Windborne long-distance migration of malaria mosquitoes in the Sahel.

Over the past two decades efforts to control malaria have halved the number of cases globally, yet burdens remain high in much of Africa and the elimination of malaria has not been achieved even in areas where extreme reductions have been sustained, such as South Africa1,2. Studies seeking to understand the paradoxical persistence of malaria in areas in which surface water is absent for 3-8 months of the year have suggested that some species of Anopheles mosquito use long-distance migration3. Here we confirm this hypothesis through aerial sampling of mosquitoes at 40-290 m above ground level and provide-to our knowledge-the first evidence of windborne migration of African malaria vectors, and consequently of the pathogens that they transmit. Ten species, including the primary malaria vector Anopheles coluzzii, were identified among 235 anopheline mosquitoes that were captured during 617 nocturnal aerial collections in the Sahel of Mali. Notably, females accounted for more than 80% of all of the mosquitoes that we collected. Of these, 90% had taken a blood meal before their migration, which implies that pathogens are probably transported over long distances by migrating females. The likelihood of capturing Anopheles species increased with altitude (the height of the sampling panel above ground level) and during the wet seasons, but variation between years and localities was minimal. Simulated trajectories of mosquito flights indicated that there would be mean nightly displacements of up to 300 km for 9-h flight durations. Annually, the estimated numbers of mosquitoes at altitude that cross a 100-km line perpendicular to the prevailing wind direction included 81,000 Anopheles gambiae sensu stricto, 6 million A. coluzzii and 44 million Anopheles squamosus. These results provide compelling evidence that millions of malaria vectors that have previously fed on blood frequently migrate over hundreds of kilometres, and thus almost certainly spread malaria over these distances. The successful elimination of malaria may therefore depend on whether the sources of migrant vectors can be identified and controlled.

Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus.

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d'Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions.

Confirmation of the presence of Anopheles stephensi among internally displaced people's camps and host communities in Aden city, Yemen.

BACKGROUND: Declines in global malaria cases and deaths since the millennium are currently challenged by multiple factors including funding limitations, limits of, and resistance to vector control tools, and also recent spread of the invasive vector species, Anopheles stephensi-especially into novel urban settings where malaria rates are typically low. Coupled with general increases in urbanization and escalations in the number of conflicts creating rapid and unplanned population displacement into temporary shelter camps within host urban areas, particularly in the Middle East and sub-Saharan Africa, increased urban malaria is a major threat to control and elimination. METHODS: Entomological monitoring surveys (targeting Aedes aegypti) of water containers across urban areas hosting internally displaced people (IDP) communities in Aden city, Yemen, were performed by The MENTOR Initiative, a non-governmental organisation. As part of these surveys in 2021 23 larvae collected and raised to adults were morphologically identified as An. stephensi. Twelve of the samples were sent to Liverpool School of Tropical Medicine for independent morphological assessment and genetic analysis by sequencing the ribosomal ITS2 region and the mitochondrial COI gene. RESULTS: All twelve samples were confirmed morphologically and by sequence comparison of the single ITS2 and COI haplotype detected to the NCBI BLAST database as An. stephensi. Phylogenetic analysis with comparable COI sequences indicated close relationship to haplotypes found in Djibouti and Ethiopia. CONCLUSION: The study results confirm the presence of An. stephensi in Yemen. Confirmation of the species in multiple urban communities hosting thousands of IDPs living in temporary shelters with widescale dependency on open water containers is of particular concern due to the vulnerability of the population and abundance of favourable breeding sites for the vector. Proactive monitoring and targeted integrated vector management are required to limit impacts in this area of typically low malaria transmission, and to prevent further the spread of An. stephensi within the region.

Advances in the genetic characterization of the malaria vector, Anopheles funestus, and implications for improved surveillance and control.

Anopheles mosquitoes present a major public health challenge in sub-Saharan Africa; notably, as vectors of malaria that kill over half a million people annually. In parts of the east and southern Africa region, one species in the Funestus group, Anopheles funestus, has established itself as an exceptionally dominant vector in some areas, it is responsible for more than 90% of all malaria transmission events. However, compared to other malaria vectors, the species is far less studied, partly due to difficulties in laboratory colonization and the unresolved aspects of its taxonomy and systematics. Control of An. funestus is also increasingly difficult because it has developed widespread resistance to public health insecticides. Fortunately, recent advances in molecular techniques are enabling greater insights into species identity, gene flow patterns, population structure, and the spread of resistance in mosquitoes. These advances and their potential applications are reviewed with a focus on four research themes relevant to the biology and control of An. funestus in Africa, namely: (i) the taxonomic characterization of different vector species within the Funestus group and their role in malaria transmission; (ii) insecticide resistance profile; (iii) population genetic diversity and gene flow, and (iv) applications of genetic technologies for surveillance and control. The research gaps and opportunities identified in this review will provide a basis for improving the surveillance and control of An. funestus and malaria transmission in Africa.

Modelling spatiotemporal trends in the frequency of genetic mutations conferring insecticide target-site resistance in African mosquito malaria vector species.

BACKGROUND: Resistance in malaria vectors to pyrethroids, the most widely used class of insecticides for malaria vector control, threatens the continued efficacy of vector control tools. Target-site resistance is an important genetic resistance mechanism caused by mutations in the voltage-gated sodium channel (Vgsc) gene that encodes the pyrethroid target-site. Understanding the geographic distribution of target-site resistance, and temporal trends across different vector species, can inform strategic deployment of vector control tools. RESULTS: We develop a Bayesian statistical spatiotemporal model to interpret species-specific trends in the frequency of the most common resistance mutations, Vgsc-995S and Vgsc-995F, in three major malaria vector species Anopheles gambiae, An. coluzzii, and An. arabiensis over the period 2005-2017. The models are informed by 2418 observations of the frequency of each mutation in field sampled mosquitoes collected from 27 countries spanning western and eastern regions of Africa. For nine selected countries, we develop annual predictive maps which reveal geographically structured patterns of spread of each mutation at regional and continental scales. The results show associations, as well as stark differences, in spread dynamics of the two mutations across the three vector species. The coverage of ITNs was an influential predictor of Vgsc allele frequencies, with modelled relationships between ITN coverage and allele frequencies varying across species and geographic regions. We found that our mapped Vgsc allele frequencies are a significant partial predictor of phenotypic resistance to the pyrethroid deltamethrin in An. gambiae complex populations. CONCLUSIONS: Our predictive maps show how spatiotemporal trends in insecticide target-site resistance mechanisms in African An. gambiae vary across individual vector species and geographic regions. Molecular surveillance of resistance mechanisms will help to predict resistance phenotypes and track their spread.

Whole-genome sequencing reveals high complexity of copy number variation at insecticide resistance loci in malaria mosquitoes.

Polymorphisms in genetic copy number can influence gene expression, coding sequence, and zygosity, making them powerful actors in the evolutionary process. Copy number variants (CNVs) are however understudied, being more difficult to detect than single-nucleotide polymorphisms. We take advantage of the intense selective pressures on the major malaria vector Anopheles gambiae, caused by the widespread use of insecticides for malaria control, to investigate the role of CNVs in the evolution of insecticide resistance. Using the whole-genome sequencing data from 1142 samples in the An. gambiae 1000 genomes project, we identified 250 gene-containing CNVs, encompassing a total of 267 genes of which 28 were in gene families linked to metabolic insecticide resistance, representing significant enrichment of these families. The five major gene clusters for metabolic resistance all contained CNVs, with 44 different CNVs being found across these clusters and multiple CNVs frequently covering the same genes. These 44 CNVs are widespread (45% of individuals carry at least one of them) and have been spreading through positive selection, indicated by their high local frequencies and extended haplotype homozygosity. Our results demonstrate the importance of CNVs in the response to selection, highlighting the urgent need to identify the contribution of each CNV to insecticide resistance and to track their spread as the use of insecticides in malaria endemic countries intensifies and as the operational deployment of next-generation bed nets targeting metabolic resistance gathers pace. Our detailed descriptions of CNVs found across the species range provide the tools to do so.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Invasive Malaria Vector Anopheles stephensi Mosquitoes in Sudan, 2016-2018.

Anopheles stephensi mosquitoes are urban malaria vectors in Asia that have recently invaded the Horn of Africa. We detected emergence of An. stephensi mosquitoes in 2 noncontiguous states of eastern Sudan. Results of mitochondrial DNA sequencing suggest the possibility of distinct invasions, potentially from a neighboring country.

Evolution of the Insecticide Target Rdl in African Anopheles Is Driven by Interspecific and Interkaryotypic Introgression.

The evolution of insecticide resistance mechanisms in natural populations of Anopheles malaria vectors is a major public health concern across Africa. Using genome sequence data, we study the evolution of resistance mutations in the resistance to dieldrin locus (Rdl), a GABA receptor targeted by several insecticides, but most notably by the long-discontinued cyclodiene, dieldrin. The two Rdl resistance mutations (296G and 296S) spread across West and Central African Anopheles via two independent hard selective sweeps that included likely compensatory nearby mutations, and were followed by a rare combination of introgression across species (from A. gambiae and A. arabiensis to A. coluzzii) and across nonconcordant karyotypes of the 2La chromosomal inversion. Rdl resistance evolved in the 1950s as the first known adaptation to a large-scale insecticide-based intervention, but the evolutionary lessons from this system highlight contemporary and future dangers for management strategies designed to combat development of resistance in malaria vectors.

The genetic architecture of target-site resistance to pyrethroid insecticides in the African malaria vectors Anopheles gambiae and Anopheles coluzzii.

Resistance to pyrethroid insecticides is a major concern for malaria vector control. Pyrethroids target the voltage-gated sodium channel (VGSC), an essential component of the mosquito nervous system. Substitutions in the amino acid sequence can induce a resistance phenotype. We use whole-genome sequence data from phase 2 of the Anopheles gambiae 1000 Genomes Project (Ag1000G) to provide a comprehensive account of genetic variation in the Vgsc gene across 13 African countries. In addition to known resistance alleles, we describe 20 other non-synonymous nucleotide substitutions at appreciable population frequency and map these variants onto a protein model to investigate the likelihood of pyrethroid resistance phenotypes. Thirteen of these novel alleles were found to occur almost exclusively on haplotypes carrying the known L995F kdr (knock-down resistance) allele and may enhance or compensate for the L995F resistance genotype. A novel mutation I1527T, adjacent to a predicted pyrethroid-binding site, was found in tight linkage with V402L substitutions, similar to allele combinations associated with resistance in other insect species. We also analysed genetic backgrounds carrying resistance alleles, to determine which alleles have experienced recent positive selection, and describe ten distinct haplotype groups carrying known kdr alleles. Five of these groups are observed in more than one country, in one case separated by over 3000 km, providing new information about the potential for the geographical spread of resistance. Our results demonstrate that the molecular basis of target-site pyrethroid resistance in malaria vectors is more complex than previously appreciated, and provide a foundation for the development of new genetic tools for insecticide resistance management.

Associated patterns of insecticide resistance in field populations of malaria vectors across Africa.

The development of insecticide resistance in African malaria vectors threatens the continued efficacy of important vector control methods that rely on a limited set of insecticides. To understand the operational significance of resistance we require quantitative information about levels of resistance in field populations to the suite of vector control insecticides. Estimation of resistance is complicated by the sparsity of observations in field populations, variation in resistance over time and space at local and regional scales, and cross-resistance between different insecticide types. Using observations of the prevalence of resistance in mosquito species from the Anopheles gambiae complex sampled from 1,183 locations throughout Africa, we applied Bayesian geostatistical models to quantify patterns of covariation in resistance phenotypes across different insecticides. For resistance to the three pyrethroids tested, deltamethrin, permethrin, and λ-cyhalothrin, we found consistent forms of covariation across sub-Saharan Africa and covariation between resistance to these pyrethroids and resistance to DDT. We found no evidence of resistance interactions between carbamate and organophosphate insecticides or between these insecticides and those from other classes. For pyrethroids and DDT we found significant associations between predicted mean resistance and the observed frequency of kdr mutations in the Vgsc gene in field mosquito samples, with DDT showing the strongest association. These results improve our capacity to understand and predict resistance patterns throughout Africa and can guide the development of monitoring strategies.

Rapid selection of a pyrethroid metabolic enzyme CYP9K1 by operational malaria control activities.

Since 2004, indoor residual spraying (IRS) and long-lasting insecticide-impregnated bednets (LLINs) have reduced the malaria parasite prevalence in children on Bioko Island, Equatorial Guinea, from 45% to 12%. After target site-based (knockdown resistance; kdr) pyrethroid resistance was detected in 2004 in Anopheles coluzzii (formerly known as the M form of the Anopheles gambiae complex), the carbamate bendiocarb was introduced. Subsequent analysis showed that kdr alone was not operationally significant, so pyrethroid-based IRS was successfully reintroduced in 2012. In 2007 and 2014-2015, mass distribution of new pyrethroid LLINs was undertaken to increase the net coverage levels. The combined selection pressure of IRS and LLINs resulted in an increase in the frequency of pyrethroid resistance in 2015. In addition to a significant increase in kdr frequency, an additional metabolic pyrethroid resistance mechanism had been selected. Increased metabolism of the pyrethroid deltamethrin was linked with up-regulation of the cytochrome P450 CYP9K1. The increase in resistance prompted a reversion to bendiocarb IRS in 2016 to avoid a resurgence of malaria, in line with the national Malaria Control Program plan.

Using sibship reconstructions to understand the relationship between larval habitat productivity and oviposition behaviour in Kenyan Anopheles arabiensis.

BACKGROUND: Strategies for combatting residual malaria by targeting vectors outdoors are gaining importance as the limitations of primary indoor interventions are reached. Strategies to target ovipositing females or her offspring are broadly applicable because all mosquitoes require aquatic habitats for immature development irrespective of their biting or resting preferences. Oviposition site selection by gravid females is frequently studied by counting early instar larvae in habitats; an approach which is valid only if the number of larvae correlates with the number of females laying eggs. This hypothesis was tested against the alternative, that a higher abundance of larvae results from improved survival of a similar or fewer number of families. METHODS: In a controlled experiment, 20 outdoor artificial ponds were left uncovered for 4 days to allow oviposition by wild mosquitoes, then covered with netting and first and second instar larvae sampled daily. Natural Anopheles habitats of two different types were also identified, and all visible larvae sampled. All larvae were identified to species, and most samples of the predominant species, Anopheles arabiensis, were genotyped using microsatellites for sibling group reconstructions using two contrasting softwares, BAPS and COLONY. RESULTS: In the ponds, the number of families reconstructed by each software significantly predicted larval abundance (BAPS R2 = 0.318, p = 0.01; COLONY R2 = 0.476, p = 0.001), and suggested that around 50% of females spread larvae across multiple ponds (skip oviposition). From natural habitats, the mean family size again predicted larval abundance using BAPS (R2 = 0.829, p = 0.017) though not using COLONY (R2 = 0.218, p = 0.68), but both softwares once more suggested high rates of skip oviposition (in excess of 50%). CONCLUSION: This study shows that, whether in closely-located artificial habitats or natural breeding sites, higher early instar larval densities result from more females laying eggs in these sites. These results provide empirical support for use of early instar larval abundance as an index for oviposition site preference. Furthermore, the sharing of habitats by multiple females and the high skip-oviposition rate in An. arabiensis suggest that larviciding by auto-dissemination of insecticide may be successful.

Insecticide resistance in Anopheles gambiae from the northern Democratic Republic of Congo, with extreme knockdown resistance (kdr) mutation frequencies revealed by a new diagnostic assay.

BACKGROUND: Mutations in the voltage-gated sodium channel at codon 1014 confer knock-down resistance (kdr) to pyrethroids in a wide range of insects. Anopheles gambiae exhibits two mutant alleles at codon 1014, serine and phenylalanine; and both are now widespread across Africa. Existing screening methods only allow for one resistant allele to be detected per assay. A new locked nucleic acid (LNA) qPCR assay was developed for the simultaneous detection of both mutant alleles and the wild type allele in a single assay. This tri-allelic detection assay was assessed as part of a study of the insecticide resistance in An. gambiae sensu stricto (s.s.) in the previously un-sampled area of Nord Ubangi, Democratic Republic of the Congo. METHODS: Samples from three sites were tested for insecticide susceptibility using WHO bioassays, with and without the synergist PBO preceding pyrethroid exposures, and were subsequently analysed for frequency and resistance-association of the Vgsc-1014 and Vgsc-N1575Y mutations. Results from the LNA-kdr 1014 assay were compared to results from standard TaqMan-kdr assays. RESULTS: Anopheles gambiae sensu lato (s.l.) was by far the predominant vector captured (84%), with only low frequencies of Anopheles funestus s.l. (9%) detected in Nord Ubangi. Molecular identification found An. gambiae s.s. to be the principal vector (99%) although Anopheles coluzzii was detected at very low frequency. Anopheles gambiae were susceptible to the carbamate insecticide bendiocarb, but resistant to DDT and to the pyrethroids permethrin and deltamethrin. Susceptibility to both pyrethroids was partially restored with prior exposure to PBO suggesting likely involvement of metabolic resistance. Anopheles gambiae s.s. was homozygous for kdr resistant alleles with both the L1014F and L1014S mutations present, and the N1575Y polymorphism was present at low frequency. The LNA-kdr assay simultaneously detected both resistant alleles and gave results entirely consistent with those from the two TaqMan-kdr assays. CONCLUSION: This study provides rare data on insecticide resistance and mechanisms in Anopheles from the centre of Africa, with the first detection of N1575Y. Nord Ubangi populations of An. gambiae s.s. show insecticide resistance mediated by both metabolic mechanisms and Vgsc mutations. The LNA-kdr assay is particularly suitable for use in populations in which both 1014S and 1014F kdr alleles co-occur and provides robust results, with higher throughput and at a quarter of the cost of TaqMan assays.

CYP6 P450 enzymes and ACE-1 duplication produce extreme and multiple insecticide resistance in the malaria mosquito Anopheles gambiae.

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.

The last bastion? X chromosome genotyping of Anopheles gambiae species pair males from a hybrid zone reveals complex recombination within the major candidate 'genomic island of speciation'.

Speciation with gene flow may be aided by reduced recombination helping to build linkage between genes involved in the early stages of reproductive isolation. Reduced recombination on chromosome X has been implicated in speciation within the Anopheles gambiae complex, species of which represent the major Afrotropical malaria vectors. The most recently diverged, morphologically indistinguishable, species pair, A. gambiae and Anopheles coluzzii, ubiquitously displays a 'genomic island of divergence' spanning over 4 Mb from chromosome X centromere, which represents a particularly promising candidate region for reproductive isolation genes, in addition to containing the diagnostic markers used to distinguish the species. Very low recombination makes the island intractable for experimental recombination studies, but an extreme hybrid zone in Guinea Bissau offers the opportunity for natural investigation of X-island recombination. SNP analysis of chromosome X hemizygous males revealed: (i) strong divergence in the X-island despite a lack of autosomal divergence; (ii) individuals with multiple-recombinant genotypes, including likely double crossovers and localized gene conversion; (iii) recombination-driven discontinuity both within and between the molecular species markers, suggesting that the utility of the diagnostics is undermined under high hybridization. The largely, but incompletely protected nature of the X centromeric genomic island is consistent with a primary candidate area for accumulation of adaptive variants driving speciation with gene flow, while permitting some selective shuffling and removal of genetic variation.

Does insecticide resistance contribute to heterogeneities in malaria transmission in The Gambia?

BACKGROUND: Malaria hotspots, areas with consistently higher than average transmission, may become increasingly common as malaria declines. This phenomenon, currently observed in The Gambia, may be caused by several factors, including some related to the local vectors, whose contribution is poorly understood. METHODS: Using WHO susceptibility bioassays, insecticide resistance status was determined in vector populations sampled from six pairs of villages across The Gambia, each pair contained a low and high prevalence village. RESULTS: Three vector species were observed (23.5% Anopheles arabiensis, 31.2% Anopheles gambiae, 43.3% Anopheles coluzzii and 2.0% An. coluzzii × An. gambiae hybrids). Even at a fine scale, significant differences in species composition were detected within village pairs. Resistance to both DDT and deltamethrin was more common in An. gambiae, most markedly in the eastern part of The Gambia and partly attributable to differing frequencies of resistance mutations. The Vgsc-1014F target site mutation was strongly associated with both DDT (OR = 256.7, (95% CI 48.6-6374.3, p < 0.001) and deltamethrin survival (OR = 9.14, (95% CI 4.24-21.4, p < 0.001). A second target site mutation, Vgsc-1575Y, which co-occurs with Vgsc-1014F, and a metabolic marker of resistance, Gste2-114T, conferred additional survival benefits to both insecticides. DDT resistance occurred significantly more frequently in villages with high malaria prevalence (p = 0.025) though this did not apply to deltamethrin resistance. CONCLUSION: Whilst causality of relationships requires further investigation, variation in vector species and insecticide resistance in The Gambia is associated with malaria endemicity; with a notably higher prevalence of infection and insecticide resistance in the east of the country. In areas with heterogeneous malaria transmission, the role of the vector should be investigated to guide malaria control interventions.

Limited genomic divergence between intraspecific forms of Culex pipiens under different ecological pressures.

BACKGROUND: Divergent selection can be a major driver of ecological speciation. In insects of medical importance, understanding the speciation process is both of academic interest and public health importance. In the West Nile virus vector Culex pipiens, intraspecific pipiens and molestus forms vary in ecological and physiological traits. Populations of each form appear to share recent common ancestry but patterns of genetic differentiation across the genome remain unknown. Here, we undertook an AFLP genome scan on samples collected from both sympatric and allopatric populations from Europe and the USA to quantify the extent of genomic differentiation between the two forms. RESULTS: The forms were clearly differentiated but each exhibited major population sub-structuring between continents. Divergence between pipiens and molestus forms from USA was higher than in both inter- and intra-continental comparisons with European samples. The proportion of outlier loci between pipiens and molestus (≈3 %) was low but consistent in both continents, and similar to those observed between sibling species of other mosquito species which exhibit contemporary gene flow. Only two of the outlier loci were shared between inter-form comparisons made within Europe and USA. CONCLUSION: This study supports the molestus and pipiens status as distinct evolutionary entities with low genomic divergence. The low number of shared divergent loci between continents suggests a relatively limited number of genomic regions determining key typological traits likely to be driving incipient speciation and/or adaptation of molestus to anthropogenic habitats.

Contemporary evolution of resistance at the major insecticide target site gene Ace-1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae.

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine-serine substitution at codon 119 of the Ace-1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace-1 119S haplotype, whereas 119G diversity was high overall but very low at non-synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace-1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace-1 gene, whereas 119G alleles were unduplicated. Ace-1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace-1, emphasizing the need to integrate CNV analysis into genome scans for selection.

Insecticide resistance profile of Anopheles gambiae from a phase II field station in Cové, southern Benin: implications for the evaluation of novel vector control products.

BACKGROUND: Novel indoor residual spraying (IRS) and long-lasting insecticidal net (LLIN) products aimed at improving the control of pyrethroid-resistant malaria vectors have to be evaluated in Phase II semi-field experimental studies against highly pyrethroid-resistant mosquitoes. To better understand their performance it is necessary to fully characterize the species composition, resistance status and resistance mechanisms of the vector populations in the experimental hut sites. METHODS: Bioassays were performed to assess phenotypic insecticide resistance in the malaria vector population at a newly constructed experimental hut site in Cové, a rice growing area in southern Benin, being used for WHOPES Phase II evaluation of newly developed LLIN and IRS products. The efficacy of standard WHOPES-approved pyrethroid LLIN and IRS products was also assessed in the experimental huts. Diagnostic genotyping techniques and microarray studies were performed to investigate the genetic basis of pyrethroid resistance in the Cové Anopheles gambiae population. RESULTS: The vector population at the Cové experimental hut site consisted of a mixture of Anopheles coluzzii and An. gambiae s.s. with the latter occurring at lower frequencies (23 %) and only in samples collected in the dry season. There was a high prevalence of resistance to pyrethroids and DDT (>90 % bioassay survival) with pyrethroid resistance intensity reaching 200-fold compared to the laboratory susceptible An. gambiae Kisumu strain. Standard WHOPES-approved pyrethroid IRS and LLIN products were ineffective in the experimental huts against this vector population (8-29 % mortality). The L1014F allele frequency was 89 %. CYP6P3, a cytochrome P450 validated as an efficient metabolizer of pyrethroids, was over-expressed. CONCLUSION: Characterizing pyrethroid resistance at Phase II field sites is crucial to the accurate interpretation of the performance of novel vector control products. The strong levels of pyrethroid resistance at the Cové experimental hut station make it a suitable site for Phase II experimental hut evaluations of novel vector control products, which aim for improved efficacy against pyrethroid-resistant malaria vectors to WHOPES standards. The resistance genes identified can be used as markers for further studies investigating the resistance management potential of novel mixture LLIN and IRS products tested at the site.