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The versatility of cytochrome P450 reductase ( CPR ) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR ( AgCPR ), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR ( AfCPR ) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR , e.g., the coordinates of the second FAD stacking residue AfCPR -Y 456 differ from that of AgCPR -His 456 . While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR , the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b , capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.

Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi-omics approach, followed-up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted sequencing of candidate resistance genes detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNPs were in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where SNPs in the P450 CYP9K1 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited reduced genetic diversity in Uganda underlying an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid change in CYP9K1 was fixed in Ugandan mosquitoes but not in other An. funestus populations. This study sheds further light on the evolution of metabolic resistance in a major malaria vector by implicating more genes and variants that can be used to design field-applicable markers to better track resistance Africa-wide., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Leucine-rich repeat proteins and antimicrobial peptides are the key components of the innate immune response to Plasmodium and other microbial pathogens in Anopheles mosquitoes. The APL1 gene of the malaria vector Anopheles funestus has exceptional levels of non-synonymous polymorphism across the range of An. funestus, with an average π n of 0.027 versus a genome-wide average of 0.002, and π n is consistently high in populations across Africa. Elevated APL1 diversity was consistent between the independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide resistance was observed. Although lacking the diversity of APL1 , two further mosquito innate-immunity genes of the gambicin anti-microbial peptide family had π n /π s ratios greater than one, possibly driven by either positive or balancing selection. The cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, a result discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogues, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. In conclusion, we hypothesize that higher gene expression of APL1 than its paralogues is correlated with a more open chromatin formation, which enhances gene conversion and elevated diversity at this locus.

Resistance is threatening the effectiveness of insecticide-based interventions in use for malaria control. Pinpointing genes associated with resistance is crucial for evidence-based resistance management targeting the major malaria vectors. Here, a combination of RNA-seq based genome-wide transcriptional analysis and RNA-silencing in vivo functional validation were used to identify key insecticide resistance genes associated with DDT and DDT/permethrin cross-resistance across Africa. A cluster of glutathione-S-transferase from epsilon group were found to be overexpressed in resistant populations of Anopheles funestus across Africa including GSTe1 [Cameroon (fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)], GSTe2 [Cameroon (4.47), Ghana (7.52), Malawi (2.13)], GSTe3 [Cameroon (2.49), Uganda (2.60)], GSTe4 in Ghana (3.47), GSTe5 [Ghana (2.94), Malawi (2.26)], GSTe6 [Cameroun (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)] and GSTe7 (2.39) in Ghana. Validation of GSTe genes expression profiles by qPCR confirmed that the genes are differentially expressed across Africa with a greater overexpression in DDT-resistant mosquitoes. RNAi-based knock-down analyses supported that five GSTe genes are playing a major role in resistance to pyrethroids (permethrin and deltamethrin) and DDT in An. funestus , with a significant recovery of susceptibility observed when GSTe2 , 3 , 4 , 5 and GSTe6 were silenced. These findings established that GSTe3 , 4 , 5 and 6 contribute to DDT resistance and should be further characterized to identify their specific genetic variants, to help design DNA-based diagnostic assays, as previously done for the 119F-GSTe2 mutation. This study highlights the role of GSTe s in the development of resistance to insecticides in malaria vectors and calls for actions to mitigate this resistance.

Increased levels of insecticide resistance in major malaria vectors such as Anopheles funestus threaten the effectiveness of insecticide-based control programmes. Understanding the landscape features impacting the spread of resistance makers is necessary to design suitable resistance management strategies. Here, we examined the influence of the highest mountain in West Africa (Mount Cameroon; 4095 m elevation) on the spread of metabolic and target-site resistance alleles in An . funestus populations. Vector composition varied across the four localities surveyed along the altitudinal cline with major vectors exhibiting high parity rate (80.5%). Plasmodium infection rates ranged from 0.79% ( An. melas ) to 4.67% ( An. funestus ). High frequencies of GSTe2 R (67-81%) and Rdl R (49-90%) resistance alleles were observed in An. funestus throughout the study area, with GSTe2 R frequency increasing with altitude, whereas the opposite is observed for Rdl R . Patterns of genetic diversity and population structure analyses revealed high levels of polymorphisms with 12 and 16 haplotypes respectively for GSTe2 and Rdl . However, the reduced diversity patterns of resistance allele carriers revealed signatures of positive selection on the two genes across the study area irrespective of the altitude. Despite slight variations associated with the altitude, the spread of resistance alleles suggest that control strategies could be implemented against malaria vectors across mountainous landscapes.

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide-based interventions. Here, we deciphered the role of a 6.5-kb structural variation (SV) in driving cytochrome P450-mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole-genome pooled sequencing detected an intergenic 6.5-kb SV between duplicated CYP6P9a/b P450s in pyrethroid-resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5-fold higher activity (p < .0001) for the SV- carrying fragment than the SV- free one. Quantitative real-time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV- (1.7- to 2-fold) and homozygotes SV-/SV- (4-to 5-fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV-/SV-; odds ratio [OR] = 2,079.4, p < .001). The 6.5-kb SV is present at high frequency in southern Africa (80%-100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid-treated nets (OR 27.7; p < .0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote-resistant at the three loci (SV+/CYP6P9a&#95;R/CYP6P9b&#95;R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid-based nets., (© 2020 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Background: Understanding the mechanisms used by Anopheles mosquitoes to survive insecticide exposure is key to manage existing insecticide resistance and develop more suitable insecticide-based malaria vector control interventions as well as other alternative integrated tools. To this regard, the molecular basis of permethrin, DDT and dieldrin resistance in Anopheles funestus (sensu stricto) at Akaka-Remo was investigated., Methods: Bioassays were conducted on 3-5-day-old adult An. funestus (s.s.) mosquitoes for permethrin, DDT and dieldrin susceptibility test. The molecular mechanisms of mosquito resistance to these insecticides were investigated using microarray and reverse transcriptase PCR techniques. The voltage-gated sodium channel region of mosquitoes was also screened for the presence of knockdown resistance mutations (kdr west and east) by sequencing method., Results: Anopheles funestus (s.s.) population was resistant to permethrin (mortality rate of 68%), DDT (mortality rate of 10%) and dieldrin (mortality rate of 8%) insecticides. Microarray and RT-PCR analyses revealed the overexpression of glutathione S-transferase genes, cytochrome P450s, esterase, trypsin and cuticle proteins in resistant mosquitoes compared to control. The GSTe2 was the most upregulated detoxification gene in permethrin-resistant (FC = 44.89), DDT-resistant (FC = 57.39) and dieldrin-resistant (FC = 41.10) mosquitoes compared to control population (FC = 22.34). The cytochrome P450 gene, CYP6P9b was also upregulated in both permethrin- and DDT-resistant mosquitoes. The digestive enzyme, trypsin (hydrolytic processes) and the cuticle proteins (inducing cuticle thickening leading to reduced insecticides penetration) also showed high involvement in insecticide resistance, through their overexpression in resistant mosquitoes compared to control. The kdr east and west were absent in all mosquitoes analysed, suggesting their non-involvement in the observed mosquito resistance., Conclusions: The upregulation of metabolic genes, especially the GSTe2 and trypsin, as well as the cuticle proteins is driving insecticide resistance of An. funestus (s.s.) population. However, additional molecular analyses, including functional metabolic assays of these genes as well as screening for a possible higher cuticular hydrocarbon and lipid contents, and increased procuticle thickness in resistant mosquitoes are needed to further describe their distinct roles in mosquito resistance.

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improving control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweeps, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes., Competing Interests: The authors declare that they have no competing interests.

The Nigerian Government is scaling up the distribution of insecticide-treated bed nets for malaria control, but the lack of surveillance data, especially in the Sudan/Sahel region of the country, may hinder targeting priority populations. Here, the vectorial role and insecticide resistance profile of a population of a major malaria vector Anopheles funestus sensu stricto from Sahel of Nigeria was characterised. An. funestus s.s. was the only vector found, with a high human blood index (100%) and a biting rate of 5.3/person/night. High Plasmodium falciparum infection was discovered (sporozoite rate = 54.55%). The population is resistant to permethrin (mortality = 48.30%, LT 50 = 65.76 min), deltamethrin, DDT (dichlorodiphenyltrichloroethane) and bendiocarb, with mortalities of 29.44%, 56.34% and 54.05%, respectively. Cone-bioassays established loss of efficacy of the pyrethroid-only long-lasting insecticidal nets (LLINs); but 100% recovery of susceptibility was obtained for piperonylbutoxide (PBO)-containing PermaNet ® 3.0. Synergist bioassays with PBO and diethyl maleate recovered susceptibility, implicating CYP450s (permethrin mortality = 78.73%, χ 2 = 22.33, P < 0.0001) and GSTs (DDT mortality = 81.44%, χ 2 = 19.12, P < 0.0001). A high frequency of 119F GSTe2 mutation (0.84) was observed (OR = 16, χ 2 = 3.40, P = 0.05), suggesting the preeminent role of metabolic resistance. These findings highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in Nigeria.

Growing insecticide resistance in malaria vectors is threatening the effectiveness of insecticide-based interventions, including Long Lasting Insecticidal Nets (LLINs). However, the impact of metabolic resistance on the effectiveness of these tools remains poorly characterized. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F- GSTe2 ), we established that GST-mediated resistance is reducing the efficacy of LLINs against Anopheles funestus . Hut trials performed in Cameroon revealed that Piperonyl butoxide (PBO)-based nets induced a significantly higher mortality against pyrethroid resistant An. funestus than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control. Genotyping the L119F- GSTe2 mutation revealed that, for permethrin-based nets, 119F- GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes (OR = 11.7; p < 0.01). Furthermore, GSTe2 -resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR = 2.1; p < 0.01) and PermaNet 3.0 (side) (OR = 30.1; p < 0.001) but not for Olyset Plus. This study shows that the efficacy of PBO-based nets (e.g., blood feeding inhibition) against pyrethroid resistant malaria vectors could be impacted by other mechanisms including GST-mediated metabolic resistance not affected by the synergistic action of PBO. Mosaic LLINs incorporating a GST inhibitor (diethyl maleate) could help improve their efficacy in areas of GST-mediated resistance.

Elucidating the genetic basis of metabolic resistance to insecticides in malaria vectors is crucial to prolonging the effectiveness of insecticide-based control tools including long lasting insecticidal nets (LLINs). Here, we show that cis-regulatory variants of the cytochrome P450 gene, CYP6P9b, are associated with pyrethroid resistance in the African malaria vector Anopheles funestus. A DNA-based assay is designed to track this resistance that occurs near fixation in southern Africa but not in West/Central Africa. Applying this assay we demonstrate, using semi-field experimental huts, that CYP6P9b-mediated resistance associates with reduced effectiveness of LLINs. Furthermore, we establish that CYP6P9b combines with another P450, CYP6P9a, to additively exacerbate the reduced efficacy of insecticide-treated nets. Double homozygote resistant mosquitoes (RR/RR) significantly survive exposure to insecticide-treated nets and successfully blood feed more than other genotypes. This study provides tools to track and assess the impact of multi-gene driven metabolic resistance to pyrethroids, helping improve resistance management.

Background: Insecticide resistance poses a serious threat to insecticide-based interventions in Africa. There is a fear that resistance escalation could jeopardize malaria control efforts. Monitoring of cases of aggravation of resistance intensity and its impact on the efficacy of control tools is crucial to predict consequences of resistance., Methods: The resistance levels of an Anopheles funestus population from Palmeira, southern Mozambique, were characterized and their impact on the efficacy of various insecticide-treated nets established., Results: A dramatic loss of efficacy of all long-lasting insecticidal nets (LLINs), including piperonyl butoxide (PBO)-based nets (Olyset Plus), was observed. This An. funestus population consistently (2016, 2017, and 2018) exhibited a high degree of pyrethroid resistance. Molecular analyses revealed that this resistance escalation was associated with a massive overexpression of the duplicated cytochrome P450 genes CYP6P9a and CYP6P9b, and also the fixation of the resistance CYP6P9a&#95;R allele in this population in 2016 (100%) in contrast to 2002 (5%). However, the low recovery of susceptibility after PBO synergist assay suggests that other resistance mechanisms could be involved., Conclusions: The loss of efficacy of pyrethroid-based LLINs with and without PBO is a concern for the effectiveness of insecticide-based interventions, and action should be taken to prevent the spread of such super-resistance., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Metabolic resistance to insecticides is threatening malaria control in Africa. However, the extent to which it impacts malaria transmission remains unclear. Here, we investigated the association between a marker of glutathione S-transferase mediated metabolic resistance and Plasmodium infection in field population of Anopheles funestus s.s. in comparison to the A296S-RDL target site mutation. The 119F-GSTe2 resistant allele was present in southern (Obout) (56%) and central (Mibellon) (25%) regions of Cameroon whereas the 296S-RDL resistant allele was detected at 98.5% and 15% respectively. The whole mosquito Plasmodium and sporozoite infection rates were 57% and 14.8% respectively in Obout (n = 508) and 19.7% and 5% in Mibellon (n = 360). No association was found between L119F-GSTe2 genotypes and whole mosquito infection status. However, when analyzing oocyst and sporozoite infection rates separately, the resistant homozygote 119F/F genotype was significantly more associated with Plasmodium infection in Obout than both heterozygote (OR = 2.5; P = 0.012) and homozygote susceptible (L/L119) genotypes (OR = 2.10; P = 0.013). In contrast, homozygote RDL susceptible mosquitoes (A/A296) were associated more frequently with Plasmodium infection than other genotypes (OR = 4; P = 0.03). No additive interaction was found between L119F and A296S. Sequencing of the GSTe2 gene showed no association between the polymorphism of this gene and Plasmodium infection. Glutathione S-transferase metabolic resistance is potentially increasing the vectorial capacity of resistant An. funestus mosquitoes. This could result in a possible exacerbation of malaria transmission in areas of high GSTe2-based metabolic resistance to insecticides.

Metabolic resistance to insecticides such as pyrethroids in mosquito vectors threatens control of malaria in Africa. Unless it is managed, recent gains in reducing malaria transmission could be lost. To improve monitoring and assess the impact of insecticide resistance on malaria control interventions, we elucidated the molecular basis of pyrethroid resistance in the major African malaria vector, Anopheles funestus We showed that a single cytochrome P450 allele ( CYP6P9a &#95;R) in A. funestus reduced the efficacy of insecticide-treated bednets for preventing transmission of malaria in southern Africa. Expression of key insecticide resistance genes was detected in populations of this mosquito vector throughout Africa but varied according to the region. Signatures of selection and adaptive evolutionary traits including structural polymorphisms and cis-regulatory transcription factor binding sites were detected with evidence of selection due to the scale-up of insecticide-treated bednet use. A cis-regulatory polymorphism driving the overexpression of the major resistance gene CYP6P9a allowed us to design a DNA-based assay for cytochrome P450-mediated resistance to pyrethroid insecticides. Using this assay, we tracked the spread of pyrethroid resistance and found that it was almost fixed in mosquitoes from southern Africa but was absent from mosquitoes collected elsewhere in Africa. Furthermore, a field study in experimental huts in Cameroon demonstrated that mosquitoes carrying the resistance CYP6P9a&#95;R allele survived and succeeded in blood feeding more often than did mosquitoes that lacked this allele. Our findings highlight the need to introduce a new generation of insecticide-treated bednets for malaria control that do not rely on pyrethroid insecticides., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Despite the increased report of insecticide resistance in malaria vectors, its impact on mosquito's life-traits after exposure to insecticide-treated nets remains under investigated. Here, we assessed the effects of exposure to PermaNet 2.0 on several life traits of An. gambiae s.l. and An. funestus s.l. field mosquitoes in Cameroon., Methodology: Female Anopheles mosquitoes were collected indoor using electric aspirators in southern Cameroon (Obout) in 2016. After assessing the resistance status of F1 from the field collected-mosquitoes, progeny of the first generation (An. funestus s.l.) and seventh generation (An. gambiae s.l.) were used to assess the long-term effect of exposure to PermaNet 2.0 on several life-traits of these vectors (longevity, blood feeding ability, fecundity and fertility) in comparison to untreated net. In addition, the L119F-GSTe2 mutation associated with DDT/pyrethroids resistance in An. funestus was genotyped to assess its association with increased life-span post-exposure., Principal Findings: Both An. funestus and An. gambiae were resistant to pyrethroids and DDT with a greater level in the latter. Pyrethroid-only nets PermaNet 2.0 (17.5% mortality) and Olyset (0% mortality) exhibited a significantly reduced efficacy against An. funestus in contrast to a greater efficacy for PBO-based Nets Olyset Plus (65% mortality), PermaNet 3.0 top (100% mortality). In both species, mosquitoes that survived exposure to PermaNet 2.0 exhibited a significantly reduced longevity than those non-exposed (6.95 days vs 12.46 for An. funestus P<0.001; 8.87 vs 11.25 days for An. gambiae; P<0.001). However, no significant difference was observed for blood feeding and fecundity in both species. In addition, molecular analysis of the L119F-GSTe2 mutation revealed that this mutation is associated with an increase in the chance of surviving after exposure to this net in An. funestus., Conclusions: These results show that although the PermaNet 2.0 presents a reduced efficacy against resistant populations, it remains efficient after exposure by reducing the life expectancy of the vectors which could contribute in the reduction of malaria incidence., Competing Interests: The authors have declared that no competing interests exist.

Metabolic resistance to insecticides threatens malaria control. However, little is known about its fitness cost in field populations of malaria vectors, thus limiting the design of suitable resistance management strategies. Here, we assessed the association between the glutathione S -transferase GSTe2-mediated metabolic resistance and life-traits of natural populations of Anopheles funestus . A total of 1200 indoor resting blood-fed female An. funestus (F₀) were collected in Mibellon, Cameroon (2016/2017), and allowed to lay eggs individually. Genotyping of F1 mosquitoes for the L119F-GSTE2 mutation revealed that L/L119-homozygote susceptible (SS) mosquitoes significantly laid more eggs than heterozygotes L119F-RS (odds ratio (OR) = 2.06; p < 0.0001) and homozygote resistant 119F/F-RR (OR = 2.93; p < 0.0001). L/L119-SS susceptible mosquitoes also showed the higher ability for oviposition than 119F/F-RR resistant (OR = 2.68; p = 0.0002) indicating a reduced fecundity in resistant mosquitoes. Furthermore, L119F-RS larvae developed faster (nine days) than L119F-RR and L119F-SS (11 days) (X² = 11.052; degree of freedom (df) = 4; p = 0.02) suggesting a heterozygote advantage effect for larval development. Interestingly, L/L119-SS developed faster than 119F/F-RR (OR = 5.3; p < 0.0001) revealing an increased developmental time in resistant mosquitoes. However, genotyping and sequencing revealed that L119F-RR mosquitoes exhibited a higher adult longevity compared to RS (OR > 2.2; p < 0.05) and SS (OR > 2.1; p < 0.05) with an increased frequency of GSTe2-resistant haplotypes in mosquitoes of D30 after adult emergence. Additionally, comparison of the expression of GSTe2 revealed a significantly increased expression from D1-D30 after emergence of adults (Anova test (F) = 8; df= 3; p = 0.008). The negative association between GSTe2 and some life traits of An. funestus could facilitate new resistance management strategies. However, the increased longevity of GSTe2-resistant mosquitoes suggests that an increase in resistance could exacerbate malaria transmission.

Background: Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. In order to implement suitable insecticide resistance management strategies, it is necessary to understand the underlying mechanisms involved. To achieve this, the molecular basis of permethrin and DDT resistance in the principal malaria vector, Anopheles funestus from inland Benin (Kpome), was investigated., Results: Here, using a microarray-based genome-wide transcription and qRT-PCR analysis, we showed that metabolic resistance mechanisms through over-expression of cytochrome P450 and glutathione S-transferase genes (GSTs) are a major contributor to DDT and permethrin resistance in Anopheles funestus from Kpome. The GSTe2 gene was the most upregulated detoxification gene in both DDT- [fold-change (FC: 16.0)] and permethrin-resistant (FC: 18.1) mosquitoes suggesting that upregulation of this gene could contribute to DDT resistance and cross-resistance to permethrin. CYP6P9a and CYP6P9b genes that have been previously associated with pyrethroid resistance were also significantly overexpressed with FC 5.4 and 4.8, respectively, in a permethrin resistant population. Noticeably, the GSTs, GSTd1-5 and GSTd3, were more upregulated in DDT-resistant than in permethrin-resistant Anopheles funestus suggesting these genes are more implicated in DDT resistance. The absence of the L1014F or L1014S kdr mutations in the voltage-gated sodium channel gene coupled with the lack of directional selection at the gene further supported that knockdown resistance plays little role in this resistance., Conclusions: The major role played by metabolic resistance to pyrethroids in this An. funestus population in Benin suggests that using novel control tools combining the P450 synergist piperonyl butoxide (PBO), such as PBO-based bednets, could help manage the growing pyrethroid resistance in this malaria vector in Benin.

Malaria remains the main cause of mortality and morbidity in the Central African Republic. However, the main malaria vectors remain poorly characterised, preventing the design of suitable control strategies. Here, we characterised the patterns and mechanisms of insecticide resistance in three important vectors from Bangui. Mosquitoes were collected indoors, using electrical aspirators in July 2016 in two neighborhoods at Bangui. WHO bioassays performed, using F 2 An. gambiae sensu lato (s.l.), revealed a high level of resistance to type I (permethrin) and II (deltamethrin) pyrethroids and dichlorodiphenyltrichloroethane (< 3% mortality). Molecular analysis revealed the co-occurrence of Anopheles coluzzii (56.8 %) and An. gambiae s.s. (43.2%) within the An. gambiae complex. Anopheles funestus s.s. was the sole species belonging to An. funestus group. Both kdr-w (40% of homozygotes and 60% of heterozygotes/kdr-w/wild type) and kdr-e (37.5% of heterozygotes) mutations were found in An. gambiae. Contrariwise, only the kdr-w (9.5% homozygotes and 85.7% of heterozygotes) was detected in An. coluzzii. Quantitative RT-PCR showed that CYP6M2 and CYP6P3 are not upregulated in An. coluzzii from Bangui. Analysis of the sodium channel gene revealed a reduced diversity in An. coluzzii and An. gambiae s.s. In An. funestus s.s., the pyrethroid/DDT GSTe2 L119F resistance allele was detected at high frequency (54.7%) whereas a very low frequency for Rdl was observed. Polymorphism analysis of GSTe2 and GABA receptor gene in An. funestus revealed the presence of one resistant haplotype for each gene. This study provides baseline information to help guide current and future malaria vector control interventions in CAR.

Background: Malaria vectors are increasingly developing resistance to insecticides across Africa. The impact of such resistance on the continued effectiveness of insecticide-based interventions remains unclear due to poor characterization of vector populations. This study reports the characterization of malaria vectors at Mibellon, a selected site in Cameroon for experimental hut study, including species composition, Plasmodium infection rate, resistance profiles and mechanisms., Methods: Indoor resting blood-fed Anopheles mosquitoes were collected from houses at Mibellon in 2017 and forced to lay eggs to generate F 1 adult mosquitoes. Insecticides susceptibility bioassays were performed on the F 1 adult mosquitoes following the WHO protocol to assess resistance profile to insecticides. The molecular basis of resistance and Plasmodium infection rate were investigated using TaqMan genotyping., Results: Anopheles funestus sensu stricto (s.s.) was predominant in Mibellon (80%) followed by Anopheles gambiae s.s. (20%). High levels of resistance to pyrethroids and organochlorides were observed for both species. Moderate resistance was observed against bendiocarb (carbamate) in both species, but relatively higher in An. gambiae s.s. In contrast, full susceptibility was recorded for the organophosphate malathion. The PBO synergist assays with permethrin and deltamethrin revealed a significant recovery of the susceptibility in Anopheles funestus s.s. population (48.8 to 98.1% mortality and 38.3 to 96.5% mortality, respectively). The DDT/pyrethroid 119F-GSTe2 resistant allele (28.1%) and the dieldrin 296S-RDL resistant (9.7%) were detected in An. funestus s.s. The high pyrethroid/DDT resistance in An. gambiae correlated with the high frequency of 1014F knockdown resistance allele (63.9%). The 1014S-kdr allele was detected at low frequency (1.97%). The Plasmodium infection rate was 20% in An. gambiae, whereas An. funestus exhibited an oocyst rate of 15 and 5% for the sporozoite rate., Conclusion: These results highlight the increasing spread of insecticide resistance and the challenges that control programmes face to maintain the continued effectiveness of insecticide-based interventions.

Background: The epidemiological profiles of vector-borne diseases, such as malaria, are strongly associated with landscape components. The reduction of malaria burden in endemic and epidemic regions mainly depends on knowledge of the malaria-transmitting mosquito species, populations and behavioural characteristics, as well as malaria exposure risks. This work aimed at carrying out a holistic study in order to characterise Anopheles species in relation to human malaria in seven wetlands along the lower section of the volcanic chain of Cameroon., Results: Eight malaria vectors: Anopheles arabiensis, An. coluzzii, An. funestus (s.s.), An. gambiae, An. hancocki, An. melas, An. nili and An. ziemanni, were found biting humans. Anopheles gambiae was widespread; however, it played a secondary role in the Ndop plain where An. ziemmani was the primary vector species (79.2%). Anophelines were more exophagic (73.6%) than endophagic (26.4%), showing a marked nocturnal activity (22:00-4:00 h) for An. coluzzii and An. gambiae while An. funestus (s.s.) was mostly caught between 1:00 and 6:00 h and An. ziemanni having an early evening biting behaviour (18:00-00:00 h). Female Anopheles were mostly observed to have relative high parity rates (≥ 70%), with the exception of the Meanja site where species parity varies from 46 to 55%. Overall, the transmission level was low with entomological inoculation rates estimated to 0.7 infected bites per person per month (ib/p/mth) in Tiko and Ndop, 1.4 ib/p/mth in Mamfe and 2.24 ib/p/mth in Santchou., Conclusions: The present study represents detailed Anopheles vector characterisation from an understudied area along the volcanic chain of Cameroon with endemic malaria transmission. The significant differences in bionomics and Anopheles species distribution within the studied wetlands, highlights the importance of providing baseline data and an opportunity to assess the outcome of ongoing malaria control interventions in the country.

Resistance to pyrethroids (the ingredients in bed net insecticides) in the major malaria vector Anopheles funestus is threatening recent gains in the fight against malaria. Here, we established the role of an over-expressed P450, A. funestus CYP6AA1 in insecticides resistance. Transcription profiling of CYP6AA1 across Africa using microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that it is significantly more over-expressed in southern African populations compared to West (Benin) and East African (Uganda). Heterologous expression in Escherichia coli coupled with metabolism assays demonstrated that CYP6AA1 metabolises type I (permethrin) and type II (deltamethrin) pyrethroids, as well as bendiocarb (a carbamate). Transgenic Drosophila melanogaster flies over-expressing CYP6AA1 were significantly more resistant to pyrethroid insecticides, permethrin and deltamethrin compared with control flies not expressing the gene, validating the role of this gene in pyrethroid resistance. In silico modelling and docking simulations predicted the intermolecular receptor-ligand interactions which allow this P450 to metabolise the pyrethroids and bendiocarb. Validation of CYP6AA1 as a pyrethroid resistance gene makes it possible to monitor the spread of resistance in the field where this P450 is over-expressed. Its potential cross-resistance role makes it necessary to monitor the gene closely to inform control programs on molecular basis of multiple resistance in the field.

Accounting for approximately 11% of all malaria cases, the Democratic Republic of the Congo (DRC) is central to malaria elimination efforts. To support vector control interventions in DRC, we characterized the dynamics and impact of insecticide resistance in major malaria vectors in 2015. High Plasmodium infection rates were recorded in Anopheles gambiae and Anopheles funestus, with Plasmodium falciparum predominant over Plasmodium malariae. Both mosquito species exhibited high and multiple resistance to major public health insecticide classes. The extremely high resistance to permethrin and DDT (dichlorodiphenyltrichloroethane) in An. gambiae (low mortalities after 6 hours exposure) is worrisome, and is supported by a reduced insecticidal effect of bed nets against both mosquito species in laboratory tests. Metabolic and target site insensitivity mechanisms are driving this resistance in An. gambiae, but only the former was observed in An. funestus. These findings highlight the urgent need for actions to prolong the effectiveness of insecticide-based interventions in DRC., (© The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Aedes aegypti and Ae. albopictus are the major epidemic vectors of several arbovirus diseases such as yellow fever, dengue, Zika and chikungunya worldwide. Both Aedes vectors are presents in Cameroon; however, knowledge on the dynamic of the distribution of these species across cities and their resistance profile to insecticide are limited. Here, we assessed the current distribution of Ae. aegypti and Ae. albopictus in Yaoundé, the Capital City, established the resistance profile to insecticides and explored the resistance mechanisms involved., Methods: Immature stages of Aedes were sampled in several breeding sites in December 2015 (dry season) and June 2016 (rainy season) in three central neighborhoods and four peripheral neighborhoods and reared to adult stage. The G0 adults were used for molecular identification and genotyping of F1534C mutation in Ae. aegypti. Bioassays and piperonyl butoxide (PBO) assays were carried out according to WHO guidelines., Results: Analysis revealed that both species Ae. aegypti and Ae. albopictus are present in all prospected sites in Yaounde. However, in the dry season Ae. aegypti is most abundant in neighborhoods located in downtown. In contrast, Ae. albopictus was found most prevalent in suburbs whatever the season and in downtown during the rainy season. Bioassay analysis showed that both Ae. aegypti and Ae. albopictus, are resistant to 0.05% deltamethrin, 0.1% bendiocarb and 4% dichlorodiphenyltrichloroethane (DDT). A decreased of susceptibility to 0.75% permethrin and a full susceptibility to malathion 5% was observed. The mortality rate was increased after pre-exposure to synergist PBO. None of Ae. aegypti assayed revealed the presence of F1534C mutation., Conclusion: These findings are useful to planning vector control programme against arbovirus vectors in Cameroon and can be used as baseline in Africa where data on Aedes resistance is very scarce to plan further works.

Pyrethroid resistance in malaria vector, An. funestus is increasingly reported across Africa, threatening the sustainability of pyrethroid-based control interventions, including long lasting insecticidal nets (LLINs). Managing this problem requires understanding of the molecular basis of the resistance from different regions of the continent, to establish whether it is being driven by a single or independent selective events. Here, using a genome-wide transcription profiling of pyrethroid resistant populations from southern (Malawi), East (Uganda), and West Africa (Benin), we investigated the molecular basis of resistance, revealing strong differences between the different African regions. The duplicated cytochrome P450 genes ( CYP6P9a and CYP6P9b ) which were highly overexpressed in southern Africa are not the most upregulated in other regions, where other genes are more overexpressed, including GSTe2 in West (Benin) and CYP9K1 in East (Uganda). The lack of directional selection on both CYP6P9a and CYP6P9b in Uganda in contrast to southern Africa further supports the limited role of these genes outside southern Africa. However, other genes such as the P450 CYP9J11 are commonly overexpressed in all countries across Africa. Here, CYP9J11 is functionally characterized and shown to confer resistance to pyrethroids and moderate cross-resistance to carbamates (bendiocarb). The consistent overexpression of GSTe2 in Benin is coupled with a role of allelic variation at this gene as GAL4-UAS transgenic expression in Drosophila flies showed that the resistant 119F allele is highly efficient in conferring both DDT and permethrin resistance than the L119. The heterogeneity in the molecular basis of resistance and cross-resistance to insecticides in An. funestus populations throughout sub-Saharan African should be taken into account in designing resistance management strategies., (Copyright © 2017 Riveron et al.)

Background: Dengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance., Methodology/principal Findings: Genome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise pyrethroids, DDT and bendiocarb., Conclusion/significance: The predominant over-expression of cytochrome P450s suggests that synergist-based (PBO) control tools could be utilised to improve control of this major dengue vector across Malaysia., Competing Interests: The authors have declared that no competing interests exist.

Background.  Malaria remains an important public health issue in Benin, with Anopheles gambiae s.l. and Anopheles funestus s.s being the predominant vectors. This study was designed to generate information on An. funestus distribution, molecular speciation, Plasmodium infection rate and insecticide susceptibility status across Benin. Methods.  Mosquito samples were collected from December 2014 to January 2016 in 46 localities in Benin. These samples were mapped and An. funestus collected were speciated to the molecular level. Plasmodium infection rate was determined using a Taqman assay and susceptibility to insecticides was assessed using the WHO guidelines. The genotyping of the L119F- Gste2 mutation was also carried out.  Results.   An. funestus was found in 8 out of the 46 localities surveyed with a high presence in Tanongou (wet Sudanese ecological zone), Kpome, Doukonta and Pahou (sub-equatorial ecological zone). Molecular identifications revealed that only An. funestus s.s was present in southern Benin, whereas in Tanongou (northern Benin) An. funestus s.s. and An. leesoni were found in sympatry at proportions of 77.7% and 22.3% respectively. Plasmodium infection rate of An. funestus was higher in southern Benin at a range of 13 to 18% compared to 5.6% recorded in Tanongou. High DDT (8±0.5%) and permethrin (11±0.5%) resistance were observed in Doukonta, Kpome and Pahou, contrasting with relatively low resistance profiles: mortality-DDT=90±3.18% and mortality-permethrin=100% in Tanongou. Genotyping analysis revealed  high frequency  of the resistant 119F allele in the South (Kpome and Doukonta) compared to the North (Tanongou).  Discussion and Conclusion.  The high presence of   An. funestus in the South compared to the North  could be due to favorable environmental and climatic conditions found in both regions. A significant Plasmodium infection rate was recorded across the country. A high resistance profile was recorded in the southern Benin; this raises the need for further investigations on resistance selection factors., Competing Interests: No competing interests were disclosed.

Background: Knowing the extent and spread of insecticide resistance in malaria vectors is vital to successfully manage insecticide resistance in Africa. This information in the main malaria vector, Anopheles funestus sensu stricto, is completely lacking in the most populous country in Africa, Nigeria. This study reports the insecticide susceptibility status and the molecular basis of resistance of An. funestus as well as its involvement in malaria transmission in Akaka-Remo, a farm settlement village in southwest Nigeria., Results: Plasmodium infection analysis using TaqMan protocol coupled with a nested PCR revealed an infection rate of 8% in An. funestus s.s. from Akaka-Remo. WHO susceptibility tests showed this species has developed multiple resistance to insecticides in the study area. Anopheles funestus s.s. population in Akaka-Remo is highly resistant to organochlorines: dieldrin (8%) and DDT (10%). Resistance was also observed against pyrethroids: permethrin (68%) and deltamethrin (87%), and the carbamate bendiocarb (84%). Mortality rate with DDT slightly increased (from 10 to 30%, n = 45) after PBO pre-exposure indicating that cytochrome P450s play little role in DDT resistance while high mortalities were recorded after PBO pre-exposure with permethrin (from 68 to 100%, n = 70) and dieldrin (from 8 to 100%, n = 48) suggesting the implication of P450s in the observed permethrin and dieldrin resistance. High frequencies of resistant allele, 119F in F 0 (77%) and F 1 (80% in resistant and 72% in susceptible) populations with an odd ratio of 1.56 (P = 0.1859) show that L119F-GSTe2 mutation is almost fixed in the population. Genotyping of the A296S-RDL mutation in both F 0 and F 1 samples shows an association with dieldrin resistance with an odd ratio of 81 (P < 0.0001) (allelic frequency (R) = 76% for F 0 ; for F 1 , 90 and 10% were observed in resistant and susceptible populations, respectively) as this mutation is not yet fixed in the population., Conclusion: The study reports multiple insecticide resistance in An. funestus from Akaka Remo. It is, therefore, necessary to pay more attention to this major malaria vector for effective malaria control in Nigeria.

Background: Despite the recent progress in establishing the patterns of insecticide resistance in the major malaria vector Anopheles funestus, Central African populations of this species remain largely uncharacterised. To bridge this important gap and facilitate the implementation of suitable control strategies against this vector, we characterised the resistance patterns of An. funestus population from northern Cameroon., Methods and Findings: Collection of indoor-resting female mosquitoes in Gounougou (northern Cameroon) in 2012 and 2015 revealed a predominance of An. funestus during dry season. WHO bioassays performed using F1 An. funestus revealed that the population was multiple resistant to several insecticide classes including pyrethroids (permethrin, deltamethrin, lambda-cyhalothrin and etofenprox), carbamates (bendiocarb) and organochlorines (DDT and dieldrin). However, a full susceptibility was observed against the organophosphate malathion. Bioassays performed with 2015 collection revealed that resistance against pyrethroids and DDT is increasing. PBO synergist assays revealed a significant recovery of susceptibility for all pyrethroids but less for DDT. Analysis of the polymorphism of a portion of the voltage-gated sodium channel gene (VGSC) revealed the absence of the L1014F/S kdr mutation but identified 3 novel amino acid changes I877L, V881L and A1007S. However, no association was established between VGSC polymorphism and pyrethroid/DDT resistance. The DDT resistant 119F-GSTe2 allele (52%) and the dieldrin resistant 296S-RDL allele (45%) were detected in Gounougou. Temporal analysis between 2006, 2012 and 2015 collections revealed that the 119F-GSTe2 allele was relatively stable whereas a significant decrease is observed for 296S-RDL allele., Conclusion: This multiple resistance coupled with the temporal increased in resistance intensity highlights the need to take urgent measures to prolong the efficacy of current insecticide-based interventions against An. funestus in this African region., Competing Interests: The authors have declared that no competing interests exist.

Background: Understanding the dynamics of insecticide resistance in African malaria vectors is crucial for successful implementation of resistance management strategies in the continent. This study reports a high and multiple insecticide resistance in Anopheles funestus from southern Ghana which could compromise the Malaria Operational Plan in this country, if not tackled. Adult Anopheles mosquitoes were collected in Obuasi and Adawukwa, in southern Ghana. Plasmodium infection rates, susceptibility to the main insecticides used in public health and the molecular basis of insecticide resistance were established., Results: An. funestus (sensu stricto) (s.s.) was the predominant mosquito species found resting inside the houses in Obuasi, while at Adawukwa it was found together with An. coluzzii. Parasite rates were high in An. funestus (s.s.) populations from both localities, with Plasmodium infection rates greater than 12.5 %. Both, An. funestus (s.s.) and An. coluzzii, from the two sites exhibited high resistance to the insecticide from various classes including the pyrethroids, carbamates and DDT, but remained fully susceptible to the organophosphates. A preliminary characterization of the underlying molecular mechanisms of resistance in An. funestus (s.s.) populations from both sites revealed that CYP6P9a, CYP6P9b, CYP6M7 and GSTe2 genes are upregulated, markedly higher in Obuasi (between 3.35 and 1.83 times) than in Adawukwa population. The frequency of L119F-GSTe2 and A296S-RDL resistance markers were also higher in Obuasi (42.5 and 68.95 % higher), compared with An. funestus (s.s.) populations from Adawukwa. These findings suggest that the similar resistance pattern observed in both An. funestus (s.s.) populations are driven by different mechanisms., Conclusions: Resistance to multiple insecticides in public health use is present in malaria vectors from Ghana with major resistance genes already operating in the field. This should be taken into consideration in the design of resistance management strategies to avoid operational failure.

Background: Knowledge on the spread and distribution of insecticide resistance in major malaria vectors such as Anopheles funestus is key to implement successful resistance management strategies across Africa. Here, by assessing the susceptibility status of an inland population of An. funestus Giles (Kpome) and investigating molecular basis of resistance, we show that multiple resistance and consistent plasmodium infection rate are present in Anopheles funestus populations from Kpome., Methods: The insecticide susceptibility level of collected Anopheles funestus was assessed. Synergist (PBO) was used to screen resistance mechanisms. The TaqMan technique was used for genotyping of insecticide resistant alleles and detecting plasmodium infection levels. The nested PCR was used to further assess the plasmodium infection rate., Results: The TaqMan analysis of plasmodial infections revealed an infection rate (18.2 %) of An. funestus in this locality. The WHO bioassays revealed a multiple phenotypic resistance profile for An. funestus in Kpome. This population is highly resistant to pyrethroids (permethrin and deltamethrin), organochlorines (DDT), and carbamates (bendiocarb). A reduced susceptibility was observed with dieldrin. Mortalities did not vary after pre-exposure to PBO for DDT indicating that cytochrome P450s play little role in DDT resistance in Kpome. In contrast, we noticed, a significant increase in mortalities when PBO was combined to permethrin suggesting the direct involvement of P450s in pyrethroid resistance. A high frequency of the L119F-GSTe2 DDT resistance marker was observed in the wild DDT resistant population (9 %RS and 91 %RR) whereas the A296S mutation was detected at a low frequency (1 %RS and 99 %SS)., Conclusion: The presence of multiple resistance in An. funestus populations in the inland locality of Kpome is established in this study as recently documented in the costal locality of Pahou. Data from both localities suggest that resistance could be widespread in Benin and this highlights the need for further studies to assess the geographical distribution of insecticide resistance across Benin and neighboring countries as well as a more comprehensive analysis of the resistance mechanisms involved.

Background: Anopheles funestus is one of the major malaria vectors in tropical Africa, notably in Senegal. The highly anthropophilic and endophilic behaviours of this mosquito make it a good target for vector control operations through the use of insecticide treated nets, long-lasting insecticide nets and indoor residual spraying. However, little is known about patterns of resistance to insecticides and the underlying resistance mechanisms in field populations of this vector in Senegal., Methods: Here, we assessed the susceptibility status of An. funestus populations from Gankette Balla, located in northern Senegal and investigated the potential resistance mechanisms., Results: WHO bioassays indicated that An. funestus is resistant to lambda-cyhalothrin 0.05 % (74.64 % mortality), DDT 4 % (83.36 % mortality) and deltamethrin 0.05 % (88.53 % mortality). Suspected resistance was observed to permethrin 0.75 % (91.19 % mortality), bendiocarb 0.1 % (94.13 % mortality) and dieldrin 4 % (96.41 % mortality). However, this population is fully susceptible to malathion 5 % (100 % mortality) and fenitrothion 1 % (100 % mortality). The microarray and qRT-PCR analysis indicated that the lambda-cyhalothrin resistance in Gankette Balla is conferred by metabolic resistance mechanisms under the probable control of cytochrome P450 genes among which CYP6M7 is the most overexpressed. The absence of overexpression of the P450 gene, CYP6P9a, indicates that the resistance mechanism in Senegal is different to that observed in southern Africa., Conclusions: This study represents the first report of pyrethroid and DDT resistance in An. funestus from Senegal and shows that resistance to insecticides is not only confined to An. gambiae as previously thought. Therefore, urgent action should be taken to manage the resistance in this species to ensure the continued effectiveness of malaria control.

Carbamates are increasingly used for vector control notably in areas with pyrethroid resistance. However, a cross-resistance between these insecticides in major malaria vectors such as Anopheles funestus could severely limit available resistance management options. Unfortunately, the molecular basis of such cross-resistance remains uncharacterized in An. funestus, preventing effective resistance management. Here, using a genomewide transcription profiling, we revealed that metabolic resistance through upregulation of cytochrome P450 genes is driving carbamate resistance. The P450s CYP6P9a, CYP6P9b and CYP6Z1 were the most upregulated detoxification genes in the multiple resistant mosquitoes. However, in silico docking simulations predicted CYP6Z1 to metabolize both pyrethroids and carbamates, whereas CYP6P9a and CYP6P9b were predicted to metabolize only the pyrethroids. Using recombinant enzyme metabolism and inhibition assays, we demonstrated that CYP6Z1 metabolizes bendiocarb and pyrethroids, whereas CYP6P9a and CYP6P9b metabolize only the pyrethroids. Other upregulated gene families in resistant mosquitoes included several cuticular protein genes suggesting a possible reduced penetration resistance mechanism. Investigation of the target-site resistance in acetylcholinesterase 1 (ace-1) gene detected and established the association between the new N485I mutation and bendiocarb resistance (odds ratio 7.3; P < 0.0001). The detection of multiple haplotypes in single mosquitoes after cloning suggested the duplication of ace-1. A TaqMan genotyping of the N485I in nine countries revealed that the mutation is located only in southern Africa with frequency of 10-15% suggesting its recent occurrence. These findings will help in monitoring the spread and evolution of carbamate resistance and improve the design of effective resistance management strategies to control this malaria vector., (© 2016 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

Control of Aedes albopictus, major dengue and chikungunya vector, is threatened by growing cases of insecticide resistance. The mechanisms driving this resistance remain poorly characterised. This study investigated the molecular basis of insecticide resistance in Malaysian populations of Ae. albopictus. Microarray-based transcription profiling revealed that metabolic resistance (cytochrome P450 up-regulation) and possibly a reduced penetration mechanism (consistent over-expression of cuticular protein genes) were associated with pyrethroid resistance. CYP6P12 over-expression was strongly associated with pyrethroid resistance whereas CYP6N3 was rather consistently over-expressed across carbamate and DDT resistant populations. Other detoxification genes also up-regulated in permethrin resistant mosquitoes included a glucuronosyltransferase (AAEL014279-RA) and the glutathione-S transferases GSTS1 and GSTT3. Functional analyses further supported that CYP6P12 contributes to pyrethroid resistance in Ae. albopictus as transgenic expression of CYP6P12 in Drosophila was sufficient to confer pyrethroid resistance in these flies. Furthermore, molecular docking simulations predicted CYP6P12 possessing enzymatic activity towards pyrethroids. Patterns of polymorphism suggested early sign of selection acting on CYP6P12 but not on CYP6N3. The major role played by P450 in the absence of kdr mutations suggests that addition of the synergist PBO to pyrethroids could improve the efficacy of this insecticide class and overcome resistance in field populations of Ae. albopictus.

Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control. Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre. The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val109Ile, Asp335Glu and Asn384Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.

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.

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.

Background: Metabolic resistance to insecticides is the biggest threat to the continued effectiveness of malaria vector control. However, its underlying molecular basis, crucial for successful resistance management, remains poorly characterized., Results: Here, we demonstrate that the single amino acid change L119F in an upregulated glutathione S-transferase gene, GSTe2, confers high levels of metabolic resistance to DDT in the malaria vector Anopheles funestus. Genome-wide transcription analysis revealed that GSTe2 was the most over-expressed detoxification gene in DDT and permethrin-resistant mosquitoes from Benin. Transgenic expression of GSTe2 in Drosophila melanogaster demonstrated that over-transcription of this gene alone confers DDT resistance and cross-resistance to pyrethroids. Analysis of GSTe2 polymorphism established that the point mutation is tightly associated with metabolic resistance to DDT and its geographical distribution strongly correlates with DDT resistance patterns across Africa. Functional characterization of recombinant GSTe2 further supports the role of the L119F mutation, with the resistant allele being more efficient at metabolizing DDT than the susceptible one. Importantly, we also show that GSTe2 directly metabolizes the pyrethroid permethrin. Structural analysis reveals that the mutation confers resistance by enlarging the GSTe2 DDT-binding cavity, leading to increased DDT access and metabolism. Furthermore, we show that GSTe2 is under strong directional selection in resistant populations, and a restriction of gene flow is observed between African regions, enabling the prediction of the future spread of this resistance., Conclusions: This first DNA-based metabolic resistance marker in mosquitoes provides an essential tool to track the evolution of resistance and to design suitable resistance management strategies.

Background: Although the An. funestus group conceals one of the major malaria vectors in Africa, little is known about the dynamics of members of this group across the continent. Here, we investigated the species composition, infection rate and susceptibility to insecticides of this species group in Uganda., Methods: Indoor resting blood-fed Anopheles adult female mosquitoes were collected from 3 districts in Uganda. Mosquitoes morphologically belonging to the An. funestus group were identified to species by PCR. The sporozoite infection rates were determined by TaqMan and a nested PCR. Susceptibility to major insecticides was assessed using WHO bioassays. The potential role of four candidate resistance genes was assessed using qRT-PCR., Results: An. funestus s.s. and An. parensis, were the only members of the An. funestus group identified. Both species were sympatric in Masindi (North-West), whereas only An. parensis was present in Mityana (Central) and Ntungamo (South-West). The Plasmodium falciparum infection detected in An. parensis (4.2%) by TaqMan could not be confirmed by nested PCR, whereas the 5.3% infection in An. funestus s.s. was confirmed. An. parensis was susceptible to most insecticides, however, a moderate resistance was observed against deltamethrin and DDT. In the sympatric population of Masindi, resistance was observed to pyrethroids (permethrin and deltamethrin) and DDT, but all the resistant mosquitoes belonged to An. funestus s.s. No significant over-expression was observed for the four P450 candidate genes CYP6M7, CYP9K1, CYP6P9 and CYP6AA4 between deltamethrin resistant and control An. parensis. However, when compared with the susceptible FANG An. funestus s.s strain, the CYP9K1 is significantly over-expressed in An. parensis (15-fold change; P < 0.001), suggesting it could play a role in the deltamethrin resistance., Conclusion: The contrasting infection rates and insecticide susceptibility profiles of both species highlights the importance of accurate species identification for successful vector control programs.

Host plant shifts of herbivorous insects may be a first step toward sympatric speciation and can create new pests of agriculturally important crops; however, the molecular mechanisms that mediate this process are poorly understood. Certain races of the polyphagous aphid Myzus persicae have recently adapted to feed on tobacco (Myzus persicae nicotianae) and show a reduced sensitivity to the plant alkaloid nicotine and cross-resistance to neonicotinoids a class of synthetic insecticides widely used for control. Here we show constitutive overexpression of a cytochrome P450 (CYP6CY3) allows tobacco-adapted races of M. persicae to efficiently detoxify nicotine and has preadapted them to resist neonicotinoid insecticides. CYP6CY3, is highly overexpressed in M. persicae nicotianae clones from three continents compared with M. persicae s.s. and expression level is significantly correlated with tolerance to nicotine. CYP6CY3 is highly efficient (compared with the primary human nicotine-metabolizing P450) at metabolizing nicotine and neonicotinoids to less toxic metabolites in vitro and generation of transgenic Drosophila expressing CYP6CY3 demonstrate that it confers resistance to both compounds in vivo. Overexpression of CYP6CY3 results from the expansion of a dinucleotide microsatellite in the promoter region and a recent gene amplification, with some aphid clones carrying up to 100 copies. We conclude that the mutations leading to overexpression of CYP6CY3 were a prerequisite for the host shift of M. persicae to tobacco and that gene amplification and microsatellite polymorphism are evolutionary drivers in insect host adaptation.

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.

Pyrethroid resistance in Anopheles funestus is threatening malaria control in Africa. Elucidation of underlying resistance mechanisms is crucial to improve the success of future control programs. A positional cloning approach was used to identify genes conferring resistance in the uncharacterised rp2 quantitative trait locus (QTL) previously detected in this vector using F6 advanced intercross lines (AIL). A 113 kb BAC clone spanning rp2 was identified and sequenced revealing a cluster of 15 P450 genes and one salivary protein gene (SG7-2). Contrary to A. gambiae, AfCYP6M1 is triplicated in A. funestus, while AgCYP6Z2 orthologue is absent. Five hundred and sixty-five new single nucleotide polymorphisms (SNPs) were identified for genetic mapping from rp2 P450s and other genes revealing high genetic polymorphisms with one SNP every 36 bp. A significant genotype/phenotype association was detected for rp2 P450s but not for a cluster of cuticular protein genes previously associated with resistance in A. gambiae. QTL mapping using F6 AIL confirms the rp2 QTL with an increase logarithm of odds score of 5. Multiplex gene expression profiling of 15 P450s and other genes around rp2 followed by individual validation using qRT-PCR indicated a significant overexpression in the resistant FUMOZ-R strain of the P450s AfCYP6Z1, AfCYP6Z3, AfCYP6M7 and the glutathione-s-transferase GSTe2 with respective fold change of 11.2, 6.3, 5.5 and 2.8. Polymorphisms analysis of AfCYP6Z1 and AfCYP6Z3 identified amino acid changes potentially associated with resistance further indicating that these genes are controlling the pyrethroid resistance explained by the rp2 QTL. The characterisation of this rp2 QTL significantly improves our understanding of resistance mechanisms in A. funestus.