel eukaryotic taxa. Using sequence similarity matching and de novo phylogenetic placement, the fraction of unassigned sequences was reduced to an average of 4%, and many unclassified OTUs were assigned as relatives of known taxa. A novel taxon of the genus Ophryocystis in the phylum Apicomplexa (which also includes Plasmodium) is widespread in Anopheles larvae from East and West Africa. Notably, Ophryocystis is present at fluctuating abundance among larval breeding sites, consistent with the expected pattern of an epidemic pathogen. Species richness of the eukaryotic microbiome was not significantly different across sites from East to West Africa, while species richness of the prokaryotic microbiome was significantly lower in West Africa. Laboratory colonies of Anopheles coluzzii harbor 26 eukaryotic OTUs, of which 38% (n = 10) are shared with wild populations, while 16 OTUs are unique to the laboratory colonies. Genetically distinct An. coluzzii colonies co-housed in the same facility maintain different prokaryotic microbiome profiles, suggesting a persistent host genetic influence on microbiome composition. These results provide a foundation to understand the role of the Anopheles eukaryotic microbiome in vector immunity and pathogen transmission. We hypothesize that prevalent apicomplexans such as Ophryocystis associated with Anopheles could induce interference or competition against Plasmodium within the vector. This and other members of the eukaryotic microbiome may offer candidates for new vector control tools.

Anopheles ecology, genetics and malaria transmission in northern Cambodia.

In the Greater Mekong Subregion, malaria cases have significantly decreased but little is known about the vectors or mechanisms responsible for residual malaria transmission. We analysed a total of 3920 Anopheles mosquitoes collected during the rainy and dry seasons from four ecological settings in Cambodia (villages, forested areas near villages, rubber tree plantations and forest sites). Using odor-baited traps, 81% of the total samples across all sites were collected in cow baited traps, although 67% of the samples attracted by human baited traps were collected in forest sites. Overall, 20% of collected Anopheles were active during the day, with increased day biting during the dry season. 3131 samples were identified morphologically as 14 different species, and a subset was also identified by DNA amplicon sequencing allowing determination of 29 Anopheles species. The investigation of well characterized insecticide mutations (ace-1, kdr, and rdl genes) indicated that individuals carried mutations associated with response to all the different classes of insecticides. There also appeared to be a non-random association between mosquito species and insecticide resistance with Anopheles peditaeniatus exhibiting nearly fixed mutations. Molecular screening for Plasmodium sp. presence indicated that 3.6% of collected Anopheles were positive, most for P. vivax followed by P. falciparum. These results highlight some of the key mechanisms driving residual human malaria transmission in Cambodia, and illustrate the importance of diverse collection methods, sites and seasons to avoid bias and better characterize Anopheles mosquito ecology in Southeast Asia.

Leucine-Rich Immune Factor APL1 Is Associated With Specific Modulation of Enteric Microbiome Taxa in the Asian Malaria Mosquito Anopheles stephensi.

The commensal gut microbiome is contained by the enteric epithelial barrier, but little is known about the degree of specificity of host immune barrier interactions for particular bacterial taxa. Here, we show that depletion of leucine-rich repeat immune factor APL1 in the Asian malaria mosquito Anopheles stephensi is associated with higher midgut abundance of just the family Enterobacteraceae, and not generalized dysbiosis of the microbiome. The effect is explained by the response of a narrow clade containing two main taxa related to Klebsiella and Cedecea. Analysis of field samples indicate that these two taxa are recurrent members of the wild Anopheles microbiome. Triangulation using sequence and functional data incriminated relatives of C. neteri and Cedecea NFIX57 as candidates for the Cedecea component, and K. michiganensis, K. oxytoca, and K.sp. LTGPAF-6F as candidates for the Klebsiella component. APL1 presence is associated with host ability to specifically constrain the abundance of a narrow microbiome clade of the Enterobacteraceae, and the immune factor may promote homeostasis of this clade in the enteric microbiome for host benefit.

Gene copy number and function of the APL1 immune factor changed during Anopheles evolution.

BACKGROUND: The recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined. METHODS: The APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis. RESULTS: APL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus. CONCLUSIONS: APL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa.

Correction to: De novo profiling of RNA viruses in Anopheles malaria vector mosquitoes from forest ecological zones in Senegal and Cambodia.

Following the publication of this article [1], the authors reported that the original shading in columns 3 and 4 of Table 3, which indicated the presence or absence of viruses in each library, had been removed during typesetting.

De novo profiling of RNA viruses in Anopheles malaria vector mosquitoes from forest ecological zones in Senegal and Cambodia.

BACKGROUND: Mosquitoes are colonized by a large but mostly uncharacterized natural virome of RNA viruses, and the composition and distribution of the natural RNA virome may influence the biology and immunity of Anopheles malaria vector populations. RESULTS: Anopheles mosquitoes were sampled in malaria endemic forest village sites in Senegal and Cambodia, including Anopheles funestus, Anopheles gambiae group sp., and Anopheles coustani in Senegal, and Anopheles hyrcanus group sp., Anopheles maculatus group sp., and Anopheles dirus in Cambodia. The most frequent mosquito species sampled at both study sites are human malaria vectors. Small and long RNA sequences were depleted of mosquito host sequences, de novo assembled and clustered to yield non-redundant contigs longer than 500 nucleotides. Analysis of the assemblies by sequence similarity to known virus families yielded 115 novel virus sequences, and evidence supports a functional status for at least 86 of the novel viral contigs. Important monophyletic virus clades in the Bunyavirales and Mononegavirales orders were found in these Anopheles from Africa and Asia. The remaining non-host RNA assemblies that were unclassified by sequence similarity to known viruses were clustered by small RNA profiles, and 39 high-quality independent contigs strongly matched a pattern of classic RNAi processing of viral replication intermediates, suggesting they are entirely undescribed viruses. One thousand five hundred sixty-six additional high-quality unclassified contigs matched a pattern consistent with Piwi-interacting RNAs (piRNAs), suggesting that strand-biased piRNAs are generated from the natural virome in Anopheles. To functionally query piRNA effect, we analyzed piRNA expression in Anopheles coluzzii after infection with O'nyong nyong virus (family Togaviridae), and identified two piRNAs that appear to display specifically altered abundance upon arbovirus infection. CONCLUSIONS: Anopheles vectors of human malaria in Africa and Asia are ubiquitously colonized by RNA viruses, some of which are monophyletic but clearly diverged from other arthropod viruses. The interplay between small RNA pathways, immunity, and the virome may represent part of the homeostatic mechanism maintaining virome members in a commensal or nonpathogenic state, and could potentially influence vector competence.

Correction: An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

[This corrects the article DOI: 10.1371/journal.ppat.1005306.].

Composition and genetics of malaria vector populations in the Central African Republic.

BACKGROUND: In many African countries malaria has declined sharply due to a synergy of actions marked by the introduction of vector control strategies, but the disease remains a leading cause of morbidity and mortality in Central African Republic (CAR). An entomological study was initiated with the aim to characterize the malaria vectors in Bangui, the capital of CAR, and determine their vector competence. METHODS: A cross-sectional entomological study was conducted in 15 sites of the district of Bangui, the capital of CAR, in September-October 2013 and a second collection was done in four of those sites between November and December 2013. Mosquitoes were collected by human landing catch (HLC) indoors and outdoors and by pyrethrum spray catch of indoor-resting mosquitoes. Mosquitoes were analysed for species and multiple other attributes, including the presence of Plasmodium falciparum circumsporozoite protein or DNA, blood meal source, 2La inversion karyotype, and the L1014F kdr insecticide resistance mutation. RESULTS: Overall, 1292 anophelines were analysed, revealing a predominance of Anopheles gambiae and Anopheles funestus, with a small fraction of Anopheles coluzzii. Molecular typing of the An. gambiae complex species showed that An. gambiae was predominant (95.7 %) as compared to An. coluzzii (2.1 %), and Anopheles arabiensis was not present. In some areas the involvement of secondary vectors, such as Anopheles coustani, expands the risk of infection. By HLC sampling, An. funestus displayed a stronger endophilic preference than mosquitoes from the An. gambiae sister taxa, with a mean indoor-capture rate of 54.3 % and 67.58 % for An. gambiae sister taxa and An. funestus, respectively. Human biting rates were measured overall for each of the species with 28 or 29 bites/person/night, respectively. Both vectors displayed a strong human feeding preference as determined by blood meal source, which was not different between the different sampling sites. An. coustani appears to be highly exophilic, with 92 % of HLC samples captured outdoors. The mean CSP rate in head-thorax sections of all Anopheles was 5.09 %, and was higher in An. gambiae s.l. (7.4 %) than in An. funestus (3.3 %). CSP-positive An. coustani were also detected in outdoor HLC samples. In the mosquitoes of the An. gambiae sister taxa the kdr-w mutant allele was nearly fixed, with 92.3 % resistant homozygotes, and no susceptible homozygotes detected. CONCLUSIONS: This study collected data on anopheline populations in CAR, behaviour of vectors and transmission levels. Further studies should investigate the biting behaviour and susceptibility status of the anophelines to different insecticides to allow the establishment of appropriate vector control based on practical entomological knowledge.

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes.

Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O'nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.

Genetic Structure of a Local Population of the Anopheles gambiae Complex in Burkina Faso.

Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa. Population heterogeneities for ecological and behavioral attributes expand and stabilize malaria transmission over space and time, and populations may change in response to vector control, urbanization and other factors. There is a need for approaches to comprehensively describe the structure and characteristics of a sympatric local mosquito population, because incomplete knowledge of vector population composition may hinder control efforts. To this end, we used a genome-wide custom SNP typing array to analyze a population collection from a single geographic region in West Africa. The combination of sample depth (n = 456) and marker density (n = 1536) unambiguously resolved population subgroups, which were also compared for their relative susceptibility to natural genotypes of Plasmodium falciparum malaria. The population subgroups display fluctuating patterns of differentiation or sharing across the genome. Analysis of linkage disequilibrium identified 19 new candidate genes for association with underlying population divergence between sister taxa, A. coluzzii (M-form) and A. gambiae (S-form).

An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with broad specificity a rarity.

Association mapping by pooled sequencing identifies TOLL 11 as a protective factor against Plasmodium falciparum in Anopheles gambiae.

BACKGROUND: The genome-wide association study (GWAS) techniques that have been used for genetic mapping in other organisms have not been successfully applied to mosquitoes, which have genetic characteristics of high nucleotide diversity, low linkage disequilibrium, and complex population stratification that render population-based GWAS essentially unfeasible at realistic sample size and marker density. METHODS: We designed a novel mapping strategy for the mosquito system that combines the power of linkage mapping with the resolution afforded by genetic association. We established founder colonies from West Africa, controlled for diversity, linkage disequilibrium and population stratification. Colonies were challenged by feeding on the infectious stage of the human malaria parasite, Plasmodium falciparum, mosquitoes were phenotyped for parasite load, and DNA pools for phenotypically similar mosquitoes were Illumina sequenced. Phenotype-genotype mapping was carried out in two stages, coarse and fine. RESULTS: In the first mapping stage, pooled sequences were analysed genome-wide for intervals displaying relativereduction in diversity between phenotype pools, and candidate genomic loci were identified for influence upon parasite infection levels. In the second mapping stage, focused genotyping of SNPs from the first mapping stage was carried out in unpooled individual mosquitoes and replicates. The second stage confirmed significant SNPs in a locus encoding two Toll-family proteins. RNAi-mediated gene silencing and infection challenge revealed that TOLL 11 protects mosquitoes against P. falciparum infection. CONCLUSIONS: We present an efficient and cost-effective method for genetic mapping using natural variation segregating in defined recent Anopheles founder colonies, and demonstrate its applicability for mapping in a complex non-model genome. This approach is a practical and preferred alternative to population-based GWAS for first-pass mapping of phenotypes in Anopheles. This design should facilitate mapping of other traits involved in physiology, epidemiology, and behaviour.

The kdr-bearing haplotype and susceptibility to Plasmodium falciparum in Anopheles gambiae: genetic correlation and functional testing.

BACKGROUND: 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. 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. METHODS: Mosquitoes 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. RESULTS: A 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. CONCLUSIONS: Genetic 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.

Genome analysis of a major urban malaria vector mosquito, Anopheles stephensi.

BACKGROUND: Anopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions. The type form of the species is responsible for the majority of urban malaria transmission across its range. RESULTS: Here, we report the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing. Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis. Comparisons between An. stephensi and An. gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes. An. stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An. gambiae. We also identify a number of Y-chromosome contigs and BACs. Interspersed repeats constitute 7.1% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs. RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism. CONCLUSIONS: The genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector. Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution. RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions.

Equivalent susceptibility of Anopheles gambiae M and S molecular forms and Anopheles arabiensis to Plasmodium falciparum infection in Burkina Faso.

BACKGROUND: The Anopheles gambiae sensu lato (s.l.) species complex in Burkina Faso consists of Anopheles arabiensis, and molecular forms M and S of Anopheles gambiae sensu stricto (s.s.). Previous studies comparing the M and S forms for level of infection with Plasmodium falciparum have yielded conflicting results. METHODS: Mosquito larvae were sampled from natural pools, reared to adulthood under controlled conditions, and challenged with natural P. falciparum by experimental feeding with blood from gametocyte carriers. Oocyst infection prevalence and intensity was determined one week after infection. DNA from carcasses was genotyped to identify species and molecular form. RESULTS: In total, 7,400 adult mosquitoes grown from wild-caught larvae were challenged with gametocytes in 29 experimental infections spanning four transmission seasons. The overall infection prevalence averaged 40.7% for A. gambiae M form, 41.4% for A. gambiae S form, and 40.1% for A. arabiensis. There was no significant difference in infection prevalence or intensity between the three population groups. Notably, infection experiments in which the population groups were challenged in parallel on the same infective blood displayed less infection difference between population groups, while infections with less balanced composition of population groups had lower statistical power and displayed apparent differences that fluctuated more often from the null average. CONCLUSION: The study clearly establishes that, at the study site in Burkina Faso, there is no difference in genetic susceptibility to P. falciparum infection between three sympatric population groups of the A. gambiae s.l. complex. Feeding the mosquito groups on the same infective blood meal greatly increases statistical power. Conversely, comparison of the different mosquito groups between, rather than within, infections yields larger apparent difference between mosquito groups, resulting from lower statistical power and greater noise, and could lead to false-positive results. In making infection comparisons between population groups, it is more accurate to compare the different groups after feeding simultaneously upon the same infective blood.

Evidence for population-specific positive selection on immune genes of Anopheles gambiae.

Host-pathogen interactions can be powerful drivers of adaptive evolution, shaping the patterns of molecular variation at the genes involved. In this study, we sequenced alleles from 28 immune-related loci in wild samples of multiple genetic subpopulations of the African malaria mosquito Anopheles gambiae, obtaining unprecedented sample sizes and providing the first opportunity to contrast patterns of molecular evolution at immune-related loci in the recently discovered GOUNDRY population to those of the indoor-resting M and S molecular forms. In contrast to previous studies that focused on immune genes identified in laboratory studies, we centered our analysis on genes that fall within a quantitative trait locus associated with resistance to Plasmodium falciparum in natural populations of A. gambiae. Analyses of haplotypic and genetic diversity at these 28 loci revealed striking differences among populations in levels of genetic diversity and allele frequencies in coding sequence. Putative signals of positive selection were identified at 11 loci, but only one was shared by two subgroups of A. gambiae. Striking patterns of linkage disequilibrium were observed at several loci. We discuss these results with respect to ecological differences among these strata as well as potential implications for disease transmission.

Diverged alleles of the Anopheles gambiae leucine-rich repeat gene APL1A display distinct protective profiles against Plasmodium falciparum.

Functional studies have demonstrated a role for the Anopheles gambiae APL1A gene in resistance against the human malaria parasite, Plasmodium falciparum. Here, we exhaustively characterize the structure of the APL1 locus and show that three structurally different APL1A alleles segregate in the Ngousso colony. Genetic association combined with RNAi-mediated gene silencing revealed that APL1A alleles display distinct protective profiles against P. falciparum. One APL1A allele is sufficient to explain the protective phenotype of APL1A observed in silencing experiments. Epitope-tagged APL1A isoforms expressed in an in vitro hemocyte-like cell system showed that under assay conditions, the most protective APL1A isoform (APL1A(2)) localizes within large cytoplasmic vesicles, is not constitutively secreted, and forms only one protein complex, while a less protective isoform (APL1A(1)) is constitutively secreted in at least two protein complexes. The tested alleles are identical to natural variants in the wild A. gambiae population, suggesting that APL1A genetic variation could be a factor underlying natural heterogeneity of vector susceptibility to P. falciparum.