Video augmentation of the WHO cone assay to quantify mosquito behavioural responses to insecticide-treated nets.

BACKGROUND: Insecticide-treated nets (ITNs) using pyrethroids have been the main vector control tools deployed in malaria endemic countries and are responsible for the dramatic reduction in African malaria cases in the early 2000s. The World Health Organization (WHO) cone test was designed to assess the rapid toxicity effects of pyrethroid exposure on mosquito vectors but has yielded no insights beyond 60-min knockdown and 24-h mortality. As dual-active-ingredient (AI) ITNs become more widespread, bioassays that can provide realistic assessment of single- and dual-treated ITNs (i.e. nets with more than one active ingredient) are urgently needed. METHODS: We present an augmentation of the cone test that enables accurate quantification of vector behavioural responses (specifically movement, spatial and temporal occupancy) to ITNs using video recording and bespoke software that uses background segmentation methods to detect spatial changes in the movement of mosquitoes within the cone. Four strains of Anopheles gambiae sensu lato (s.l.) were exposed to four ITNs (PermaNet 2.0, PermaNet 3.0, Olyset Net, Interceptor G2) and untreated nets in these modified cone tests. Life history data (post-exposure blood-feeding, blood meal weight, longevity) for individual mosquitoes were recorded. RESULTS: All mosquitoes responded to the presence of ITNs, spending from 1.48 to 3.67 times more time in the upper region of the cone, depending on the ITN type. Of all ITNs, PermaNet 2.0 provoked the smallest change in behavioural response. Activity in the cone influenced observed post-exposure longevity, and in resistant strains exposed to Interceptor G2, the higher the activity, the greater the risk of dying, as long as the proportion of activity at the net surface was less than 50%. All ITNs inhibited blood-feeding, and smaller blood meals were taken when mosquitoes fed. CONCLUSIONS: The additional mosquito behaviour data obtained by using this modification to the WHO cone test provides unique insight into the innate responses of different mosquito strains on untreated nets and the entomological mode of action of ITNs, important evidence when evaluating ITN characteristics.

A closer look at the WHO cone bioassay: video analysis of the hidden effects of a human host on mosquito behaviour and insecticide contact.

BACKGROUND: The WHO cone test is one of three tests currently used to evaluate the efficacy of insecticide-treated bed nets (ITNs). It generates two test outputs, knockdown and 24-h mortality, both indicative of immediate toxicity but that reveal little about the nature of mosquito and ITN interaction or how results translate to real-world settings. METHODS: A human arm held 5 mm behind the net surface acted as a host attractant during cone tests and a smartphone was used to capture mosquito behaviour in the cone. Post-exposure blood feeding and survival for nine days were recorded; ingested blood meal size was determined by measuring excreted haematin. Four strains of Anopheles gambiae (insecticide susceptible: Kisumu and N'gousso; insecticide resistant: Banfora and VK7) were tested with and without the host attractant using untreated, Permanet 2.0 and Olyset nets. Video recordings were scan sampled every five seconds to record mosquito positions on either the net, in flight or in contact with the cone. Generalized estimating equations were used to analyse all data except survival within nine days which was analysed using Weighted Cox Regression. RESULTS: Net contact was the most frequently recorded behaviour in all Anopheles spp. strains on all nets. Adding the human host as attractant triggered excitatory behaviours: in all strains, the magnitude of net contact was significantly decreased compared to tests without a host. ITN exposure altered the observed behaviour of the two susceptible strains, which exhibited a decreased response to the host during ITN tests. The resistant strains did not alter their behaviour during ITN tests. Significantly less net contact was observed during Olyset Net tests compared to Permanet 2.0. The host presence affected survival after exposure: Banfora and VK7 mosquitoes exposed to Permanet 2.0 with a host lived longer compared to tests performed without a host. However, mosquitoes that blood-fed and survived long enough to digest the blood meal did not exhibit significantly reduced longevity regardless of the presence of the host attractant. CONCLUSIONS: Simple modifications to the WHO cone test and extension of post-test monitoring beyond the current 24 h enable detailed behavioural characterizations of individual ITNs to be compiled. The effects observed from testing with a host and including blood feeding suggest that more representative estimates of true of ITN efficacy are gained with these modifications than when using the current testing protocol.

Candidate-gene based GWAS identifies reproducible DNA markers for metabolic pyrethroid resistance from standing genetic variation in East African Anopheles gambiae.

Metabolic resistance to pyrethroid insecticides is widespread in Anopheles mosquitoes and is a major threat to malaria control. DNA markers would aid predictive monitoring of resistance, but few mutations have been discovered outside of insecticide-targeted genes. Isofemale family pools from a wild Ugandan Anopheles gambiae population, from an area where operational pyrethroid failure is suspected, were genotyped using a candidate-gene enriched SNP array. Resistance-associated SNPs were detected in three genes from detoxification superfamilies, in addition to the insecticide target site (the Voltage Gated Sodium Channel gene, Vgsc). The putative associations were confirmed for two of the marker SNPs, in the P450 Cyp4j5 and the esterase Coeae1d by reproducible association with pyrethroid resistance in multiple field collections from Uganda and Kenya, and together with the Vgsc-1014S (kdr) mutation these SNPs explained around 20% of variation in resistance. Moreover, the >20 Mb 2La inversion also showed evidence of association with resistance as did environmental humidity. Sequencing of Cyp4j5 and Coeae1d detected no resistance-linked loss of diversity, suggesting selection from standing variation. Our study provides novel, regionally-validated DNA assays for resistance to the most important insecticide class, and establishes both 2La karyotype variation and humidity as common factors impacting the resistance phenotype.

Local selection in the presence of high levels of gene flow: Evidence of heterogeneous insecticide selection pressure across Ugandan Culex quinquefasciatus populations.

BACKGROUND: Culex quinquefasciatus collected in Uganda, where no vector control interventions directly targeting this species have been conducted, was used as a model to determine if it is possible to detect heterogeneities in selection pressure driven by insecticide application targeting other insect species. METHODOLOGY/PRINCIPAL FINDINGS: Population genetic structure was assessed through microsatellite analysis, and the impact of insecticide pressure by genotyping two target-site mutations, Vgsc-1014F of the voltage-gated sodium channel target of pyrethroid and DDT insecticides, and Ace1-119S of the acetylcholinesterase gene, target of carbamate and organophosphate insecticides. No significant differences in genetic diversity were observed among populations by microsatellite markers with HE ranging from 0.597 to 0.612 and low, but significant, genetic differentiation among populations (FST = 0.019, P = 0.001). By contrast, the insecticide-resistance markers display heterogeneous allelic distributions with significant differences detected between Central Ugandan (urban) populations relative to Eastern and Southwestern (rural) populations. In the central region, a frequency of 62% for Vgsc-1014F, and 32% for the Ace1-119S resistant allele were observed. Conversely, in both Eastern and Southwestern regions the Vgsc-1014F alleles were close to fixation, whilst Ace1-119S allele frequency was 12% (although frequencies may be underestimated due to copy number variation at both loci). CONCLUSIONS/SIGNIFICANCE: Taken together, the microsatellite and both insecticide resistance target-site markers provide evidence that in the face of intense gene flow among populations, disjunction in resistance frequencies arise due to intense local selection pressures despite an absence of insecticidal control interventions targeting Culex.

Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus.

Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for Vgsc CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ≈10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for Vgsc CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of Vgsc CN in wild populations and the elucidation of association between the Vgsc CN and insecticide resistance.

Spatial repellents and malaria transmission in an endemic area of Cambodia with high mosquito net usage.

BACKGROUND: The spread of artemisinin resistant malaria from SE Asia to the rest of the world remains a threat that will only be ended by eliminating malaria from the region. Novel control approaches are required to mitigate this threat. Spatial repellents (SR) are one such approach. We therefore conducted a multiple cross-over experiment from April 2013 - April 2014, in which all houses in one of two villages in Mondolkiri Province, Cambodia were alternately supplied with an emanator of the spatial repellent metofluthrin per 30 m3 of protected area to cover all potential peridomestic areas where people might spend their time before sleeping. Emanators were replaced every month for a three-month period. MATERIAL AND METHODS: Mosquito densities were simultaneously monitored in each village for two weeks every month using six CDC light-traps/night run from 18.00 to 07.00 hrs inside bedrooms and malaria prevalence, seroconversion and gSG6 protein rates assessed from prevalence surveys. After emanators were installed in the first village they were installed in the second village for a further three-month period and following that were again used in the initial village for a further three months. Surveys were undertaken before the initial installation of the emanators and at each cross-over point. RESULTS: Anopheles dirus densities were highest in houses closest to the forest. Transmission rates were low even before the application of the emanators. Perhaps due to the low levels of malaria transmission in Mondolkiri no significant relationships were found in Plasmodium cases or seroconversion rates between villages, surveys or by intervention. Adult males, who might spend more time unprotected in the forest at night, appeared to be at greater risk of becoming infected with P. falciparum malaria as compared to women or young children. CONCLUSION: At the malaria transmission levels present in Mondolkiri the metofluthrin emanators evaluated had no observable effect on malaria prevalence. This may be due to confounding by low prevalence rates.

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.

A significant association between deltamethrin resistance, Plasmodium falciparum infection and the Vgsc-1014S resistance mutation in Anopheles gambiae highlights the epidemiological importance of resistance markers.

BACKGROUND: The success of malaria vector control is threatened by widespread pyrethroid insecticide resistance. However, the extent to which insecticide resistance impacts transmission is unclear. The objective of this study was to examine the association between the DDT/pyrethroid knockdown resistance mutation Vgsc-1014S, commonly termed kdr, and infection with Plasmodium falciparum sporozoites in Anopheles gambiae. METHODS: WHO standard methods were used to characterize susceptibility of wild female mosquitoes to 0.05 % deltamethrin. PCR-based molecular diagnostics were used to identify mosquitoes to species and to genotype at the Vgsc-L1014S locus. ELISAs were used to detect the presence of P. falciparum sporozoites and for blood meal identification. RESULTS: Anopheles mosquitoes were resistant to deltamethrin with mortality rates of 77.7 % [95 % CI 74.9-80.3 %]. Of 545 mosquitoes genotyped 96.5 % were A. gambiae s.s. and 3.5 % were Anopheles arabiensis. The Vgsc-1014S mutation was detected in both species. Both species were predominantly anthropophagic. In A. gambiae s.s., Vgsc-L1014S genotype was significantly associated with deltamethrin resistance (χ2 = 11.2; p < 0.001). The P. falciparum sporozoite infection rate was 4.2 %. There was a significant association between the presence of sporozoites and Vgsc-L1014S genotype in A. gambiae s.s. (χ2 = 4.94; p = 0.026). CONCLUSIONS: One marker, Vgsc-1014S, was associated with insecticide resistance and P. falciparum infection in wild-caught mixed aged populations of A. gambiae s.s. thereby showing how resistance may directly impact transmission.

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.

Contemporary gene flow between wild An. gambiae s.s. and An. arabiensis.

BACKGROUND: In areas where the morphologically indistinguishable malaria mosquitoes Anopheles gambiae Giles and An. arabiensis Patton are sympatric, hybrids are detected occasionally via species-diagnostic molecular assays. An. gambiae and An. arabiensis exhibit both pre- and post-reproductive mating barriers, with swarms largely species-specific and male F1 (first-generation) hybrids sterile. Consequently advanced-stage hybrids (back-crosses to parental species), which would represent a route for potentially-adaptive introgression, are expected to be very rare in natural populations. Yet the use of one or two physically linked single-locus diagnostic assays renders them indistinguishable from F1 hybrids and levels of interspecific gene flow are unknown. METHODS: We used data from over 350 polymorphic autosomal SNPs to investigate post F1 gene flow via patterns of genomic admixture between An. gambiae and An. arabiensis from eastern Uganda. Simulations were used to investigate the statistical power to detect hybrids with different levels of crossing and to identify the hybrid category significantly admixed genotypes could represent. RESULTS: A range of admixture proportions were detected for 11 field-collected hybrids identified via single-locus species-diagnostic PCRs. Comparison of admixture data with simulations indicated that at least seven of these hybrids were advanced generation crosses, with backcrosses to each species identified. In addition, of 36 individuals typing as An. gambiae or An. arabiensis that exhibited outlying admixture proportions, ten were identified as significantly mixed backcrosses, and at least four of these were second or third generation crosses. CONCLUSIONS: Our results show that hybrids detected using standard diagnostics will often be hybrid generations beyond F1, and that in our study area around 5% (95% confidence intervals 3%-9%) of apparently 'pure' species samples may also be backcrosses. This is likely an underestimate because of rapidly-declining detection power beyond the first two backcross generations. Post-F1 gene flow occurs at a far from inconsequential rate between An. gambiae and An. arabiensis, and, especially for traits under strong selection, could readily lead to adaptive introgression of genetic variants relevant for vector control.

Genetic basis of pyrethroid resistance in a population of Anopheles arabiensis, the primary malaria vector in Lower Moshi, north-eastern Tanzania.

BACKGROUND: Pyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, consequently, studies are only just beginning to unravel the genes involved. Permethrin resistance in An. arabiensis in Lower Moshi, Tanzania has been linked to elevated levels of both P450 monooxygenases and ß-esterases. We have conducted a gene expression study to identify specific genes linked with metabolic resistance in the Lower Moshi An. arabiensis population. METHODS: Microarray experiments employing an An. gambiae whole genome expression chip were performed on An. arabiensis, using interwoven loop designs. Permethrin-exposed survivors were compared to three separate unexposed mosquitoes from the same or a nearby population. A subsection of detoxification genes were chosen for subsequent quantitative real-time PCR (qRT-PCR). RESULTS: Microarray analysis revealed significant over expression of 87 probes and under expression of 85 probes (in pairwise comparisons between permethrin survivors and unexposed sympatric and allopatric samples from Dar es Salaam (controls). For qRT-PCR we targeted over expressed ABC transporter genes (ABC '2060'), a glutathione-S-transferase, P450s and esterases. Design of efficient, specific primers was successful for ABC '2060'and two P450s (CYP6P3, CYP6M2). For the CYP4G16 gene, we used the primers that were previously used in a microarray study of An. arabiensis from Zanzibar islands. Over expression of CYP4G16 and ABC '2060' was detected though with contrasting patterns in pairwise comparisons between survivors and controls. CYP4G16 was only up regulated in survivors, whereas ABC '2060' was similar in survivors and controls but over expressed in Lower Moshi samples compared to the Dar es Salaam samples. Increased transcription of CYP4G16 and ABC '2060' are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. CONCLUSIONS: Increased transcription of a P450 (CYP4G16) and an ABC transporter (ABC 2060) are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Our study provides replication of CYP4G16 as a candidate gene for pyrethroid resistance in An. arabiensis, although its role may not be in detoxification, and requires further investigation.

Gene flow-dependent genomic divergence between Anopheles gambiae M and S forms.

Anopheles gambiae sensu stricto exists as two often-sympatric races termed the M and S molecular forms, characterized by fixed differences at an X-linked marker. Extreme divergence between M and S forms at pericentromeric "genomic islands" suggested that selection on variants therein could be driving interform divergence in the presence of ongoing gene flow, but recent work has detected much more widespread genomic differentiation. Whether such genomic islands are important in reproductive isolation or represent ancestral differentiation preserved by low recombination is currently unclear. A critical test of these competing hypotheses could be provided by comparing genomic divergence when rates of recent introgression vary. We genotyped 871 single nucleotide polymorphisms (SNPs) in A. gambiae sensu stricto from locations of M and S sympatry and allopatry, encompassing the full range of observed hybridization rates (0-25%). M and S forms were readily partitioned based on genomewide SNP variation in spite of evidence for ongoing introgression that qualitatively reflects hybridization rates. Yet both the level and the heterogeneity of genomic divergence varied markedly in line with levels of introgression. A few genomic regions of differentiation between M and S were common to each sampling location, the most pronounced being two centromere-proximal speciation islands identified previously but with at least one additional region outside of areas expected to exhibit reduced recombination. Our results demonstrate that extreme divergence at genomic islands does not simply represent segregating ancestral polymorphism in regions of low recombination and can be resilient to substantial gene flow. This highlights the potential for islands comprising a relatively small fraction of the genome to play an important role in early-stage speciation when reproductive isolation is limited.

Cryptic diversity within the major trypanosomiasis vector Glossina fuscipes revealed by molecular markers.

BACKGROUND: The tsetse fly Glossina fuscipes s.l. is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three G. fuscipes subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units. PRINCIPAL FINDINGS: The nuclear ribosomal Internal transcribed spacer 1 (ITS1) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies G. f. fuscipes or G. f. quanzensis. Instead, the most strongly supported monophyletic group was comprised of flies sampled from Ethiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of G. fuscipes s.l., and was useful for deriving the interrelationship of closely related populations. CONCLUSION/SIGNIFICANCE: We propose that the morphological classification alone is not used to classify populations of G. fuscipes for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme.

Association mapping of insecticide resistance in wild Anopheles gambiae populations: major variants identified in a low-linkage disequilbrium genome.

BACKGROUND: Association studies are a promising way to uncover the genetic basis of complex traits in wild populations. Data on population stratification, linkage disequilibrium and distribution of variant effect-sizes for different trait-types are required to predict study success but are lacking for most taxa. We quantified and investigated the impacts of these key variables in a large-scale association study of a strongly selected trait of medical importance: pyrethroid resistance in the African malaria vector Anopheles gambiae. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped ≈1500 resistance-phenotyped wild mosquitoes from Ghana and Cameroon using a 1536-SNP array enriched for candidate insecticide resistance gene SNPs. Three factors greatly impacted study power. (1) Population stratification, which was attributable to co-occurrence of molecular forms (M and S), and cryptic within-form stratification necessitating both a partitioned analysis and genomic control. (2) All SNPs of substantial effect (odds ratio, OR>2) were rare (minor allele frequency, MAF<0.05). (3) Linkage disequilibrium (LD) was very low throughout most of the genome. Nevertheless, locally high LD, consistent with a recent selective sweep, and uniformly high ORs in each subsample facilitated significant direct and indirect detection of the known insecticide target site mutation kdr L1014F (OR≈6; P<10(-6)), but with resistance level modified by local haplotypic background. CONCLUSION: Primarily as a result of very low LD in wild A. Gambiae, LD-based association mapping is challenging, but is feasible at least for major effect variants, especially where LD is enhanced by selective sweeps. Such variants will be of greatest importance for predictive diagnostic screening.

Towards a genetic map for Anopheles albimanus: identification of microsatellite markers and a preliminary linkage map for chromosome 2.

Fifty microsatellite loci were identified in the malaria vector Anopheles albimanus. Markers segregating in F2 progeny of crosses between laboratory strains of An. albimanus were used to construct a preliminary genetic map. More than 300 progeny were genotyped, but the resolution of the map was limited by the lack of polymorphisms in the microsatellite alleles. A robust linkage map for chromosome 2 was established, and additional markers were assigned to the third and X chromosomes by linkage to morphological markers of known physical location. Additional non-informative microsatellite sequences are provided including some showing similarity to those of An. gambiae. This study significantly increases the number of genetic markers available for An. albimanus and provides useful tools for population genetics and genetic mapping studies in this important malaria vector.

High, clustered, nucleotide diversity in the genome of Anopheles gambiae revealed through pooled-template sequencing: implications for high-throughput genotyping protocols.

BACKGROUND: Association mapping approaches are dependent upon discovery and validation of single nucleotide polymorphisms (SNPs). To further association studies in Anopheles gambiae we conducted a major resequencing programme, primarily targeting regions within or close to candidate genes for insecticide resistance. RESULTS: Using two pools of mosquito template DNA we sequenced over 300 kbp across 660 distinct amplicons of the An. gambiae genome. Comparison of SNPs identified from pooled templates with those from individual sequences revealed a very low false positive rate. False negative rates were much higher and mostly resulted from SNPs with a low minor allele frequency. Pooled-template sequencing also provided good estimates of SNP allele frequencies. Allele frequency estimation success, along with false positive and negative call rates, improved significantly when using a qualitative measure of SNP call quality. We identified a total of 7062 polymorphic features comprising 6995 SNPs and 67 indels, with, on average, a SNP every 34 bp; a high rate of polymorphism that is comparable to other studies of mosquitoes. SNPs were significantly more frequent in members of the cytochrome p450 mono-oxygenases and carboxy/cholinesterase gene-families than in glutathione-S-transferases, other detoxification genes, and control genomic regions. Polymorphic sites showed a significantly clustered distribution, but the degree of SNP clustering (independent of SNP frequency) did not vary among gene families, suggesting that clustering of polymorphisms is a general property of the An. gambiae genome. CONCLUSION: The high frequency and clustering of SNPs has important ramifications for the design of high-throughput genotyping assays based on allele specific primer extension or probe hybridisation. We illustrate these issues in the context of the design of Illumina GoldenGate assays.

Mapping a quantitative trait locus (QTL) conferring pyrethroid resistance in the African malaria vector Anopheles funestus.

BACKGROUND: Pyrethroid resistance in Anopheles funestus populations has led to an increase in malaria transmission in southern Africa. Resistance has been attributed to elevated activities of cytochrome P450s but the molecular basis underlying this metabolic resistance is unknown. Microsatellite and SNP markers were used to construct a linkage map and to detect a quantitative trait locus (QTL) associated with pyrethroid resistance in the FUMOZ-R strain of An. funestus from Mozambique. RESULTS: By genotyping 349 F2 individuals from 11 independent families, a single major QTL, rp1, at the telomeric end of chromosome 2R was identified. The rp1 QTL appears to present a major effect since it accounts for more than 60% of the variance in susceptibility to permethrin. This QTL has a strong additive genetic effect with respect to susceptibility. Candidate genes associated with pyrethroid resistance in other species were physically mapped to An. funestus polytene chromosomes. This showed that rp1 is genetically linked to a cluster of CYP6 cytochrome P450 genes located on division 9 of chromosome 2R and confirmed earlier reports that pyrethroid resistance in this strain is not associated with target site mutations (knockdown resistance). CONCLUSION: We hypothesize that one or more of these CYP6 P450s clustered on chromosome 2R confers pyrethroid resistance in the FUMOZ-R strain of An. funestus.

An integrated genetic and physical map for the malaria vector Anopheles funestus.

We have constructed a genetic map of the major African malaria vector, Anopheles funestus, using genetic markers segregating in F(2) progeny from crosses between two strains colonized from different field sites. Genotyping was performed on 174 progeny from three families using 33 microsatellite markers, a single RFLP, and 15 single nucleotide polymorphism (SNP) loci. Four linkage groups were resolved and these were anchored to chromosomes X and 2 and chromosomal arms 3R and 3L by comparison with a physical map of this species. Five markers were linked to the X chromosome, 16 markers to chromosome 2, and 10 and 11 markers to chromosomal arms 3R and 3L, respectively. This significantly increases the number of chromosomally defined genetic markers for this species and will facilitate the identification of genes controlling epidemiologically important traits such as resistance to insecticides or vector competence.