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Resistance to pyrethroids, the sole insecticide class recommended for treating bed nets, threatens the control of major malaria vectors, including Anopheles funestus Effective management of resistance requires an understanding of the dynamics and mechanisms driving resistance. Here, using genome-wide transcription and genetic diversity analyses, we show that a shift in the molecular basis of pyrethroid resistance in southern African populations of this species is associated with a restricted gene flow. Across the most highly endemic and densely populated regions in Malawi, An. funestus is resistant to pyrethroids, carbamates, and organochlorides. Genome-wide microarray-based transcription analysis identified overexpression of cytochrome P450 genes as the main mechanism driving this resistance. The most up-regulated genes include cytochrome P450s (CYP) CYP6P9a, CYP6P9b and CYP6M7. However, a significant shift in the overexpression profile of these genes was detected across a south/north transect, with CYP6P9a and CYP6P9b more highly overexpressed in the southern resistance front and CYP6M7 predominant in the northern front. A genome-wide genetic structure analysis of southern African populations of An. funestus from Zambia, Malawi, and Mozambique revealed a restriction of gene flow between populations, in line with the geographical variation observed in the transcriptomic analysis. Genetic polymorphism analysis of the three key resistance genes, CYP6P9a, CYP6P9b, and CYP6M7, support barriers to gene flow that are shaping the underlying molecular basis of pyrethroid resistance across southern Africa. This barrier to gene flow is likely to impact the design and implementation of resistance management strategies in the region., Competing Interests: The authors declare no conflict of interest.

Background: Establishing the extent, geographical distribution and mechanisms of insecticide resistance in malaria vectors is a prerequisite for resistance management. Here, we report a widespread distribution of insecticide resistance in the major malaria vector An. funestus across Uganda and western Kenya under the control of metabolic resistance mechanisms., Methodology/principal Findings: Female An. funestus collected throughout Uganda and western Kenya exhibited a Plasmodium infection rate between 4.2 to 10.4%. Widespread resistance against both type I (permethrin) and II (deltamethrin) pyrethroids and DDT was observed across Uganda and western Kenya. All populations remain highly susceptible to carbamate, organophosphate and dieldrin insecticides. Knockdown resistance plays no role in the pyrethroid and DDT resistance as no kdr mutation associated with resistance was detected despite the presence of a F1021C replacement. Additionally, no signature of selection was observed on the sodium channel gene. Synergist assays and qRT-PCR indicated that metabolic resistance plays a major role notably through elevated expression of cytochrome P450s. DDT resistance mechanisms differ from West Africa as the L119F-GSTe2 mutation only explains a small proportion of the genetic variance to DDT resistance., Conclusion: The extensive distribution of pyrethroid and DDT resistance in East African An. funestus populations represents a challenge to the control of this vector. However, the observed carbamate and organophosphate susceptibility offers alternative solutions for resistance management.

Background: Pyrethroid resistance in the major malaria vector Anopheles funestus is rapidly expanding across Southern Africa. It remains unknown whether this resistance has a unique origin with the same molecular basis or is multifactorial. Knowledge of the origin, mechanisms and evolution of resistance are crucial to designing successful resistance management strategies., Results: Here, we established the resistance profile of a Zambian An. funestus population at the northern range of the resistance front. Similar to other Southern African populations, Zambian An. funestus mosquitoes are resistant to pyrethroids and carbamate, but in contrast to populations in Mozambique and Malawi, these insects are also DDT resistant. Genome-wide microarray-based transcriptional profiling and qRT-PCR revealed that the cytochrome P450 gene CYP6M7 is responsible for extending pyrethroid resistance northwards. Indeed, CYP6M7 is more over-expressed in Zambia [fold-change (FC) 37.7; 13.2 for qRT-PCR] than CYP6P9a (FC15.6; 8.9 for qRT-PCR) and CYP6P9b (FC11.9; 6.5 for qRT-PCR), whereas CYP6P9a and CYP6P9b are more highly over-expressed in Malawi and Mozambique. Transgenic expression of CYP6M7 in Drosophila melanogaster coupled with in vitro assays using recombinant enzymes and assessments of kinetic properties demonstrated that CYP6M7 is as efficient as CYP6P9a and CYP6P9b in conferring pyrethroid resistance. Polymorphism patterns demonstrate that these genes are under contrasting selection forces: the exceptionally diverse CYP6M7 likely evolves neutrally, whereas CYP6P9a and CYP6P9b are directionally selected. The higher variability of CYP6P9a and CYP6P9b observed in Zambia supports their lesser role in resistance in this country., Conclusion: Pyrethroid resistance in Southern Africa probably has multiple origins under different evolutionary forces, which may necessitate the design of different resistance management strategies.

Pyrethroid insecticides are critical for malaria control in Africa. However, resistance to this insecticide class in the malaria vector Anopheles funestus is spreading rapidly across Africa, threatening the success of ongoing and future malaria control programs. The underlying resistance mechanisms driving the spread of this resistance in wild populations remain largely unknown. Here, we show that increased expression of two tandemly duplicated P450 genes, CYP6P9a and CYP6P9b, is the main mechanism driving pyrethroid resistance in Malawi and Mozambique, two southern African countries where this insecticide class forms the mainstay of malaria control. Genome-wide transcription analysis using microarray and quantitative RT-PCR consistently revealed that CYP6P9a and CYP6P9b are the two genes most highly overexpressed (>50-fold; q < 0.01) in permethrin-resistant mosquitoes. Transgenic expression of CYP6P9a and CYP6P9b in Drosophila melanogaster demonstrated that elevated expression of either of these genes confers resistance to both type I (permethrin) and type II (deltamethrin) pyrethroids. Functional characterization of recombinant CYP6P9b confirmed that this protein metabolized both type I (permethrin and bifenthrin) and type II (deltamethrin and Lambda-cyhalothrin) pyrethroids but not DDT. Variability analysis identified that a single allele of each of these genes is predominantly associated with pyrethroid resistance in field populations from both countries, which is suggestive of a single origin of this resistance that has since spread across the region. Urgent resistance management strategies should be implemented in this region to limit a further spread of this resistance and minimize its impact on the success of ongoing malaria control programs.

The impact of insecticide resistance on insect-borne disease programs is difficult to quantify. The possibility of eliminating malaria in high-transmission settings is heavily dependent on effective vector control reducing disease transmission rates. Pyrethroids are the dominant insecticides used for malaria control, with few options for their replacement. Their failure will adversely affect our ability to control malaria. Pyrethroid resistance has been selected in Malawi over the last 3 y in the two major malaria vectors Anopheles gambiae and Anopheles funestus, with a higher frequency of resistance in the latter. The resistance in An. funestus is metabolically based and involves the up-regulation of two duplicated P450s. The same genes confer resistance in Mozambican An. funestus, although the levels of up-regulation differ. The selection of resistance over 3 y has not increased malaria transmission, as judged by annual point prevalence surveys in 1- to 4-y-old children. This is true in areas with long-lasting insecticide-treated nets (LLINs) alone or LLINs plus pyrethroid-based insecticide residual spraying (IRS). However, in districts where IRS was scaled up, it did not produce the expected decrease in malaria prevalence. As resistance increases in frequency from this low initial level, there is the potential for vector population numbers to increase with a concomitant negative impact on control efficacy. This should be monitored carefully as part of the operational activities in country.

The Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (∼ 1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.

Resistance to pyrethroids, the sole insecticide class recommended for treating bed nets, threatens the control of major malaria vectors, including Anopheles funestus. Effective management of resistance requires an understanding of the dynamics and mechanisms driving resistance. Here, using genome-wide transcription and genetic diversity analyses, we show that a shift in the molecular basis of pyrethroid resistance in southern African populations of this species is associated with a restricted gene flow. Across the most highly endemic and densely populated regions in Malawi, An. funestus is resistant to pyrethroids, carbamates, and organochlorides. Genome-wide microarraybased transcription analysis identified overexpression of cytochrome P450 genes as the main mechanism driving this resistance. The most up-regulated genes include cytochrome P450s (CYP) CYP6P9a, CYP6P9b and CYP6M7. However, a significant shift in the overexpression profile of these genes was detected across a south/north transect, with CYP6P9a and CYP6P9b more highly overexpressed in the southern resistance front and CYP6M7 predominant in the northern front. A genome- wide genetic structure analysis of southern African populations of An. funestus from Zambia, Malawi, and Mozambique revealed a restriction of gene flow between populations, in line with the geographical variation observed in the transcriptomic analysis. Genetic polymorphism analysis of the three key resistance genes, CYP6P9a, CYP6P9b, and CYP6M7, support barriers to gene flow that are shaping the underlying molecular basis of pyrethroid resistance across southern Africa. This barrier to gene flow is likely to impact the design and implementation of resistance management strategies in the region. [ABSTRACT FROM AUTHOR]

Background: Deciphering the dynamics and evolution of insecticide resistance in malaria vectors is crucial for successful vector control. This study reports an increase of resistance intensity and a rise of multiple insecticide resistance in Anopheles funestus in Malawi leading to reduced bed net efficacy. Methods: Anopheles funestus group mosquitoes were collected in southern Malawi and the species composition, Plasmodium infection rate, susceptibility to insecticides and molecular bases of the resistance were analysed. Results: Mosquito collection revealed a predominance of An. funestus group mosquitoes with a high hybrid rate (12.2 %) suggesting extensive species hybridization. An. funestus sensu stricto was the main Plasmodium vector (4.8 % infection). Consistently high levels of resistance to pyrethroid and carbamate insecticides were recorded and had increased between 2009 and 2014. Furthermore, the 2014 collection exhibited multiple insecticide resistance, notably to DDT, contrary to 2009. Increased pyrethroid resistance correlates with reduced efficacy of bed nets (<5 % mortality by Olyset® net), which can compromise control efforts. This change in resistance dynamics is mirrored by prevalent resistance mechanisms, firstly with increased over-expression of key pyrethroid resistance genes (CYP6Pa/b and CYP6M7) in 2014 and secondly, detection of the A296S-RDL dieldrin resistance mutation for the first time. However, the L119F-GSTe2 and kdr mutations were absent. Conclusions: Such increased resistance levels and rise of multiple resistance highlight the need to rapidly implement resistance management strategies to preserve the effectiveness of existing insecticide-based control interventions. [ABSTRACT FROM AUTHOR]