Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using natural Plasmodium falciparum gametocyte isolates from West-Africa.

Antimalarial drugs that can block the transmission of Plasmodium gametocytes to mosquito vectors would be highly beneficial for malaria elimination efforts. Identifying transmission-blocking drugs currently relies on evaluation of their activity against gametocyte-producing laboratory parasite strains and would benefit from a testing pipeline with genetically diverse field isolates. The aims of this study were to develop a pipeline to test drugs against P. falciparum gametocyte field isolates and to evaluate the transmission-blocking activity of a set of novel compounds. Two assays were designed so they could identify both the overall transmission-blocking activity of a number of marketed and experimental drugs by direct membrane feeding assays (DMFA), and then also discriminate between those that are active against the gametocytes (gametocyte killing or sterilizing) or those that block development in the mosquito (sporontocidal). These DMFA assays used venous blood samples from naturally infected Plasmodium falciparum gametocyte carriers and locally reared Anopheles gambiae s.s. mosquitoes. Overall transmission-blocking activity was assessed following a 24 hour incubation of compound with gametocyte infected blood (TB-DMFA). Sporontocidal activity was evaluated following addition of compound directly prior to feeding, without incubation (SPORO-DMFA); Gametocyte viability was retained during 24-hour incubation at 37°C when gametocyte infected red blood cells were reconstituted in RPMI/serum. Methylene-blue, MMV693183, DDD107498, atovaquone and P218 showed potent transmission-blocking activity in the TB-DMFA, and both atovaquone and the novel antifolate P218 were potent inhibitors of sporogonic development in the SPORO-DMA. This work establishes a pipeline for the integral use of field isolates to assess the transmission-blocking capacity of antimalarial drugs to block transmission that should be validated in future studies.

The interplay between malaria vectors and human activity accounts for high residual malaria transmission in a Burkina Faso village with universal ITN coverage.

BACKGROUND: Mosquito and human behaviour interaction is a key determinant of the maximum level of protection against malaria that can be provided by insecticide-treated nets (ITNs). Nevertheless, scant literature focuses on this interaction, overlooking a fundamental factor for efficient malaria control. This study aims to estimate malaria transmission risk in a Burkina Faso village by integrating vector biting rhythms with some key information about human habits. METHODS: Indoor/outdoor human landing catches were conducted for 16 h (16:00-08:00) during 8 nights (September 2020) in Goden village. A survey about net usage and sleeping patterns was submitted to half the households (October-December 2020). A subsample of collected specimens of Anopheles gambiae sensu lato was molecularly processed for species identification, Plasmodium detection from heads-thoraxes and L1014F pyrethroid-resistance allele genotyping. Hourly mosquito abundance was statistically assessed by GLM/GAM, and the entomological inoculation rate (EIR) was corrected for the actual ITN usage retrieved from the questionnaire. RESULTS: Malaria transmission was mainly driven by Anopheles coluzzii (68.7%) followed by A. arabiensis (26.2%). The overall sporozoite rate was 2% with L1014F estimated frequency of 0.68 (N = 1070 out of 15,201 A. gambiae s.l. collected). No major shift in mosquito biting rhythms in response to ITN or differences between indoor and outdoor catches were detected. Impressive high biting pressure (mean 30.3 mosquitoes/person/hour) was exerted from 20:00 to 06:00 with a peak at 4:00. Human survey revealed that nearly all inhabitants were awake before 20:00 and after 7:00 and at least 8.7% had no access to bednets. Adjusting for anthropological data, the EIR dropped from 6.7 to 1.2 infective bites/person/16 h. In a scenario of full net coverage and accounting only for the human sleeping patterns, the daily malaria transmission risk not targetable by ITNs was 0.69 infective bites. CONCLUSIONS: The high mosquito densities and interplay between human/vector activities means that an estimated 10% of residual malaria transmission cannot be prevented by ITNs in the village. Locally tailored studies, like the current one, are essential to explore the heterogeneity of human exposure to infective bites and, consequently, to instruct the adoption of new vector control tools strengthening individual and community protection.

Pyriproxyfen-treated bed nets reduce reproductive fitness and longevity of pyrethroid-resistant Anopheles gambiae under laboratory and field conditions.

BACKGROUND: The efficacy of insecticide-treated nets (ITNs) containing the insect growth regulator pyriproxyfen (PPF) and pyrethroid insecticides (PPF-ITNs) is being assessed in clinical trials to determine whether they provide greater protection from malaria than standard pyrethroid-treated ITNs in areas where mosquitoes are resistant to pyrethroids. Understanding the entomological mode of action of this new ITN class will aide interpretation of the results from these trials. METHODS: Anopheles gambiae sensu lato (s.l.) mosquitoes from a susceptible laboratory strain were exposed to PPF-treated netting 24 h, 6 h, and immediately prior to, or 24 h post blood feeding, and the impact on fecundity, fertility and longevity recorded. Pyrethroid-resistant populations were exposed to nets containing permethrin and PPF (PPF-ITNs) in cone bioassays and daily mortality recorded. Mosquitoes were also collected from inside houses pre- and post-distribution of PPF-ITNs in a clinical trial conduced in Burkina Faso; female An. gambiae s.l. were then assessed for fecundity and fertility. RESULTS: PPF exposure reduced the median adult lifespan of insecticide-susceptible mosquitoes by 4 to 5 days in all exposure times (p < 0.05) other than 6 h pre-blood meal and resulted in almost complete lifelong sterilization. The longevity of pyrethroid-resistant mosquitoes was also reduced by at least 5 days after exposure to PPF-ITNs compared to untreated nets, but was unaffected by exposure to standard pyrethroid only ITNs. A total of 386 blood-fed or gravid An. gambiae s.l. females were collected from five villages between 1 and 12 months before distribution of PPF-ITNs. Of these mosquitoes, 75% laid eggs and the remaining 25% appeared to have normal ovaries upon dissection. In contrast, only 8.6% of the 631 blood-fed or gravid An. gambiae s.l. collected post PPF-ITN distribution successfully oviposited; 276 (43.7%) did not oviposit but had apparently normal ovaries upon dissection, and 301 (47.7%) did not oviposit and had abnormal eggs upon dissection. Egg numbers were also significantly lower (average of 138/female prior distribution vs 85 post distribution, p < 0.05). CONCLUSION: Exposure to a mixture of PPF and pyrethroids on netting shortens the lifespan of mosquitoes and reduces reproductive output. Sterilization of vectors lasted at least one year under operational conditions. These findings suggest a longer effective lifespan of PPF-pyrethroid nets than reported previously.

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.

Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii.

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences. Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

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.

Barrier bednets target malaria vectors and expand the range of usable insecticides.

Transmission of Plasmodium falciparum malaria parasites occurs when nocturnal Anopheles mosquito vectors feed on human blood. In Africa, where malaria burden is highest, bednets treated with pyrethroid insecticide were highly effective in preventing mosquito bites and reducing transmission, and essential to achieving unprecedented reductions in malaria until 2015 (ref. 1). Since then, progress has stalled2, and with insecticidal bednets losing efficacy against pyrethroid-resistant Anopheles vectors3,4, methods that restore performance are urgently needed to eliminate any risk of malaria returning to the levels seen before their widespread use throughout sub-Saharan Africa5. Here, we show that the primary malaria vector Anopheles gambiae is targeted and killed by small insecticidal net barriers positioned above a standard bednet in a spatial region of high mosquito activity but zero contact with sleepers, opening the way for deploying many more insecticides on bednets than is currently possible. Tested against wild pyrethroid-resistant A. gambiae in Burkina Faso, pyrethroid bednets with organophosphate barriers achieved significantly higher killing rates than bednets alone. Treated barriers on untreated bednets were equally effective, without significant loss of personal protection. Mathematical modelling of transmission dynamics predicted reductions in clinical malaria incidence with barrier bednets that matched those of 'next-generation' nets recommended by the World Health Organization against resistant vectors. Mathematical models of mosquito-barrier interactions identified alternative barrier designs to increase performance. Barrier bednets that overcome insecticide resistance are feasible using existing insecticides and production technology, and early implementation of affordable vector control tools is a realistic prospect.

Influence of genetic polymorphism on transcriptional enhancer activity in the malaria vector Anopheles coluzzii.

Enhancers are cis-regulatory elements that control most of the developmental and spatial gene expression in eukaryotes. Genetic variation of enhancer sequences is known to influence phenotypes, but the effect of enhancer variation upon enhancer functional activity and downstream phenotypes has barely been examined in any species. In the African malaria vector, Anopheles coluzzii, we identified candidate enhancers in the proximity of genes relevant for immunity, insecticide resistance, and development. The candidate enhancers were functionally validated using luciferase reporter assays, and their activity was found to be essentially independent of their physical orientation, a typical property of enhancers. All of the enhancers segregated genetically polymorphic alleles, which displayed significantly different levels of functional activity. Deletion mutagenesis and functional testing revealed a fine structure of positive and negative regulatory elements that modulate activity of the enhancer core. Enhancer polymorphisms segregate in wild A. coluzzii populations in West Africa. Thus, enhancer variants that modify target gene expression leading to likely phenotypic consequences are frequent in nature. These results demonstrate the existence of naturally polymorphic A. coluzzii enhancers, which may help explain important differences between individuals or populations for malaria transmission efficiency and vector adaptation to the environment.

Unexpectedly high Plasmodium sporozoite rate associated with low human blood index in Anopheles coluzzii from a LLIN-protected village in Burkina Faso.

Despite the effectiveness of mass distribution of long-lasting insecticidal nets (LLINs) in reducing malaria transmission in Africa, in hyperendemic areas such as Burkina Faso the burden of malaria remains high. We here report the results of a 4-month survey on the feeding habits and Plasmodium infection in malaria vectors from a village in Burkina Faso one year following a national LLIN distribution programme. Low values of human blood index (HBI) observed in the major malaria vectors in the area (Anopheles coluzzii: N = 263, 20.1%; An. arabiensis: 5.8%, N = 103) are consistent with the hypothesis that LLINs reduced the availability of human hosts to mosquitoes. A regression meta-analysis of data from a systematic review of published studies reporting HBI and sporozoite rates (SR) for An. gambiae complex revealed that the observed SR values (An. coluzzii: 7.6%, N = 503; An. arabiensis: 5.3%, N = 225) are out of the ranges expected based on the low HBI observed. We hypothesize that a small fraction of inhabitants unprotected by bednets acts as a "core group" repeatedly exposed to mosquito bites, representing the major Plasmodium reservoir for the vectors, able to maintain a high risk of transmission even in a village protected by LLINs.

Predicting the likelihood and intensity of mosquito infection from sex specific Plasmodium falciparum gametocyte density.

Understanding the importance of gametocyte density on human-to-mosquito transmission is of immediate relevance to malaria control. Previous work (Churcher et al., 2013) indicated a complex relationship between gametocyte density and mosquito infection. Here we use data from 148 feeding experiments on naturally infected gametocyte carriers to show that the relationship is much simpler and depends on both female and male parasite density. The proportion of mosquitoes infected is primarily determined by the density of female gametocytes though transmission from low gametocyte densities may be impeded by a lack of male parasites. Improved precision of gametocyte quantification simplifies the shape of the relationship with infection increasing rapidly before plateauing at higher densities. The mean number of oocysts per mosquito rises quickly with gametocyte density but continues to increase across densities examined. The work highlights the importance of measuring both female and male gametocyte density when estimating the human reservoir of infection.

Transmission of molecularly undetectable circulating parasite clones leads to high infection complexity in mosquitoes post feeding.

Plasmodium falciparum malaria infections often comprise multiple distinct parasite clones. Few datasets have directly assessed infection complexity in humans and mosquitoes they infect. Examining parasites using molecular tools may provide insights into the selective transmissibility of isolates. Using capillary electrophoresis genotyping and next generation amplicon sequencing, we analysed complexity of parasite infections in human blood and in the midguts of mosquitoes that became infected in membrane feeding experiments using the same blood material in two West African settings. Median numbers of clones in humans and mosquitoes were higher in samples from Burkina Faso (4.5, interquartile range 2-8 for humans; and 2, interquartile range 1-3 for mosquitoes) than in The Gambia (2, interquartile range 1-3 and 1, interquartile range 1-3, for humans and mosquitoes, respectively). Whilst the median number of clones was commonly higher in human blood samples, not all transmitted alleles were detectable in the human peripheral blood. In both study sample sets, additional parasite alleles were identified in mosquitoes compared with the matched human samples (10-88.9% of all clones/feeding assay, n = 73 feeding assays). The results are likely due to preferential amplification of the most abundant clones in peripheral blood but confirm the presence of low density clones that produce transmissible sexual stage parasites.

Variation in natural exposure to anopheles mosquitoes and its effects on malaria transmission.

Variation in biting frequency by Anopheles mosquitoes can explain some of the heterogeneity in malaria transmission in endemic areas. In this study in Burkina Faso, we assessed natural exposure to mosquitoes by matching the genotype of blood meals from 1066 mosquitoes with blood from residents of local households. We observed that the distribution of mosquito bites exceeded the Pareto rule (20/80) in two of the three surveys performed (20/85, 76, and 96) and, at its most pronounced, is estimated to have profound epidemiological consequences, inflating the basic reproduction number of malaria by 8-fold. The distribution of bites from sporozoite-positive mosquitoes followed a similar pattern, with a small number of individuals within households receiving multiple potentially infectious bites over the period of a few days. Together, our findings indicate that heterogeneity in mosquito exposure contributes considerably to heterogeneity in infection risk and suggest significant variation in malaria transmission potential.

Examining the human infectious reservoir for Plasmodium falciparum malaria in areas of differing transmission intensity.

A detailed understanding of the human infectious reservoir is essential for improving malaria transmission-reducing interventions. Here we report a multi-regional assessment of population-wide malaria transmission potential based on 1209 mosquito feeding assays in endemic areas of Burkina Faso and Kenya. Across both sites, we identified 39 infectious individuals. In high endemicity settings, infectious individuals were identifiable by research-grade microscopy (92.6%; 25/27), whilst one of three infectious individuals in the lowest endemicity setting was detected by molecular techniques alone. The percentages of infected mosquitoes in the different surveys ranged from 0.05 (4/7716) to 1.6% (121/7749), and correlate positively with transmission intensity. We also estimated exposure to malaria vectors through genetic matching of blood from 1094 wild-caught bloodfed mosquitoes with that of humans resident in the same houses. Although adults transmitted fewer parasites to mosquitoes than children, they received more mosquito bites, thus balancing their contribution to the infectious reservoir.

Proboscis infection route of Beauveria bassiana triggers early death of Anopheles mosquito.

Entomopathogenic fungi are known to control vector mosquito populations. Thus, understanding the infection dynamics of entomopathogenic fungi is crucial for the effective control of insect pests such as mosquitoes. We investigated the dynamics of Beauveria bassiana s.l. 60-2 infection of Anopheles stephensi by exposing the mosquito to fungus-impregnated filter paper through two infection routes and then comparing the mortality and extent of infection. Fungal development was observed after using this inoculation method with both the tarsus route and the proboscis route, but early mosquito death occurred only after infection through the proboscis route. Fungal hyphae invaded almost all the tissues and organs before or after the death of the host, and fungal invasion of the brain was highly correlated with mortality. Moreover, although all mosquitoes that were alive at various time points after inoculation showed no fungal infection in the brain, fungal infection was detected in the brain in all dead mosquitoes. Our results suggest that fungal invasion of the brain represents one of the factors affecting mortality, and that the proboscis route of infection is critical for the early death of vector mosquitoes.

The Anopheles gambiae 2La chromosome inversion is associated with susceptibility to Plasmodium falciparum in Africa.

Chromosome inversions suppress genetic recombination and establish co-adapted gene complexes, or supergenes. The 2La inversion is a widespread polymorphism in the Anopheles gambiae species complex, the major African mosquito vectors of human malaria. Here we show that alleles of the 2La inversion are associated with natural malaria infection levels in wild-captured vectors from West and East Africa. Mosquitoes carrying the more-susceptible allele (2L+a) are also behaviorally less likely to be found inside houses. Vector control tools that target indoor-resting mosquitoes, such as bednets and insecticides, are currently the cornerstone of malaria control in Africa. Populations with high levels of the 2L+a allele may form reservoirs of persistent outdoor malaria transmission requiring novel measures for surveillance and control. The 2La inversion is a major and previously unappreciated component of the natural malaria transmission system in Africa, influencing both malaria susceptibility and vector behavior.

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.].

Single low dose primaquine to reduce gametocyte carriage and Plasmodium falciparum transmission after artemether-lumefantrine in children with asymptomatic infection: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: A single low dose (0.25 mg/kg) of primaquine is recommended as a gametocytocide in combination with artemisinin-based combination therapies for Plasmodium falciparum but its effect on post-treatment gametocyte circulation and infectiousness to mosquitoes has not been quantified. METHODS: In this randomised, double-blind, placebo-controlled trial, 360 asymptomatic parasitaemic children aged 2-15 years were enrolled and assigned to receive: artemether-lumefantrine (AL) and a dose of placebo; AL and a 0.25 mg/kg primaquine dose; or AL and a 0.40 mg/kg primaquine dose. On days 0, 2, 3, 7, 10 and 14, gametocytes were detected and quantified by microscopy, Pfs25 mRNA quantitative nucleic acid sequence based amplification (QT-NASBA), and quantitative reverse-transcriptase PCR (qRT-PCR). For a subset of participants, pre- and post-treatment infectiousness was assessed by mosquito feeding assays on days -1, 3, 7, 10 and 14. RESULTS: Both primaquine arms had lower gametocyte prevalences after day 3 compared to the placebo arm, regardless of gametocyte detection method. The mean (95% confidence interval) number of days to gametocyte clearance in children with patent gametocytes on day 0 (N = 150) was 19.7 (14.6 - 24.8), 7.7 (6.3 - 9.1) and 8.2 (6.7 - 9.6) for the AL-placebo, the 0.25 mg/kg primaquine dose and the 0.40 mg/kg primaquine dose arms, respectively. While 38.0% (30/79) of selected gametocytaemic individuals were infectious before treatment, only 1/251 participant, from the AL-placebo group, infected mosquitoes after treatment. CONCLUSIONS: We observed similar gametocyte clearance rates after 0.25 and 0.40 mg/kg primaquine doses. Infectivity to mosquitoes after AL was very low and absent in primaquine arms. CLINICALTRIALS. GOV REGISTRATION: NCT01935882.

Evolution of GOUNDRY, a cryptic subgroup of Anopheles gambiae s.l., and its impact on susceptibility to Plasmodium infection.

The recent discovery of a previously unknown genetic subgroup of Anopheles gambiae sensu lato underscores our incomplete understanding of complexities of vector population demographics in Anopheles. This subgroup, named GOUNDRY, does not rest indoors as adults and is highly susceptible to Plasmodium infection in the laboratory. Initial description of GOUNDRY suggested it differed from other known Anopheles taxa in surprising and sometimes contradictory ways, raising a number of questions about its age, population size and relationship to known subgroups. To address these questions, we sequenced the complete genomes of 12 wild-caught GOUNDRY specimens and compared these genomes to a panel of Anopheles genomes. We show that GOUNDRY is most closely related to Anopheles coluzzii, and the timing of cladogenesis is not recent, substantially predating the advent of agriculture. We find a large region of the X chromosome that has swept to fixation in GOUNDRY within the last 100 years, which may be an inversion that serves as a partial barrier to contemporary gene flow. Interestingly, we show that GOUNDRY has a history of inbreeding that is significantly associated with susceptibility to Plasmodium infection in the laboratory. Our results illuminate the genomic evolution of one of probably several cryptic, ecologically specialized subgroups of Anopheles and provide a potent example of how vector population dynamics may complicate efforts to control or eradicate malaria.

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.