Biology subjects

Wondji, C. S.

Publications and source records attributed to Wondji, C. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Multi-site phenotypic characterisation of Kisumu colonies for comparison of insecticide susceptibility between testing centres

Testing of insecticides and insecticide-based vector control tools relies on the use of bioassays whereby insects are exposed and mortality, or another relevant entomological endpoint, is measured. To be able to compare results between products and across time and space standard laboratory strains are used. Insecticide-susceptible strains are often used as comparators in studies involving insecticide-resistant populations. Kisumu is a long-established, widely-used insecticide-susceptible strain of Anopheles gambiae. In this study, 16 facilities across different countries exposed their Kisumu colonies aged 3-5 days to four insecticides (permethrin, alpha-cypermethrin, DDT and pirimiphos-methyl) at a range of concentrations in WHO tube assays to assess susceptibility. Insecticide-treated filter papers were made at the Liverpool School of Tropical Medicine and sent to testing centres, along with a standard protocol and testing conditions requirements, to standardise testing as much as possible. A first observation was that DDT results were affected by the use of Risella oil, the WHO-recommended carrier oil but immiscible in acetone, leading to uneven coating of papers and increased variability in bioassay results (40x variance between colonies). Bioassay data generated by the testing facilities suggest a wide range of susceptibility to different insecticides between colonies of Kisumu: LC50 values between colonies differed, though not significantly, by up to 4x for permethrin, 6x for pirimiphos-methyl, and 15x for alpha-cypermethrin. Some colonies are classified as insecticide resistant based on the WHO definition of <98% mortality on exposure to a discriminating concentration. The assumption that Kisumu is a standard susceptible strain, consistent between facilities is challenged, which has implications when comparing results between studies. We propose genotypic analysis of the colonies to identify signs of resistance markers indicative of contamination from resistant strains or wild mosquitoes, and generation of guidance to support the maintenance and monitoring of susceptible mosquito strains.

zoology↗

bioRxiv2026

Multi-centre laboratory study to determine discriminating concentrations for broflanilide and isocycloseram resistance monitoring in mosquitoes

1In 2024-2025 a multi-centre study involving seven international laboratories was conducted with the support of the World Health Organization (WHO). The aim of the study was to establish and validate discriminating concentrations (DCs) in WHO bottle bioassays for monitoring susceptibility to broflanilide and isocycloseram in Anopheles gambiae s.s., An. funestus, An. stephensi and Aedes aegypti. The following values are recommended for adoption as DCs for broflanilide: 10 {micro}g/bottle for An. gambiae and Ae. aegypti, 15 {micro}g/bottle for An. funestus, and 25 {micro}g/bottle for An. stephensi. The recommended DCs for isocycloseram are 15 {micro}g/bottle for Ae. aegypti, 30 {micro}g/bottle for An. gambiae, 50 {micro}g/bottle for An. stephensi, and 60 {micro}g/bottle for An. funestus. Based on the experiences of conducting this study, which represents the application of a generic protocol for establishing discriminating concentrations produced by the WHO, technical recommendations are made on the generation and analysis of DC data for insecticides in future.

zoology↗

bioRxiv2026

Generational selection, transcriptomics and functional characterization reveal the impact of environmental pollutants on the evolution of insecticide resistance in malaria vectors

Insecticide resistance is threatening malaria control. While the evolution and spread of resistance has been linked to scale-up in the distribution of public health insecticides, the role of environmental pollutants such as the polyaromatic hydrocarbons (PAHs) from industrial and agricultural use remains largely uncharacterized. The PAHs are potent ligands of the aryl hydrocarbon receptor (Ahr) transcription factors involved in the regulation of xenobiotic metabolizing enzymes, and potentially involved in insecticide resistance. Here, using field insecticide-resistant (Auyo) An. coluzzii and a laboratory-susceptible colony (Ngousso), we conducted a multi-generational selection experiment using naphthalene, fluorene and a mixture of both PAHs. After ten generations, the changes in susceptibility to insecticides were monitored using WHO bioassays and whole-transcriptome analysis (RNASeq) was conducted. Compared with the non-selected colony lines, PAH exposures significantly reduced pyrethroid and DDT resistance in the field population, suggesting fitness cost associated with established resistance. In contrast, Ngousso showed a significant increase in DDT resistance (p = 0.01) at the tenth generation. A significant increase in permethrin resistance was also observed at the seventh generation (p = 0.03). Several candidate genes from the major detoxification classes were overexpressed in the selected lines (including GSTe2, CYP6Z1, and CYP6P4); the most consistent were CYP6M4 and CYP4C27, as well as those from the Ahr pathway. Heterologous expression of CYP6M4 revealed its ability to metabolise pyrethroids, including permethrin, deltamethrin, and -cypermethrin, as well as PAHs (naphthalene and fluorene). These findings establish the role of environmental pollutants as additional drivers of metabolic insecticide resistance in An, coluzzii.

ecology↗

bioRxiv2026

Metabolomic changes underpinning permethrin resistance in the Anopheles gambiae malaria vector from Cameroon

Insecticide resistance reduces the effectiveness of current malaria vector control interventions. To understand the mechanisms underlying permethrin resistance in field-derived Anopheles gambiae vectors collected from Cameroon, we used Ultra-High Performance Liquid Chromatography coupled with High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS) to comprehensively analyze metabolic changes in resistant and susceptible samples to gain insight into mechanisms driven permethrin resistance. Resistant mosquitoes exhibited upregulated levels of inosine, nicotinic acid, dipeptides, amino acids, fumarate, uracil, and aldopentose. These data suggest that altered metabolic-based detoxification, as well as target site and metabolic shifts, and enhanced energy production contribute to permethrin resistance. Conversely, susceptible mosquitoes showed increased levels of N-acetyl-aspartic acid, xanthurenic acid, 2-hydroxyglutarate, 3-hydroxykynurenine, propanoylcarnitine, and L-pipecolic acid. These metabolites are associated with neurotoxicity, energy disruption, as well as tryptophan and lysine catabolism. These findings elucidate the metabolic pathways of permethrin-resistance and underscore the mechanisms that could lead to the emergence of pyrethroid cross-resistance.

systems biology↗

bioRxiv2025

A panel of CYP6Z genes drives broad-spectrum cross-resistance to public health insecticides in the major malaria vector An. gambiae s.s.

The spread of multiple and intense insecticide resistance in major African malaria vectors is jeopardising control efforts. Mitigating this threat requires deciphering its underlying molecular mechanisms. Here, by integrating transcriptomic profiling, analysis of genetic diversity and functional genomics approaches, we established the role of the CYP6Z genes in conferring multiple- and cross-resistance to insecticides. Investigation of CYP6Z gene expression and genetic diversity analyses indicate that resistance is primarily associated with transcriptional upregulation rather than fixed coding mutations since no predominant haplotype was selected. Structural characterisation reveals a flexible, promiscuous active site in CYP6Z enzymes, enabling binding of multiple insecticide classes. In vitro functional validation confirms that recombinant CYP6Z3 efficiently metabolizes deltamethrin (percentage depletion of 59%), permethrin (52%), -cypermethrin (37%), pirimiphos-methyl (36%), fenitrothion (25%), propoxur (25%), and possibly bendiocarb (17%). RNAi-mediated knockdown of CYP6Z genes in field-collected An. gambiae s.s. restores susceptibility to several insecticides including clothianidin: dsCYP6Z1 (mortality = 50.29%; p < 0.01), dsCYP6Z2 (47.28%; p < 0.01) and dsCYP6Z3 (39.12%; p < 0.05) compared to the control (mortality = 27.67%). Furthermore, transgenic expression in Drosophila melanogaster flies revealed that expression of CYP6Z genes alone confer cross-resistance to pyrethroids, organophosphates, carbamates and neonicotinoids, but increases susceptibility to the pro-insecticide chlorfenapyr: CYP6Z1 (mortality = 100%; p < 0.01), CYP6Z3 (98.18%; p < 0.01), and CYP6Z4 (98.01%; p < 0.01) vs. control (88.03%). This study establishes An. gambiae CYP6Z genes operate in concert to drive a broad-spectrum metabolic resistance, towards several classes of public health insecticides, while in contrast bioactivating chlorfenapyr to its more insecticidally active metabolite.

molecular biology↗

bioRxiv2025

Characterization of a new laboratory colony of Anopheles funestus mosquitoes established in Ifakara, Tanzania

BackgroundAnopheles funestus, a major vector of malaria in Africa, has proven difficult to colonize in laboratory settings, impeding research on its biology and control. After several attempts, our team recently succeeded in colonizing a strain of An. funestus from Tanzania (FUTAZ). The objective of this study was to analyse the key fitness and genotypic characteristics of these mosquitoes during multiple filial generations of laboratory adaptation and compare them to wild An. funestus from Tanzania and a pre-existing colony of An. funestus from Mozambique (FUMOZ). MethodsMeasures of mating success (percentage of female mosquitoes inseminated), body size (wing length), fecundity (number of eggs laid per female), and insecticide susceptibility (percentage of 24-hour mortality after exposure to insecticides) were compared between the newly established colonies of Tanzanian An. funestus (FUTAZ colonies), the long-established FUMOZ colonies, and a colony of Anopheles arabiensis maintained in the same laboratory. The maternal lineages of the An. funestus mosquitoes were investigated through a hydrolysis probe analysis of their mitochondrial DNA to identify distinct clades, I and II. Additionally, other intragenomic variations were examined through a PCR analysis of restriction fragment length polymorphisms (RFLP) on the third domain of 28S ribosomal DNA. These molecular markers were used to compare the FUTAZ colonies, FUMOZ colonies in Tanzania and South Africa, and the wild-collected An. funestus from Tanzania. ResultThe mating success and body size of FUTAZ females declined significantly from filial generations F1 to F6 relative to the founder population (F0), but then increased from F7 onwards eventually matching FUMOZ by F9. Fecundity was similar across all colonies tested. However, it took significantly longer for 50% of the females in the FUTAZ and FUMOZ colonies (over 10 days) to mate compared to females in the An. arabiensis colony (approximately 5 days). Insecticide resistance appeared to be lost during colonization, but this varied with insecticide classes. Majority of mosquitoes in the FUTAZ colony, as well as the wild-caught Tanzanian An. funestus belonged to Clade I (80.4-89.4%) and RFLP type "Y" (90.5-91.4%), while the FUMOZ colonies were mostly Clade II (65.5-88.5%) and RFLP type "MW" (90.5-91.5%). ConclusionThis study suggests that the mating success and body size of An. funestus decreases significantly during the early stages of colonization, then increase as the mosquitoes adapt to laboratory conditions. It is therefore crucial to have a large enough founder population to persist through these early generations in order to achieve stable colonization of An. funestus. The Clade and RFLP genotyping demonstrated the genetic similarities between the FUTAZ mosquitoes and wild-caught Tanzanian An. funestus, but also showed that the new colony can be distinguished from the FUMOZ colony.

ecology↗

bioRxiv2025

Genetic mapping of resistance: A QTL and associated polymorphism conferring resistance to alpha-cypermethrin in Anopheles funestus

The heavy reliance on pyrethroid-based interventions has accelerated the spread of resistance malaria vectors including Anopheles funestus, jeopardising control efforts. The efficacy of Insecticide-based interventions, especially insecticide-treated nets (ITNs), the cornerstone of malaria control and management, is threatened by the widespread resistance complicating malaria control. Alpha-cypermethrin, a type II pyrethroid, is increasingly utilised in various ITN formulations, including those combined with piperonyl butoxide (PBO) and chlorfenapyr-based Interceptor(R) G2 (IG2) nets, to enhance effectiveness against resistant mosquito populations. Therefore, understanding the molecular basis of resistance is essential to monitor and track resistance trends for an effective malaria control program. In this study, we identified a 1.4 Mb QTL on the telomeric end of the left arm of chromosome 2, conferring resistance to -cypermethrin (rap1 QTL). Different crossing schemes and sequencing approaches were explored to determine the most effective strategy. Individual-based QTL mapping performed on segregating individuals from an isofemale family identified a QTL at the F7 generation. Higher recombination density relative to the physical genome in the F7 isofemale family, with a recombination every 240 kb, facilitated the detection of a QTL compared to the F2 family (335 kb/cM). Additionally, we exploited bulk segregate analysis (BSA) between susceptible and resistant phenotypes from the F7 isofemale family and an F7 mixed cross-family to perform cost-effective and rapid QTL-mapping discovery. The strongest signal in both independent BSA analyses overlaps with the rap1 QTL, further supporting its role in -cypermethrin resistance. The known resistant alleles of the cytochrome P450 CYP6P9a and 6.5-kb structural variant within the rap1 QTL strongly correlate with survival to -cypermethrin. In this study, we validated that previously developed DNA-based assays, originally designed to monitor permethrin resistance, are effective for tracking resistance to -cypermethrin as well. Additionally, we identified candidate variants that can serve as reliable markers for monitoring -cypermethrin resistance. Author SummaryIn the study we used genetic crosses between resistant and susceptible Anopleles funestus colonies to identify quantitative trait loci (QTL) associated with the resistance to -cypermethrin. We have identified a QTL on the left arm of chromosome 2 conferring resistance to -cypermethrin (rap1 QTL). Different crossing schemes and sequencing approaches were explored to determine the most effective and cost-efficient strategy to map candidate loci associated with resistance. In this study we assessed the efficiency and the cost-effectiveness of Bulk segregant analysis (BSA) mapping to detect candidate loci associated with resistance. BSA analysis enabled the detection of polymorphism in candidate regions that could serve as potential SNP-based marker to track resistance to -cypermethrin. Previously developed SNP-based markers to track resistance to permethrin in known resistant alleles show a strongly correlation with survival to -cypermethrin, suggesting shared mechanisms my underly resistance to both type I (permethrin) and type II (-cypermethrin) pyrethroids.

genomics↗

bioRxiv2025

Significant variations in tolerance to clothianidin and pirimiphos-methyl in Anopheles gambiae and Anopheles funestus populations during a dramatic malaria resurgence despite sustained indoor residual spraying in Uganda

A dramatic malaria resurgence occurred in areas of Uganda between 2020 and 2022 coincident with the switch to clothianidin-based formulations for indoor residual spraying. During the resurgence, Anopheles funestus numbers increased but when an alternative insecticide, pirimiphos methyl, was reintroduced in 2023, both malaria cases and An. funestus mosquito density fell. In this study, we investigated possible causes of the resurgence by assessing; 1) whether sufficient quantities of insecticide were sprayed; 2) the residual insecticide bioefficacy against wild mosquitoes and; 3) the insecticide susceptibility of vector populations using standard test tube assays and wall cone assays. In 2023, after adjusting for extraction efficiency, 70-80% of the houses had optimal residual concentrations of insecticides (clothianidin >0.3g/m2; pirimiphos methyl >0.5g/m2) with significant variations between sampling rounds and wall types. Mud walls had the lowest residual concentration of insecticides, and the lowest observed mortality in wall cone assays, compared to fired bricks with plaster/cement/paint. In the studies of residual bio efficacy, by World Health Organization (WHO) definitions, An. funestus showed resistance to clothianidin (<80% mortality) up to 11 months and susceptibility to pirimiphos methyl (>90% mortality) when exposed to wall surfaces up to 7 months post-spray. In WHO tube tests, variations were observed in susceptibility to clothianidin in An. funestus populations using dose- and time-response assays (80-98% mortality). In 2022, An. gambiae was largely susceptible to the clothianidin-based formulation Sumishield (85-90% mortality) although the levels dropped slightly in 2023 (60-85% mortality) mainly in mud and pole houses. In contrast, An. gambiae was mildly susceptible to the pirimiphos methyl-based formulation Actellic ([~]80% mortality) and time response assays showed An. gambiae populations had very low knockdown and mortality at lower exposure time compared to An. funestus. Regression models showed a positive association between residual insecticide concentration (RIC) and mortality in houses sprayed with Sumishield but not Actellic houses. Despite the possible variations observed in spray operations, the study revealed that An. funestus exhibited a higher tolerance to clothianidin-based formulations compared to An. gambiae, and this might have driven the malaria resurgence observed in Uganda. However, there are signals of An. gambiae resistance to pirimiphos-methyl which will require further investigation and monitoring.

zoology↗

bioRxiv2024

Genomic diversity of the African malaria vector Anopheles funestus

Anopheles funestus s.s. is a formidable human malaria vector across sub-Saharan Africa. To understand how the species is evolving, especially in response to malaria vector control, we sequenced 656 modern specimens (collected 2014-2018) and 45 historic specimens (collected 1927-1967) from 16 African countries. We find high levels of genetic variation with clear and stable continental patterns. Six segregating inversions might be involved in adaptation of local ecotypes. Strong recent signals of selection centred on canonical insecticide resistance genes are shared by multiple populations. A promising gene drive target in An. gambiae is highly conserved in An. funestus. This work represents a significant advance in our understanding of the genetic diversity and population structure of An. funestus and will enable smarter targeted malaria control.

genomics↗

bioRxiv2024

Genome-wide association studies unveil major genetic loci driving insecticide resistance in Anopheles funestus in four eco-geographical settings across Cameroon

Insecticide resistance is jeopardising malaria control efforts in Africa. Deciphering the evolutionary dynamics of mosquito populations country-wide is essential for designing effective and sustainable national and subnational tailored strategies to accelerate malaria elimination efforts. Here, we employed genome-wide association studies through pooled template sequencing to compare four eco-geographically different populations of the major vector, Anopheles funestus, across a South North transect in Cameroon, aiming to identify genomic signatures of adaptive responses to insecticides. Our analysis revealed limited population structure within Northern and Central regions (FST<0.02), suggesting extensive gene flow, while populations from the Littoral/Coastal region exhibited more distinct genetic patterns (FST>0.049). Greater genetic differentiation was observed at known resistance-associated loci, resistance-to-pyrethroids 1 (rp1) (2R chromosome) and CYP9 (X chromosome), with varying signatures of positive selection across populations. Allelic variation between variants underscores the pervasive impact of selection pressures, with rp1 variants more prevalent in Central and Northern populations (FST>0.3), and the CYP9 associated variants more pronounced in the Littoral/Coastal region (FST =0.29). Evidence of selective sweeps was supported by negative Tajimas D and reduced genetic diversity in all populations, particularly in Central (Elende) and Northern (Tibati) regions. Genomic variant analysis identified novel missense mutations and signatures of complex genomic alterations such as duplications, deletions, transposable element (TE) insertions, and chromosomal inversions, all associated with selective sweeps. A 4.3 kb TE insertion was fixed in all populations with Njombe Littoral/Coastal population, showing higher frequency of CYP9K1 (G454A), a known resistance allele and TE upstream compared to elsewhere. Our study uncovered regional variations in insecticide resistance candidate variants, emphasizing the need for a streamlined DNA-based diagnostic assay for genomic surveillance across Africa. These findings will contribute to the development of tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon. Author SummaryDespite the widespread use of vector control tools to combat malaria in Cameroon, the disease burden remains high, particularly affecting children and pregnant women. This persistent burden is linked to intense resistance in malaria vectors, mainly driven by the overexpression of metabolic insecticide resistance genes. The evolutionary response of mosquito populations to both control interventions and agricultural environmental stimuli across Cameroon is not well understood. Understanding these dynamics is crucial for developing effective and sustainable strategies for malaria elimination country-wide. Here, we performed a genome-wide survey of Anopheles funestus across four eco-geographic regions in Cameroon, revealing limited population structure between the northern and southern regions. For the first time in Cameroon, we observed the emergence and widespread of two known resistance-related loci, rp1 and CYP9 loci. Additionally, we identified both known and novel replacement polymorphisms, along with complex signatures of genomic alterations such as large insertions and duplications, linked to selective sweeps. Notably, a 4.3kb structural variant was completely fixed in all regions, while the CYP9K1 resistant allele (A454A) was fixed only in the littoral/coastal region but remained under selection elsewhere highlighting the importance of designing a tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon.

genomics↗

bioRxiv2024

A single mutation G454A in P450 CYP9K1 drives pyrethroid resistance in the major malaria vector Anopheles funestus reducing bed net efficacy

Metabolic resistance to pyrethroids is jeopardizing the effectiveness of insecticide-based interventions against malaria. The complexity of the Africa-wide spatio-temporal evolution of the molecular basis of this resistance, the major genetic drivers should be detected to improve resistance management. Here, we demonstrated that a single amino acid change G454A in the cytochrome P450 CYP9K1 drives pyrethroid resistance in Anopheles funestus vector in East and Central Africa. Polymorphism analysis revealed drastic reduction of diversity of the CYP9K1 gene in Uganda (2014) with the selection of a predominant haplotype (90%), exhibited a G454A mutation. However, 6 years later (2020) the Ugandan 454A-CYP9K1 haplotype was also predominant in Cameroon (84.6%), but absent in Malawi (Southern Africa) and Ghana (West Africa). In vitro comparative heterologous metabolism assays revealed that the mutant-type 454A-CYP9K1 (R) allele metabolises type II pyrethroid (deltamethrin) better than the wild-type G454-CYP9K1 (S) allele. Transgenic Drosophila melanogaster flies expressing the mutant-type 454A-CYP9K1 allele were significantly more resistant to both type I and II pyrethroids than the flies expressing the wild-type G454-CYP9K1 allele. Genotyping with a newly designed DNA-based diagnostic assay targeting the G454A replacement revealed that this mutation is strongly associated with pyrethroid resistance as mosquitoes surviving pyrethroid exposure were significantly more homozygote resistant (Odds ratio = 567, P<0.0001). Furthermore, Cone test and experimental hut trials showed that 454A-CYP9K1 reduces the efficacy of LLINs. The resistant allele (454A) is under directional selection in Eastern and Central Africa, present but not strongly selected in Southern Africa and at very low frequency in West Africa. This study reveals the rapid spread of P450-based metabolic pyrethroid resistance driven by CYP9K1, greatly reducing the efficacy of pyrethroid-based control tools. The new DNA-based assay designed here will add to the toolbox to monitor resistance in the field and improve resistance management strategies. Author SummaryThe complex molecular basis and genetic drivers of metabolic resistance in malaria vectors should be detected to improve resistance management. Here, we established that allelic variation by a single mutation G454A in P450 CYP9K1 enzyme drives pyrethroid resistance in Anopheles funestus. Drastic reduction of diversity was noted in Ugandan female An. funestus samples collected in 2014, with a major haplotype (454A) already fixed but absent in other African regions. However, this Ugandan 454A-CYP9K1 haplotype was highly selected within 6 years in An. funestus samples from Cameroon (Central Africa), but still absent in Ghana (West Africa) and Malawi (Southern Africa). Metabolism assays revealed that the 454A-resistant allele metabolized pyrethroid better than the susceptible G454 allele and driving higher pyrethroid resistance in transgenic Drosophila melanogaster flies. DNA-based diagnostics designed around the G454A-CYP9K1 marker strongly correlates with pyrethroid resistance, reducing bed net efficacy indicating that this assay should be added to the toolbox to monitor this 454A-CYP9K1 resistance which is rapidly spreading in An. funestus mosquito populations from Eastern and Central Africa.

genetics↗

bioRxiv2024

Two highly selected mutations in the tandemly duplicated CYP6P4a and CYP6P4b drive pyrethroid resistance in Anopheles funestus

Gaining a comprehensive understanding of the genetic mechanisms underlying insecticide resistance in malaria vectors is crucial for optimising the effectiveness of insecticide-based vector control methods and developing diagnostic tools for resistance management. Considering the heterogeneity of metabolic resistance in major malaria vectors, the implementation of tailored resistance management strategies is essential for successful vector control. In this study, we provide evidence demonstrating that two highly selected mutations in the tandemly duplicated cytochrome P450 genes namely CYP6P4a and CYP6P4b, are driving pyrethroid insecticide resistance in the major malaria vector Anopheles funestus, in West Africa. Through a continent-wide polymorphism survey, we observed heightened indications of directional selection in both genes between 2014 and 2021. By conducting in vitro insecticide metabolism assays with recombinant enzymes expressed from both genes, we established that mutant alleles under selection exhibit higher metabolic efficiency compared to their wild-type counterparts. Furthermore, using the GAL4-UAS transgenic system, we demonstrated that transgenic Drosophila melanogaster flies overexpressing mutant alleles displayed an increased resistance to pyrethroids. These findings were in agreement with in silico characterisation, which highlighted changes in enzyme active site architecture that enhance the affinity of mutant alleles for type I and II pyrethroids. Furthermore, we developed two DNA-based assays capable of detecting the CYP6P4a-M220I and CYP6P4b-D284E mutations, showing their current confinement to West Africa. Genotype/phenotype correlation analyses revealed that these markers are strongly associated with resistance to types I and II pyrethroids and combine to drastically reduce the efficacy of pyrethroid bednets. Overall, our study makes available two field-applicable insecticide resistance molecular markers that will help in the monitoring and better management of insecticide resistance in West Africa. TeaserTwo field-applicable diagnostic tools for detecting metabolic resistance in Anopheles funestus to enhance insecticide resistance management in West Africa.

genetics↗

bioRxiv2024

Contrasting patterns of Asaia sp association with Plasmodium falciparum between field collected Anopheles gambiae and Anopheles coluzzii from Cameroon

The presence of bacteria of the genus Asaia in mosquitoes makes them suitable candidates for malaria paratrangenic control. To better understand whether this bacterium could be used for malaria control, we investigated and quantified Asaia influence in An.Lgambiae sl populations naturally infected by the malaria parasite Plasmodium falciparum. Adult Anopheles mosquitoes were collected across two different eco-geographical localities in Cameroon, during the dry and wet seasons. DNA was extracted from the whole individual mosquitoes and Asaia was quantified using real-time Q-PCR by amplification of 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in same mosquitoes. The density of Asaia was quantified in 864 mosquitoes. This included 439 An. gambiae collected from Bankeng and 425 An. coluzzii collected from Gounougou. The prevalence of Asaia in An. gambiae and An. coluzzii was 88.3% and 80.9% respectively, with An. gambiae were more heavily infected by Asaia than An. coluzzii. There was also a significant difference between the infection densities of Asaia per season in the two localities. We also had a significant difference between the infection densities of Asaia according to Plasmodium infection status in the two localities. However, no correlation was observed between the amount of Asaia and P. falciparum infection. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii. Further studies as experimental infection and innate immune response are required to better investigate the correlation between Anopheles mosquitoes, Asaia and Plasmodium. ImportanceMalaria control relies mainly on insecticide-based tools. However, the effectiveness of these tools is threatened by the widespread insecticide resistance in malaria vectors highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated and quantified Asaia influence in An.Lgambiae sl populations naturally infected by the malaria parasite Plasmodium falciparum. DNA was extracted from the whole individual mosquitoes collected from two localities, and then Asaia was quantified using real-time Q-PCR by amplification of 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in same mosquitoes and correlated Plasmodium infection them with the presence of Asaia infection. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii.

microbiology↗

bioRxiv2024

A single E205D allele of a key P450 CYP6P3 is driving metabolic pyrethroid resistance in the major African malaria vector Anopheles gambiae

Deciphering the molecular drivers of insecticide resistance is paramount to extend the effectiveness of malaria vector control tools. Here, we demonstrated that the E205D amino acid change in a key metabolic resistance P450 CYP6P3 drives pyrethroid resistance in the major malaria vector, Anopheles gambiae. Spatio-temporal whole genome Poolseq analyses in Cameroon detected a major P450-linked locus on chromosome 2R beside the sodium channel locus. In vitro metabolism assays with recombinantly expressed CYP6P3 protein revealed that the catalytic efficiency of 205D was 2.5 times higher than E205 with -cypermethrin. Similar patterns were observed for permethrin. Overexpression of the 205D allele in transgenic flies confers higher more pyrethroids and carbamates resistance, compared to controls. A DNA-based assay further supported that the CYP6P3-205D variant strongly correlates with pyrethroid resistance in field populations (OR=26.4; P<0.0001) and that it reduces the efficacy of pyrethroid-only LLINs with homozygote RR genotype exhibiting significantly higher survival following PermaNet 3.0 exposure compared to the SS genotype (OR: 6.1, p = 0.0113). Furthermore, the CYP6P3-E205D combines with the kdr target-site resistance mechanisms to worsen the loss of bednet efficacy. The 205D mutation is now predominant in West and Central Africa but less abundant or absent in East and South Africa with signs of introgression with An. coluzzii in Ghana. This study highlights the importance of P450-based resistance and designs field-applicable tools to easily track the spread of metabolic resistance and assess its impact on control interventions. One Sentence Summary: The major obstacle to malaria control and elimination is the spread of parasite resistance to anti-malarial drugs, and mosquito resistance to insecticides. In this study, we identified a key point mutation E205D in the metabolic gene CYP6P3 (cytochrome P450) conferring resistance to pyrethroids by enhancing the breakdown of insecticides used for bednets impregnation. DNA-based assays were then designed and used to determine the spread of the resistance across Africa and demonstrate that the CYP6P3-205D allele works together with the knockdown resistance in the voltage-gated Sodium channel to reduce the efficacy of insecticide-treated bednets.

genomics↗

bioRxiv2022

Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector

Information on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon. Genome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases, and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies - including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (fixed). High frequencies of the epidemiologically important chromosomal inversions, 2La, 2Rb and 2Rc were observed ([~]80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2 were functionally validated. Transgenic Drosophila melanogaster expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities < 10% in 24 h). Serial deletion of the 5 intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Fork-head box L1 and c-EST putative binding sites were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, -cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which if true make it an ideal insecticide against populations of An. coluzzii overexpressing this P450. These findings will facilitate regional collaborations within the Sahel region, and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policy towards local and regional malaria pre-elimination.

genomics↗

bioRxiv2022

Xeno-monitoring of molecular drivers of artemisinin and partner drug resistance in P. falciparum populations in malaria vectors across Cameroon

BackgroundMonitoring of drug resistance in Plasmodium populations is crucial for malaria control. This has primarily been performed in humans and rarely in mosquitoes where parasites genetic recombination occurs. Here, we characterized the Plasmodium spp populations in wild Anopheles vectors by analyzing the genetic diversity of the P. falciparum kelch13 and mdr1 gene fragments implicated in artemisinin and partner drug resistance across Cameroon in three major malaria vectors. MethodsAnopheles mosquitoes were collected across nine localities in Cameroon and dissected into the head/thorax (H/T) and abdomen (Abd) after species identification. A TaqMan assay was performed to detect Plasmodium infection. Fragments of the Kelch 13 and mdr1 genes were amplified in P. falciparum positive samples and directly sequenced to assess their drug resistance polymorphisms and genetic diversity profile. ResultsThe study revealed a high Plasmodium infection rate in the major Anopheles vectors across Cameroon. Notably, An. funestus vector recorded the highest sporozoite (8.02%) and oocyst (14.41%) infection rates. A high P. falciparum sporozoite rate (80.08%) alongside epidemiological signatures of significant P. malariae (15.94%) circulation were recorded in these vectors. Low genetic diversity with six (A578S, R575I, G450R, L663L, G453D, N458D) and eight (H53H, V62L, V77E, N86Y, G102G, L132I, H143H, Y184F) point mutations were observed in the k13 and mdr1 backbones respectively. Remarkably, the R575I (4.44%) k13 and Y184F (64.2%) mdr1 mutations were the predominant variants in the P. falciparum populations. ConclusionThe emerging signal of the R575I polymorphism in the Pfk13 propeller backbone entails the regular surveillance of molecular markers to inform evidence-based policy decisions. Moreover, the high frequency of the 86N184F allele highlights concerns on the plausible decline in efficacy of artemisinin-combination therapies (ACTs); further implying that parasite genotyping from mosquitoes can provide a more relevant scale for quantifying resistance epidemiology in the field.

genomics↗

bioRxiv2021

Gene conversion explains elevated diversity in the immunity modulating APL1 gene of the malaria vector Anopheles funestus

The leucine rich repeat gene APL1 is a key component of immunity to Plasmodium and other microbial pathogens in Anopheles mosquitoes. In the malaria vector Anopheles funestus the APL1 gene has four paralogues which occur along the same chromosome arm. We show that APL1 has exceptional levels of non-synonymous polymorphism across the range of An. funestus with an average {pi}n of 0.027 versus a genome-wide average of 0.002, and {pi}n (and {pi}s) is consistently high in populations across Africa. The pattern of APL1 diversity was consistent between independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide-resistance was observed with the phenotyped target-enrichment dataset. Two further innate immunity genes of the gambicin anti-microbial peptide family had {pi}n/{pi}s ratios greater than one, possibly driven by either positive or balancing selection. Cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, an observation discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogs, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. Gene conversion at APL1 versus alternative explanations is concordant with similarly elevated diversity in APL1 and TEP1 loci in An. gambiae. In contrast, the more closely related An. stephensi which also encodes a single-copy of APL1 does not show this elevated diversity. We hypothesise that a more open chromatin formation at the APL1 locus due to higher gene expression than its paralogues enhances gene conversion, and therefore increased polymorphism, at APL1.

evolutionary biology↗

bioRxiv2021

Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa

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 deep sequencing of candidate resistance genes and genomic loci detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNP was 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 CYP9K1 P450 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 a drastic reduction of genetic diversity in Uganda, in contrast to other locations, highlighting an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid mutation located between the meander and cysteine pocket of CYP9K1 was detected in all Ugandan mosquitoes. This study sheds further light on the complex evolution of metabolic resistance in a major malaria vector, by adding further resistance genes and variants that can be used to design field applicable markers to better track this resistance Africa-wide. Author SummaryMetabolic 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 several DNA based approach to associate genomic differences between resistant and susceptible mosquitoes from several field and laboratory populations of the malaria vector Anopheles funestus. We followed-up our genomic analyses with functional validation of a candidate resistance gene in East Africa. This gene (CYP9K1) is a member of the cytochrome P450 gene-family that helps to metabolise, and thereby detoxify, pyrethroid insecticides. We show that this gene is a major driver of resistance to a specific sub-class of pyrethroid insecticides only, with moderate to no effects on other insecticides used against Anopheles funestus. We were able to link resistance in this gene to a mutation that changes the amino acid glycine to alanine that may impact how the protein-product of this gene binds to target insecticides. In addition to demonstrating the biochemical specificity of an evolutionary response, we have broadened the available pool of genes can be used to monitor the spread of insecticide resistance in this species.

genomics↗