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

The Nigerian Government is scaling up the distribution of insecticide-treated bed nets for malaria control, but the lack of surveillance data, especially in the Sudan/Sahel region of the country, may hinder targeting priority populations. Here, the vectorial role and insecticide resistance profile of a population of a major malaria vector Anopheles funestus sensu stricto from Sahel of Nigeria was characterised. An. funestus s.s. was the only vector found, with a high human blood index (100%) and a biting rate of 5.3/person/night. High Plasmodium falciparum infection was discovered (sporozoite rate = 54.55%). The population is resistant to permethrin (mortality = 48.30%, LT50 = 65.76 min), deltamethrin, DDT (dichlorodiphenyltrichloroethane) and bendiocarb, with mortalities of 29.44%, 56.34% and 54.05%, respectively. Cone-bioassays established loss of efficacy of the pyrethroid-only long-lasting insecticidal nets (LLINs); but 100% recovery of susceptibility was obtained for piperonylbutoxide (PBO)-containing PermaNet®3.0. Synergist bioassays with PBO and diethyl maleate recovered susceptibility, implicating CYP450s (permethrin mortality = 78.73%, χ2 = 22.33, P < 0.0001) and GSTs (DDT mortality = 81.44%, χ2 = 19.12, P < 0.0001). A high frequency of 119F GSTe2 mutation (0.84) was observed (OR = 16, χ2 = 3.40, P = 0.05), suggesting the preeminent role of metabolic resistance. These findings highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in Nigeria.

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

10.1101/2020.03.16.993535 ◽

2020 ◽

Author(s):

Lynda Nouage ◽

Emmanuel Elanga-Ndille ◽

Achille Binyang ◽

Magellan Tchouakui ◽

Tatiane Atsatse ◽

...

Keyword(s):

Insecticide Resistance ◽

Malaria Vector ◽

Resistance Genes ◽

Blood Meal ◽

Anopheles Funestus ◽

Blood Feeding ◽

Meal Size ◽

Metabolic Resistance ◽

Feeding Success ◽

Blood Meal Size

AbstractInsecticide 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.

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Evidence of a multiple insecticide resistance in the malaria vector Anopheles funestus in South West Nigeria

Malaria Journal ◽

10.1186/s12936-016-1615-9 ◽

2016 ◽

Vol 15 (1) ◽

Cited By ~ 31

Author(s):

Rousseau J. Djouaka ◽

Seun M. Atoyebi ◽

Genevieve M. Tchigossou ◽

Jacob M. Riveron ◽

Helen Irving ◽

...

Keyword(s):

Insecticide Resistance ◽

Malaria Vector ◽

Anopheles Funestus ◽

South West

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Transcriptomic analysis of insecticide resistance in the lymphatic filariasis vectorCulex quinquefasciatus

10.1101/589028 ◽

2019 ◽

Author(s):

Walter Fabricio Silva Martins ◽

Craig Stephen Wilding ◽

Alison Taylor Isaacs ◽

Emily Joy Rippon ◽

Karine Megy ◽

...

Keyword(s):

Insecticide Resistance ◽

Lymphatic Filariasis ◽

Fold Increase ◽

Target Site ◽

Detoxification Enzymes ◽

Site Mutation ◽

Public Health Importance ◽

Metabolic Resistance ◽

Vector Borne Diseases ◽

Bendiocarb Resistance

ABSTRACTCulex quinquefasciatusplays an important role in transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF), as well as many arboviral diseases. Currently, efforts to tackleC. quinquefasciatusvectored diseases are based on either mass drug administration (MDA) for LF, or insecticide-based interventions. Widespread and intensive insecticide usage has resulted in increased resistance in mosquito vectors, includingC. quinquefasciatus. Herein, the transcriptome profile of Ugandan bendiocarb-resistantC. quinquefasciatuswas explored to identify candidate genes associated with insecticide resistance. Resistance to bendiocarb in exposed mosquitoes was marked, with 2.04% mortality following 1h exposure and 58.02% after 4h. Genotyping of the G119SAce-1target site mutation detected a highly significant association (p<0.0001; OR=25) between resistance andAce1-119S. However, synergist assays using the P450 inhibitor PBO or the esterase inhibitor TPP resulted in markedly increased mortality (to ≈80%), suggesting a role of metabolic resistance in the resistance phenotype. Using a novel, custom 60K whole-transcriptome microarray 16 genes significantly overexpressed in resistant mosquitoes were detected, with the P450Cyp6z18showing the highest differential gene expression (>8-fold increase vs unexposed controls). These results provide evidence that bendiocarb-resistance in UgandanC. quinquefasciatusis mediated by both target-site mechanisms and over-expression of detoxification enzymes.

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A differential expression of pyrethroid resistance genes in the malaria vector Anopheles funestus across Uganda is associated with patterns of gene flow

PLoS ONE ◽

10.1371/journal.pone.0240743 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0240743

Author(s):

Maurice Marcel Sandeu ◽

Charles Mulamba ◽

Gareth D. Weedall ◽

Charles S. Wondji

Keyword(s):

Population Structure ◽

Gene Flow ◽

Genetic Structure ◽

Genetic Differentiation ◽

Insecticide Resistance ◽

Pyrethroid Resistance ◽

Anopheles Funestus ◽

Metabolic Resistance ◽

Transcription Analysis ◽

Genome Wide

Background Insecticide resistance is challenging the effectiveness of insecticide-based control interventions to reduce malaria burden in Africa. Understanding the molecular basis of insecticides resistance and patterns of gene flow in major malaria vectors such as Anopheles funestus are important steps for designing effective resistance management strategies. Here, we investigated the association between patterns of genetic structure and expression profiles of genes involved in the pyrethroid resistance in An. funestus across Uganda and neighboring Kenya. Methods Blood-fed mosquitoes An. funestus were collected across the four localities in Uganda and neighboring Kenya. A Microarray-based genome-wide transcription analysis was performed to identify the set of genes associated with permethrin resistance. 17 microsatellites markers were genotyped and used to establish patterns of genetic differentiation. Results Microarray-based genome-wide transcription profiling of pyrethroid resistance in four locations across Uganda (Arua, Bulambuli, Lira, and Tororo) and Kenya (Kisumu) revealed that resistance was mainly driven by metabolic resistance. The most commonly up-regulated genes in pyrethroid resistance mosquitoes include cytochrome P450s (CYP9K1, CYP6M7, CYP4H18, CYP4H17, CYP4C36). However, expression levels of key genes vary geographically such as the P450 CYP6M7 [Fold-change (FC) = 115.8 (Arua) vs 24.05 (Tororo) and 16.9 (Kisumu)]. In addition, several genes from other families were also over-expressed including Glutathione S-transferases (GSTs), carboxylesterases, trypsin, glycogenin, and nucleotide binding protein which probably contribute to insecticide resistance across Uganda and Kenya. Genotyping of 17 microsatellite loci in the five locations provided evidence that a geographical shift in the resistance mechanisms could be associated with patterns of population structure throughout East Africa. Genetic and population structure analyses indicated significant genetic differentiation between Arua and other localities (FST>0.03) and revealed a barrier to gene flow between Arua and other areas, possibly associated with Rift Valley. Conclusion The correlation between patterns of genetic structure and variation in gene expression could be used to inform future interventions especially as new insecticides are gradually introduced.

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Cytochrome P450 metabolic resistance (CYP6P9a) to pyrethroids imposes a fitness cost in the major African malaria vector Anopheles funestus

Heredity ◽

10.1038/s41437-020-0304-1 ◽

2020 ◽

Vol 124 (5) ◽

pp. 621-632 ◽

Cited By ~ 3

Author(s):

Magellan Tchouakui ◽

Jacob Riveron Miranda ◽

Leon M. J. Mugenzi ◽

Doumani Djonabaye ◽

Murielle J. Wondji ◽

...

Keyword(s):

Malaria Vector ◽

Management Strategy ◽

Management Strategies ◽

Resistance Management ◽

Anopheles Funestus ◽

Fitness Cost ◽

Malaria Vectors ◽

Hybrid Strain ◽

Metabolic Resistance ◽

The Impact

Abstract Metabolic resistance threatens the sustainability of pyrethroid-based malaria control interventions. Elucidating the fitness cost and potential reversal of metabolic resistance is crucial to design suitable resistance management strategies. Here, we deciphered the fitness cost associated with the CYP6P9a (P450-mediated metabolic resistance) in the major African malaria vector Anopheles funestus. Reciprocal crosses were performed between a pyrethroid susceptible (FANG) and resistant (FUMOZ-R) laboratory strains and the hybrid strains showed intermediate resistance. Genotyping the CYP6P9a-R resistance allele in oviposited females revealed that CYP6P9a negatively impacts the fecundity as homozygote susceptible mosquitoes (CYP6P9a-SS) lay more eggs than heterozygote (OR = 2.04: P = 0.01) and homozygote resistant mosquitoes. CYP6P9a also imposes a significant fitness cost on the larval development as homozygote resistant larvae (CYP6P9a-RR) developed significantly slower than heterozygote and homozygote susceptible mosquitoes (χ2 = 11.2; P = 0.0008). This fitness cost was further supported by the late pupation of homozygote resistant than susceptible mosquitoes (OR = 2.50; P < 0.01). However, CYP6P9a does not impact the longevity as no difference was observed in the life span of mosquitoes with different genotypes (χ2 = 1.6; P = 0.9). In this hybrid strain, a significant decrease of the resistant CYP6P9a-RR genotype was observed after ten generations (χ2 = 6.6; P = 0.01) suggesting a reversal of P450-based resistance in the absence of selection. This study shows that the P450-mediated metabolic resistance imposes a high fitness cost in malaria vectors supporting that a resistance management strategy based on rotation could help mitigate the impact of such resistance.

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