Biology subjects

Tchouakui, M.

Publications and source records attributed to Tchouakui, M..

7 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↗

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

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

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↗

bioRxiv2020

Influence of GST- and P450-based metabolic resistance to pyrethroids on blood feeding in the major African malaria vector Anopheles funestus

Insecticide resistance genes are often associated with pleiotropic effects on various mosquito life-history traits. However, very little information is available on the impact of insecticide resistance, especially metabolic resistance, on blood feeding process in mosquitoes. Here, using two recently detected DNA-based metabolic markers in the major malaria vector, An. funestus, we investigated how metabolic resistance genes could affect blood meal intake. After allowing both field F1 and lab F8 Anopheles funestus strains to feed on human arm for 30 minutes, we assessed the association between key parameters of blood meal process including, probing time, feeding duration, blood feeding success and blood meal size, and markers of glutathione S-transferase (L119F-GSTe2) and cytochrome P450 (CYP6P9a_R) - mediated metabolic resistance. None of the parameters of blood meal process was associated with L119F-GSTe2 genotypes. In contrast, for CYP6P9a_R, homozygote resistant mosquitoes were significantly more able to blood-feed than homozygote susceptible (OR = 3.3; CI 95%: 1.4-7.7; P =0.01) mosquitoes. Moreover, the volume of blood meal ingested by CYP6P9a-SS mosquitoes was lower than that of CYP6P9a-RS (P<0.004) and of CYP6P9a-RR (P<0.006). This suggests that CYP6P9a gene affects the feeding success and blood meal size of An. funestus. However, no correlation was found in the expression of CYP6P9a and that of genes encoding for salivary proteins involved in blood meal process. This study suggests that P450-based metabolic resistance may increase the blood feeding ability of malaria vectors and potential impacting their vectorial capacity.

molecular biology↗