Major Malaria Vector Anopheles Gambiae Research Articles

Sibling species of the major malaria vector Anopheles gambiae display divergent preferences for aquatic breeding sites in southern Nigeria

Background When integrated with insecticide-treated bed nets, larval control of Anopheles mosquitoes could fast-track reductions in the incidence of human malaria. However, larval control interventions may deliver suboptimal outcomes where the preferred breeding places of mosquito vectors are not well known. This study investigated the breeding habitat choices of Anopheles mosquitoes in southern Nigeria. The objective was to identify priority sites for mosquito larval management in selected urban and periurban locations where malaria remains a public health burden. MethodsMosquito larvae were collected in urban and periurban water bodies during the wet-dry season interface in Edo, Delta, and Anambra States. Field-collected larvae were identified based on PCR gel-electrophoresis and amplicon sequencing, while the associations between Anopheles larvae and the properties and locations of water bodies were assessed using a range of statistical methods.ResultsMosquito breeding sites were either man-made (72.09%) or natural (27.91%) and mostly drainages (48.84%) and puddles (25.58%). Anopheles larvae occurred in drainages, puddles, stream margins, and a concrete well, and were absent in drums, buckets, car tires, and a water-holding iron pan, all of which contained culicine larvae. Wild-caught Anopheles larvae comprised Anopheles coluzzii (80.51%), Anopheles gambiae sensu stricto (s.s.) (11.54%), and Anopheles arabiensis (7.95%); a species-specific PCR confirmed the absence of the invasive urban malaria vector Anopheles stephensi among field-collected larvae. Anopheles arabiensis, An. coluzzii, and An. gambiae s.s. displayed preferences for turbid, lowland, and partially sunlit water bodies, respectively. Furthermore, An. arabiensis preferred breeding sites located outside 500 m of households, whereas An. gambiae s.s. and An. coluzzii had increased detection odds in sites within 500 m of households. Anopheles gambiae s.s. and An. coluzzii were also more likely to be present in natural water bodies; meanwhile, 96.77% of An. arabiensis were in man-made water bodies. Intraspecific genetic variations were little in the dominant vector An. coluzzii, while breeding habitat choices of populations made no statistically significant contributions to these variations.ConclusionSibling malaria vectors in the An. gambiae complex display divergent preferences for aquatic breeding habitats in southern Nigeria. The findings are relevant for planning targeted larval control of An. coluzzii whose increasing evolutionary adaptations to urban ecologies are driving the proliferation of the mosquito, and An. arabiensis whose adults typically evade the effects of treated bed nets due to exophilic tendencies.

Detection of a reduced susceptibility to chlorfenapyr in the malaria vector Anopheles gambiae contrasts with full susceptibility in Anopheles funestus across Africa

New insecticides have recently been produced to help control pyrethroid-resistant malaria vectors including the pyrrole, chlorfenapyr. Monitoring the susceptibility of mosquito populations against this new product and potential cross-resistance with current insecticides is vital for better resistance management. In this study, we assessed the resistance status of the major malaria vectors Anopheles gambiae and Anopheles funestus to chlorfenapyr across Africa and explored potential cross-resistance with known pyrethroid resistance markers. Efficacy of chlorfenapyr 100 µg/ml against An. gambiae and An. funestus from five Cameroonian locations, the Democratic Republic of Congo, Ghana, Uganda, and Malawi was assessed using CDC bottle assays. Synergist assays were performed with PBO (4%), DEM (8%) and DEF (0.25%) and several pyrethroid-resistant markers were genotyped in both species to assess potential cross-resistance between pyrethroids and chlorfenapyr. Resistance to chlorfenapyr was detected in An. gambiae populations from DRC (Kinshasa) (mortality rate: 64.3 ± 7.1%) Ghana (Obuasi) (65.9 ± 7.4%), Cameroon (Mangoum; 75.2 ± 7.7% and Nkolondom; 86.1 ± 7.4). In contrast, all An. funestus populations were fully susceptible. A negative association was observed between the L1014F-kdr mutation and chlorfenapyr resistance with a greater frequency of homozygote resistant mosquitoes among the dead mosquitoes after exposure compared to alive (OR 0.5; P = 0.02) whereas no association was found between GSTe2 (I114T in An. gambiae; L119F in An. funestus) and resistance to chlorfenapyr. A significant increase of mortality to chlorfenapyr 10 µg/ml was observed in An. funestus after to PBO, DEM and DEF whereas a trend for a decreased mortality was observed in An. gambiae after PBO pre-exposure. This study reveals a greater risk of chlorfenapyr resistance in An. gambiae populations than in An. funestus. However, the higher susceptibility in kdr-resistant mosquitoes points to higher efficacy of chlorfenapyr against the widespread kdr-based pyrethroid resistance.

Single nucleotide polymorphism (SNP) in the doublesex ( dsx) gene splice sites and relevance for its alternative splicing in the malaria vector Anopheles gambiae.

Background:Malaria burden continues to be significant in tropical regions, and conventional vector control methods are faced with challenges such as insecticide resistance. To overcome these challenges, additional vector control interventions are vital and include modern genetic approaches as well as classical methods like the sterile insect technique (SIT). In the major human malaria vector Anopheles gambiae, a candidate gene favourable for sterility induction is the doublesex ( dsx) gene, involved in mosquitos' somatic sexually dimorphic traits determination. However, the pathways that trigger the signal of dsx gene exon skipping alternative splicing mechanism in anopheline mosquitoes are not well characterized. This study aims to screen the An. gambiae dsx gene splice site sequences for single-nucleotide polymorphisms (SNPs) that could be critical to its alternative splicing. Methods: Variant annotation data from Ag1000G project phase 2 was analysed, in order to identify splice-relevant SNPs within acceptor and donor splice sites of the An. gambiae dsx gene ( Agdsx). Results: SNPs were found in both donor and acceptor sites of the Agdsx. No splice-relevant SNPs were identified in the female-specific intron 4 acceptor site and the corresponding region in males. Two SNPs (rs48712947, rs48712962) were found in the female-specific donor site of exon 5. They were not specific to either males or females as the rs48712947 was found in female mosquitoes from Cameroon, and in both males and females from Burkina Faso. In the other splice sites, the intron 3 acceptor site carried the greatest abundance of SNPs. Conclusions: There were no gender association between the identified SNPs and the random distribution of these SNPs in mosquito populations. The SNPs in Agdsx splice sites are not critical for the alternative splicing. Other molecular mechanisms should be considered and investigated.

Genetic Diversity of Cytochrome P450s CYP6M2 and CYP6P4 Associated with Pyrethroid Resistance in the Major Malaria Vectors Anopheles coluzzii and Anopheles gambiae from Yaoundé, Cameroon.

Assessing the genetic diversity of metabolic resistance genes, such as cytochrome P450s, helps to understand the dynamics and evolution of resistance in the field. Here, we analyzed the polymorphisms of CYP6M2 and CYP6P4, associated with pyrethroid resistance in Anopheles coluzzii and Anopheles gambiae, to detect potential resistance markers. Field-caught resistant mosquitos and susceptible lab strains were crossed, and F4 was exposed to permethrin for 15 min and 90 min to discriminate highly susceptible (HS) and highly resistant (HR) mosquitos, respectively. Significant permethrin mortality reduction was observed after pre-exposure to PBO, suggesting the gene involvement of P450s. qPCR analysis revealed significant overexpression of CYP6M2 (FC = 19.57 [95% CI 13.96-25.18] for An. coluzzii; 10.16 [7.86-12.46] for An. gambiae) and CYP6P4 (FC = 6.73 [6.15-7.30] An. coluzzii; 23.62 [26.48-20.76] An. gambiae). Full-gene and ≈1 kb upstream were sequenced. For CYP6M2, the upstream region shows low diversity in HR and HS (overall Hd = 0.49, π = 0.018), whereas the full-gene shows allelic-variation but without evidence of ongoing selection. CYP6P4 upstream region showed a lower diversity in HR (Hd = 0.48) than HS (Hd = 0.86) of An. gambiae. These results highlighted that CYP6P4-associated resistance is potentially driven by modification in upstream region. However, further work is needed to determine the real causative variants that will help design rapid detection tools.

Single nucleotide polymorphism (SNP) in the doublesex (dsx) gene splice sites and relevance for its alternative splicing in the malaria vector Anopheles gambiae

Background: Malaria burden continues to be significant in tropical regions, and conventional vector control methods are faced with challenges such as insecticide resistance. To overcome these challenges, additional vector control interventions are vital and include modern genetic approaches as well as classical methods like the sterile insect technique (SIT). In the major human malaria vector Anopheles gambiae, a candidate gene favourable for sterility induction is the doublesex (dsx) gene, involved in mosquitos’ somatic sexually dimorphic traits determination. However, the pathways that trigger the signal of dsx gene exon skipping alternative splicing mechanism in anopheline mosquitoes are not well characterized. This study aims to screen the An. gambiae dsx gene splice site sequences for single-nucleotide polymorphisms (SNPs) that could be critical to its alternative splicing. Methods: Variant annotation data from Ag1000G project phase 2 was analysed, in order to identify splice-relevant SNPs within acceptor and donor splice sites of the An. gambiae dsx gene (Agdsx). Results: SNPs were found in both donor and acceptor sites of the Agdsx. No splice-relevant SNPs were identified in the female-specific intron 4 acceptor site and the corresponding region in males. Two SNPs (rs48712947, rs48712962) were found in the female-specific donor site of exon 5. They were not specific to either males or females as the rs48712947 was found in female mosquitoes from Cameroon, and in both males and females from Burkina Faso. In the other splice sites, the intron 3 acceptor site carried the greatest abundance of SNPs. Conclusions: There were no gender association between the identified SNPs and the random distribution of these SNPs in mosquito populations. The SNPs in Agdsx splice sites are not critical for the alternative splicing. Other molecular mechanisms should be considered and investigated.

Insecticide Resistance Profile and Mechanisms in An. gambiae s.l. from Ebolowa, South Cameroon.

Monitoring the trend of insecticide resistance and understanding associated genetic mechanisms is important for designing efficient malaria vector control strategies. This study was conducted to provide temporal data on insecticide resistance status and mechanisms in the major malaria vector Anopheles gambiae s.l. from Ebolowa, Southern Cameroon. Methods: Larvae of An. gambiae s.l. were collected from typical breeding sites throughout the city and reared to adulthood. Emerging adults were morphologically identified and WHO tube assays were performed to determine their susceptibility to carbamate, organophosphate and pyrethroid insecticides at diagnostic doses. When resistance was observed, its intensity was determined by performing WHO tube tests using 5 and 10 times the concentration of the diagnostic dose. Metabolic resistance mechanisms were investigated using insecticide-synergist assays. Sibling species of the An. gambiae complex were identified using SINE-PCR protocol. TaqMan assay was used to genotype the L1014F and L1014S kdr mutations, and the N1575Y mutation, an amplifier of the resistance conferred by the L1014F mutation. Results: Anopheles coluzzii was by far the dominant (99%) member of the An. gambiae s.l. complex in Ebolowa. The species was fully susceptible to carbamates and organophosphates, but resistant to all pyrethroid insecticides tested. Resistance was of moderate intensity for deltamethrin (mortality: 37%, 70% and 99% for 1×, 5× and 10× insecticide concentration, respectively) but rather of high intensity for permethrin (5% for 1×; 62% for 5× and 75% for 10×) and for alphacypermethrin (4.4% for 1×; 57% for 5× and 80% for 10×). Pre-exposure to the synergist PBO resulted in a full recovery of the susceptibility to delthametrin, but this was not observed for the other two pyrethroids tested. L1014S (kdr-East) and the N1575Y mutations were absent, whereas the L1014F (kdr-West) mutation was present at a high frequency (75%), showing a significant association with resistance to permethrin (OR = 3.8; 95%; CI [1.9−7.4]; p < 0.0001) and alphacypermethrin (OR = 3; 95%; CI [1.6−5.4]; p = 0.0002). Conclusion: The increased resistance of An. gambiae s.l. to pyrethroid insecticides as observed in Ebolowa poses a threat to the efficacy of LLINs used to protect populations from the bites of Anopheles mosquitoes that transmit malaria parasites. The present study further highlights the urgent need to implement resistance management strategies in order to maintain the effectiveness of insecticide-based vector control interventions and prevent a rebound in malaria-related mortality.

Advances in mosquito repellents: effectiveness of citronellal derivatives in laboratory and field trials.

Several essential oils, including citronella (lemongrass, Cymbopogon sp., Poaceae), are well-known mosquito repellents. A drawback of such products is their limited protection time resulting from the high volatility of their active components. In particular, citronella oil protects for <2 h, although formulations with fixatives can increase this time. We synthesized hydroxylated cyclic acetals of citronellal, the main component of citronella, to obtain derivatives with lower volatility and weaker odour. The crude mixture of isomers obtained in the reaction was tested under laboratory conditions for its repellency against two mosquito species, the major malaria vector Anopheles gambiae and the arbovirus vector Aedes albopictus, and found to be endowed with longer protection time with respect to DEET (N,N-diethyl-meta-toluamide) at the same concentration. Formulated products were tested in a latin square human field trial, in an area at a high density of A.albopictus for 8h from the application. We found that the performance of the citronellal derivatives mixture is comparable (95% protection for ≤3.5h) with those of the most widespread synthetic repellents DEET and Icaridin, tested at a four-fold higher doses. Modifying the hydrophilicity and volatility of natural repellents is a valuable strategy to design insect repellents with a long-lasting effect. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Single nucleotide polymorphism (SNP) in the doublesex (dsx) gene splice sites and relevance for its alternative splicing in the malaria vector Anopheles gambiae

Background: The malaria burden continues to be significant in tropical regions, and conventional vector control methods are faced with challenges such as insecticide resistance. To overcome these challenges, additional vector control interventions are vital and include modern genetic approaches as well as classical methods like the sterile insect technique (SIT). In the major human malaria vector Anopheles gambiae, a candidate gene favourable for sterility induction is the doublesex (dsx) gene, encoding somatic sexually dimorphic traits in mosquitoes. However, the mechanism that regulates the expression of this gene in anopheline mosquitoes is poorly understood. This study aimed to screen the An. gambiae dsx gene splice site sequences for single nucleotide polymorphisms (SNPs) that could be critical to its alternative splicing. Methods: Variant annotation data from Ag1000G project phase 2 was analysed, in order to identify splice-relevant SNPs within acceptor and donor splice sites of the An. gambiae dsx gene (Agdsx). Results: SNPs were found in both donor and acceptor sites of the Agdsx. No splice-relevant SNPs were identified in the female-specific intron 4 acceptor site and the corresponding region in males. Two SNPs (rs48712947, rs48712962) were found in the female-specific donor site of exon 5. They were not specific to either males or females as the rs48712947 was found in female mosquitoes from Cameroon, and in both males and females from Burkina Faso. In the other splice sites, the intron 3 acceptor site carried the greatest abundance of SNPs. Conclusions: There were no gender association between the identified SNPs and the random distribution of these SNPs in mosquito populations. The SNPs in Agdsx splice sites are not critical for the alternative splicing. Other molecular mechanisms should be considered and investigated.

2La Paracentric Chromosomal Inversion and Overexpressed Metabolic Genes Enhance Thermotolerance and Pyrethroid Resistance in the Major Malaria Vector Anopheles gambiae.

Simple SummaryWhile shifts in temperature may promote the spread of insects, exacerbating the intensity of vector-borne diseases like malaria, very high temperatures exert deleterious effects in insect vectors, forcing them to evolve/adapt genetically. These genetic adaptations may facilitate insecticide resistance through common genes involved in both processes. Here, the impact of thermal tolerance on pyrethroid resistance in the major malaria vector Anopheles gambiae s.l. from four localities spanning the savanna and sub-Sahel of northern Nigeria was investigated. In all four localities, An. coluzzii and An. gambiae s.s. were the only malaria vectors found. The populations were highly thermotolerant, with ~50% of mosquitoes in two sites surviving 44 °C. Thermotolerant larvae and adult mosquitoes (survivors of 44 °C) were significantly more resistant to permethrin, suggesting that prior heat-hardening facilitates insecticide resistance. Thermal tolerance and permethrin resistance were found to be associated with the 2La rearrangement (a form of chromosomal inversion). Transcriptional analysis revealed six major genes commonly overexpressed in the highly thermotolerant mosquitoes and those resistant to permethrin, suggesting common mechanisms involved in thermotolerance and insecticide resistance. These findings highlight challenges associated with insecticide-based malaria vector control, stressing the need to take environmental variables and other pleiotropic mechanisms into account for the choice of control measures.Changes in global temperature are impacting the spread/intensity of vector-borne diseases, including malaria, and accelerating evolutionary/adaptive changes in vector species. These changes, including chromosomal inversions and overexpression and/or changes in allele frequencies of thermotolerance-associated genes, may facilitate insecticide resistance through pleiotropy. This study investigated the impact of thermotolerance on pyrethroid resistance in four populations of the malaria vector An. gambiae s.l., from the savanna/sub-Sahel of northern Nigeria. Anopheles coluzzii and An. gambiae s.s. were the only malaria vectors found, sympatric in all the sites, with the former species predominant. High thermotolerance was observed, with no mortality at 38 °C, and LT50 of ~44 °C. Significantly high permethrin resistance was observed (mortality < 50%) in 44 °C heat-hardened (exposure to an intermediately high temperature provides protection to a more severe temperature or insecticide) larvae from two sites, BUK and Pantami, compared with the control, and heat-hardened adult females from Auyo (mortality = 3.00% ± 1.20, χ2 = 5.83, p < 0.01) compared with the control (12.00% ± 4.65). The 2La chromosomal inversion was detected at ~50% in subset of larvae and 58% in subset of adult females genotyped. A significant association was observed (OR = 7.2, p < 0.03) between permethrin resistance and the 2La/+a rearrangement compared with 2L+a/+a, in BUK larvae. For all sites, permethrin resistance correlated with 2La/a homozygosity in adult females (R = 5.02, p = 0.01). qRT-PCR identified six genes commonly induced/overexpressed, including the heat shock protein 70 (AGAP004581) which was 2468× and 5× overexpressed in heat-hardened and permethrin-resistant females, respectively; trehalose-6-phosphate synthase (AGAP008227); and the ionotropic glutamate receptor genes, IR25a (AGAP010272) and IR21a (AGAP008511). This study highlights challenges associated with insecticide-based malaria vector control, and the epidemiological significance of taking climate variables into account for the design/choice of control measures.

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

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

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential.

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite's extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R0, and find the average R0 is higher (range: 10.1%-12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.

New distribution record of Anopheles rivulorum-like from Sadiola, Mali, with notes on malaria vector insecticide resistance.

The SEMOS gold mine in Sadiola, southwestern Mali, has been implementing a malaria vector control programme for 15y using indoor residual house spraying and sporadic larval control. Periodic screening of the vector populations have been carried out over the years to provide information to the control programme, mainly on vector species present and their insecticide resistance status. The data from five entomological surveys, carried out in 2006, 2011, 2014, 2016 and 2018, are presented. Adult mosquitoes were collected resting on walls inside houses and on verandas. Insecticide susceptibility assays were carried out and mosquitoes subsequently identified by species using molecular assays. The major malaria vector mosquitoes, Anopheles gambiae and Anopheles arabiensis were abundant at each sampling period with Anopheles coluzzii and Anopheles funestus being rare or absent. Anopheles rivulorum was identified in 2006 and Anopheles leesoni in 2016. The presence of Anopheles rivulorum-like, identified for the first time in 2018, was not screened for in previous surveys. Insecticide susceptibility bioassays showed resistance in both A. gambiae and A. arabiensis to pyrethroids, carbamates and dichlorodiphenyltrichloroethane over the 12y. This is the first record of A. rivulorum-like west of Côte d'Ivoire. Resistance levels to the three classes of insecticides were variable but appeared to decrease after pyrethroids were discontinued for house spraying.

The Anopheles coluzzii microbiome and its interaction with the intracellular parasite Wolbachia

Wolbachia, an endosymbiotic alpha-proteobacterium commonly found in insects, can inhibit the transmission of human pathogens by mosquitoes. Biocontrol programs are underway using Aedes aegypti mosquitoes trans-infected with a non-natural Wolbachia strain to reduce dengue virus transmission. Less is known about the impact of Wolbachia on the biology and vectorial capacity of Anopheles mosquitoes, the vectors of malaria parasites. A naturally occurring strain of Wolbachia, wAnga, infects populations of the major malaria vectors Anopheles gambiae and Anopheles coluzzii in Burkina Faso. Previous studies found wAnga infection was negatively correlated with Plasmodium infection in the mosquito and wAnga influenced mosquito egg-laying behavior. Here, we investigate wAnga in natural populations of An. coluzzii and its interactions with other resident microbiota using targeted 16S sequencing. Though we find no major differences in microbiota composition associated with wAnga infection, we do find several taxa that correlate with the presence or absence of wAnga in female mosquitoes following oviposition, with the caveat that we could not rule out batch effects due to the unanticipated impact of wAnga on oviposition timing. These data suggest wAnga may influence or interact with the Anopheles microbiota, which may contribute to the impact of wAnga on Anopheles biology and vectorial capacity.

A high throughput multi-locus insecticide resistance marker panel for tracking resistance emergence and spread in Anopheles gambiae

The spread of resistance to insecticides in disease-carrying mosquitoes poses a threat to the effectiveness of control programmes, which rely largely on insecticide-based interventions. Monitoring mosquito populations is essential, but obtaining phenotypic measurements of resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). We found resistance association in four markers, including a novel mutation in the detoxification gene Gste2 (Gste2-119V). We also identified a duplication in Gste2 combining a resistance-associated mutation with its wild-type counterpart, potentially alleviating the costs of resistance. Finally, we describe the distribution of the multiple origins of kdr resistance, finding unprecedented diversity in the DRC. This panel represents the first step towards a quantitative genotypic model of insecticide resistance that can be used to predict resistance status in An. gambiae.

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

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

Mosquitoes cloak their legs to resist insecticides.

Malaria incidence has halved since the year 2000, with 80% of the reduction attributable to the use of insecticides. However, insecticide resistance is now widespread, is rapidly increasing in spectrum and intensity across Africa, and may be contributing to the increase of malaria incidence in 2018. The role of detoxification enzymes and target site mutations has been documented in the major malaria vector Anopheles gambiae; however, the emergence of striking resistant phenotypes suggests the occurrence of additional mechanisms. By comparing legs, the most relevant insect tissue for insecticide uptake, we show that resistant mosquitoes largely remodel their leg cuticles via enhanced deposition of cuticular proteins and chitin, corroborating a leg-thickening phenotype. Moreover, we show that resistant female mosquitoes seal their leg cuticles with higher total and different relative amounts of cuticular hydrocarbons, compared with susceptible ones. The structural and functional alterations in Anopheles female mosquito legs are associated with a reduced uptake of insecticides, substantially contributing to the resistance phenotype.

Insecticide resistance profiles of Anopheles gambiae s.l. in Togo and genetic mechanisms involved, during 3-year survey: is there any need for resistance management?

BackgroundMalaria, one of the world’s greatest public health challenges, is an endemic disease with stable transmission in Togo. Combating malaria requires an effective vector control. This study provides temporal data on insecticide resistance status in the major malaria vector Anopheles gambiae sensu lato (s.l.) from Togo.MethodsTwo to 5 days old females of An. gambiae s.l., originating from three localities (Baguida, Kovié, Kolokopé) were subjected to insecticide-impregnated papers during 3 years (2012, 2013, 2016) as follows: organochlorides (4% DDT), pyrethroids (0.05% deltamethrin, 0.75% permethrin, 0.05% lambdacyhalothrin), carbamates (0.4% bendiocarb and 0.1% propoxur), and organophosphates (5% malathion, 0.4% chlorpyrifos methyl, 1% fenitrothion) following the WHO standard protocol. Dead and surviving mosquitoes were stored separately in Eppendorf tubes containing silica gel for DNA extraction, species identification, and kdr and ace-1 genotyping.ResultsKnockdown times (KDT50 and KDT95) were high in An. gambiae s.l. The lowest KDTs were recorded at Baguida in 2013 for deltamethrin (KDT50 = 24.7, CI [22.4–27.12] and KDT95 = 90.78, CI [76.35–113.49]). No KDTs were recorded for DDT and in some instances for permethrin. In general, An. gambiae s.l. was resistant to most of the four classes of insecticides during the survey periods regardless of locality and year, except to chlorpyrifos methyl. In some instances, mosquitoes were fully susceptible to fenitrothion (Kolokopé: 100% and Kovié: 98.05%, CI [95.82–100.26]) and malathion (100% at both Kolokopé and Kovié) in 2013, and malathion only (Kolokopé; 100%) in 2016. Anopheles coluzzii, An. gambiae and Anopheles arabiensis were the three sibling species identified at the three localities with some hybrids at Baguida (2013), and Kovié (2012 and 2016), respectively. Anopheles gambiae was relatively dominant (61.6%). The kdr 1014F allele frequency was > 0.9 in most of the cases, except at Kolokopé (f (1014F) = 0.63, CI [0.55–0.71]) in 2013. The kdr 1014S allele frequency was below 0.02. The highest ace-1 frequencies were identified in An. gambiae at Baguida (2012: 0.52, CI [0.34–0.69] and 2013: 0.66, CI [0.46–0.86]).ConclusionThe resistance status is worrying in Togo and should be considered in future malaria vector resistance management programmes by decision-makers.

Rapid multiplex gene expression assays for monitoring metabolic resistance in the major malaria vector Anopheles gambiae

BackgroundMetabolic resistance of the major malaria vector Anopheles gambiae (s.l.) to insecticides is operationally significant, particularly in combination with target site resistance. However, detection of metabolic resistance is not trivial and relies on laborious bioassays, unspecific biochemical methods, or sophisticated and expensive molecular approaches using transcriptomics.MethodsRapid one-step multiplex TaqMan-probe based RT-qPCR assays were developed and optimised to measure the expression levels of genes associated with metabolic insecticide resistance in An. gambiae (s.l.). Primers and probes were designed to target the mRNA of cytochrome P450-dependent monooxygenases CYP6P3, CYP6M2, CYP9K1, CYP6P4 and CYP6Z1, and the glutathione-S-transferase GSTE2. The novel assays were validated versus gold standard methods with a range of phenotyped mosquito specimens. The assays were also tested directly on lysates of RNAlater®-preserved mosquitoes without an RNA extraction step.ResultsThe novel assays are efficient (reaction efficiencies = 95–109%), sensitive (covering a > 10.0 Ct range with R2 values > 0.99), specific (TaqMan chemistry), reproducible (%CV = 4.46–12.07%), as well as readily expandable to capture additional loci as they evolve or to cover additional species. The assays were successfully validated in terms of expression levels against standard two-step singleplex qPCR assays (overall % difference = -17.6%, 95% CI = -38.7–3.43%) and microarrays, using laboratory strains and field-caught samples. The assays can also be applied directly on lysates of mosquito specimens, without RNA extraction or DNase treatment.ConclusionsThe novel multiplex assays for monitoring the levels of major detoxification genes and metabolic resistance in An. gambiae (s.l.) are simple to perform, robust and rapid. They may complement current diagnostic assays to provide evidence-based and operationally relevant information for insecticide resistance management.

Evolution of host preference in anthropophilic mosquitoes

BackgroundInsecticide-treated bed nets (ITNs) have played a large role in reducing the burden of malaria. There is concern however regarding the potential of the mass distributions and use of ITNs to select for insecticide and behavioural resistance in mosquito populations. A key feature of the vectorial capacity of the major sub-Saharan African malaria vector Anopheles gambiae sensu stricto (s.s.) is its tendency to feed almost exclusively on humans. Here, an evolutionary model is used to investigate the potential for ITNs to select for increased zoophily in this highly anthropophilic species and how this is influenced by ecological and operational conditions.ResultsThe evolution of a single trait, namely the tendency to accept cattle as hosts, is modelled in mosquito populations which initially only bite humans. Thus, the conditions under which a resource specialist would broaden its diet and become a generalist are investigated. The results indicate that in the absence of insecticide-treated nets, host specialization in mosquitoes is either driven toward human specialization (when humans are more abundant than alternative hosts), or displays evolutionary bistability. The latter implies that the evolutionary endpoint relies on the initial trait value of the population. Bed nets select for increased zoophily while in use. When ITNs are removed, whether or not the population reverts to anthropophagic or zoophagic behaviour depends on whether the intervention had been maintained sufficiently long to drive the population past the evolutionarily unstable point.ConclusionsThe use of ITNs is likely to select for an increase in the biting preference for cattle. Bed nets may thus alter the population composition of major vector species in a manner that has positive epidemiological ramifications. Whether populations are set on a trajectory toward increased zoophily following the cessation of intense bed net usage in an area depends on the composition of host communities as well as operational conditions. This has potential implications for bed net campaigns, particularly with an eye toward scaling down interventions following interruption of transmission. Further research on malaria mosquito feeding behaviour is warranted to explore the conditions under which such adaptive shifts may actually occur in the field.

Contributions of cuticle permeability and enzyme detoxification to pyrethroid resistance in the major malaria vector Anopheles gambiae

To tackle the problem of insecticide resistance, all resistance mechanisms need to be studied. This study investigated the involvement of the cuticle in pyrethroid resistance in a strain of Anopheles gambiae, MRS, free of kdr mutations. Bioassays revealed MRS to be resistant to pyrethroids and DDT, indicated by increasing knockdown times and resistance ratios. Moreover, biochemical analysis indicated that metabolic resistance based on enhanced CYP450 activity may also play a role. Insecticide penetration assays showed that there were significantly lower amounts of insecticide in the MRS strain than in the susceptible control. Analysis of the levels of the selected transcripts by qPCR showed that CYP6M2, a major pyrethroid metaboliser, CYP4G16, a gene implicated in resistance via its contribution to the biosynthesis of elevated epicuticular hydrocarbons that delay insecticide uptake, and the cuticle genes CPAP3-E and CPLCX1 were upregulated after insecticide exposure. Other metabolic (CYP6P3, GSTe2) and cuticle (CPLCG3, CPRs) genes were also constitutively upregulated. Microscopic analysis showed that the cuticle layers of the MRS strain were significantly thicker than those of the susceptible strain. This study allowed us to assess the contribution made by the cuticle and metabolic mechanisms to pyrethroid resistance in Anopheles gambiae without target-site mutations.