Bionomics and insecticides resistance profiling of malaria vectors at a selected site for experimental hut trials in central Cameroon.

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 adult mosquitoes. Insecticides susceptibility bioassays were performed on the F 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

Exposure to the insecticide-treated bednet PermaNet 2.0 reduces the longevity of the wild African malaria vector Anopheles funestus but GSTe2-resistant mosquitoes lives longer

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 south-ern 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. fu-nestus 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 effi-cacy 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 longev-ity 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 fecun-dity 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 vec-tors which could contribute in the reduction of malaria incidence

The P450 CYP6Z1 confers carbamate/pyrethroid cross-resistance in a major African malaria vector beside a novel carbamate-insensitive N485I acetylcholinesterase-1 mutation

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 genome-wide transcription profiling, we revealed that metabolic resistance through up-regulation of cytochrome P450 genes is driving carbamate resistance. The P450s CYP6P9a, CYP6P9b and CYP6Z1 were the most up-regulated detoxification genes in the multiple resistant mosquitoes. However, in silico docking simulations predicted CYP6Z1 to metabolise both pyrethroids and carbamates, whereas CYP6P9a and CYP6P9b were predicted to metabolise only the pyrethroids. Using recombinant enzyme metabolism and inhibition assays we demonstrated that CYP6Z1 metabolizes bendiocarb and pyrethroids, whereas CYP6P9a and CYP6P9b metabolise only the pyrethroids. Other up-regulated 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

Multiple insecticide resistance in the major malaria vector Anopheles funestus in southern Ghana: implications for malaria control

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

Investigating molecular basis of lambda-cyhalothrin resistance in an Anopheles funestus population from Senegal.

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

The Cytochrome P450 gene CYP6P12 confers pyrethroid resistance in kdr-free Malaysian populations of the dengue vector Aedes albopictus

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

An Africa-wide genomic evolution of insecticide resistance in the malaria vector Anopheles funestus involves selective sweeps, copy number variations, gene conversion and transposons

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 improve 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 sweep, 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

Insecticide Resistance in Malaria Vectors: An Update at a Global Scale

Malaria remains the deadliest vector-borne disease in the world. With nearly half of the world’s population at risk, 216 million people suffered from malaria in 2016, with over 400,000 deaths, mainly in sub-Saharan Africa. Important global efforts have been made to eliminate malaria leading to significant reduction in malaria cases and mortality in Africa by 42% and 66%, respectively. Early diagnosis, improved drug therapies and better health infrastructure are key components, but this extraordinary success is mainly due the use of long-lasting insecticidal nets (LLINs) and indoor residual sprayings (IRS) of insecticide. Unfortunately, the emergence and spread of resistance in mosquito populations against insecticides is jeopardising the effectiveness of the most efficient malaria control interventions. To help establish suitable resistance management strategies, it is vital to better understand the distribution of resistance, its mechanisms and impact on effectiveness of control interventions and malaria transmission. In this chapter, we present the current status of insecticide resistance worldwide in main malaria vectors as well as its impact on malaria transmission, and discuss the molecular mechanisms and future perspectives

Multiple Insecticide Resistance in the Malaria Vector Anopheles funestus from Northern Cameroon Is Mediated by Metabolic Resistance Alongside Potential Target Site Insensitivity Mutations

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

Influence of a Major Mountainous Landscape Barrier (Mount Cameroon) on the Spread of Metabolic (GSTe2) and Target-Site (Rdl) Resistance Alleles in the African Malaria Vector Anopheles funestus

Abstract: 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 GSTe2R (67–81%) and RdlR (49–90%) resistance alleles were observed in An. funestus throughout the study area, with GSTe2R frequency increasing with altitude, whereas the opposite is observed for RdlR. 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