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Novel insecticides were recently introduced to counter pyrethroid resistance threats in African malaria vectors. To prolong their effectiveness, potential cross-resistance from promiscuous pyrethroid metabolic resistance mechanisms must be elucidated. Here, we demonstrate that the duplicated P450s CYP6P9a/-b, proficient pyrethroid metabolizers, reduce neonicotinoid efficacy in Anopheles funestus while enhancing the potency of chlorfenapyr. Transgenic expression of CYP6P9a/-b in Drosophila confirmed that flies expressing both genes were significantly more resistant to neonicotinoids than controls, whereas the contrasting pattern was observed for chlorfenapyr. This result was also confirmed by RNAi knockdown experiments. In vitro expression of recombinant CYP6P9a and metabolism assays established that it significantly depletes both clothianidin and chlorfenapyr, with metabolism of chlorfenapyr producing the insecticidally active intermediate metabolite tralopyril. This study highlights the risk of cross-resistance between pyrethroid and neonicotinoid and reveals that chlorfenapyr-based control interventions such as Interceptor G2 could remain efficient against some P450-based resistant mosquitoes., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Growing resistance is reported to carbamate insecticides in malaria vectors in Cameroon. However, the contribution of acetylcholinesterase ( Ace-1 ) to this resistance remains uncharacterised. Here, we established that the G119S mutation is driving resistance to carbamates in Anopheles gambiae populations from Cameroon. Insecticide bioassay on field-collected mosquitoes from Bankeng, a locality in southern Cameroon, showed high resistance to the carbamates bendiocarb (64.8% ± 3.5% mortality) and propoxur (55.71% ± 2.9%) but a full susceptibility to the organophosphate fenitrothion. The TaqMan genotyping of the G119S mutation in field-collected adults revealed the presence of this resistance allele (39%). A significant correlation was observed between the Ace-1 R and carbamate resistance at allelic ((bendiocarb; odds ratio (OR) = 75.9; p < 0.0001) and (propoxur; OR = 1514; p < 0.0001)) and genotypic (homozygote resistant vs. homozygote susceptible (bendiocarb; OR = 120.8; p < 0.0001) and (propoxur; OR = 3277; p < 0.0001)) levels. Furthermore, the presence of the mutation was confirmed by sequencing an Ace-1 portion flanking codon 119. The cloning of this fragment revealed a likely duplication of Ace-1 in Cameroon as mosquitoes exhibited at least three distinct haplotypes. Phylogenetic analyses showed that the predominant Ace-1 R allele is identical to that from West Africa suggesting a recent introduction of this allele in Central Africa from the West. The spread of this Ace-1 R represents a serious challenge to future implementation of indoor residual spraying (IRS)-based interventions using carbamates or organophosphates in Cameroon., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

D7 family proteins are among the most expressed salivary proteins in mosquitoes. They facilitate blood meal intake of the mosquito by scavenging host amines that induce vasoconstriction, platelet aggregation and pain. Despite this important role, little information is available on the impact of insecticide resistance on the regulation of D7 proteins and consequently on the blood feeding success. In this study, real-time quantitative polymerase chain reaction (qPCR) analyses were performed to investigate how pyrethroid resistance could influence the expression of genes encoding D7 family proteins in Anopheles gambiae and Anopheles funestus s.s. mosquitoes from Elon in the Central Cameroon. Out of 328 collected mosquitoes, 256 were identified as An. funestus sl and 64 as An. gambiae sl . Within the An. funestus group, An. funestus s.s . was the most abundant species (95.95%) with An. rivulorum, An. parensis and An. rivulorum -like also detected. All An. gambiae s.l mosquitoes were identified as An. gambiae. High levels of pyrethroid resistance were observed in both An. gambiae and An. funestus mosquitoes. RT-qPCR analyses revealed a significant overexpression of two genes encoding D7 proteins, D7r3 and D7r4 , in pyrethroids resistant An. funestus . However, no association was observed between the polymorphism of these genes and their overexpression. In contrast, overall D7 salivary genes were under-expressed in pyrethroid resistant An. gambiae . This study provides preliminary evidences that pyrethroid resistance could influence blood meal intake through over-expression of D7 proteins although future studies will help establishing potential impact on vectorial capacity., Competing Interests: The authors declare no conflict of interest. The sponsors of this study had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of this manuscript and in the decision to publish it.

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. [ABSTRACT FROM AUTHOR]

Background: Insecticide resistance threatens the effectiveness of malaria vector control, calling for an urgent need to design suitable resistance management strategies. Here, we established the resistance profiling of an Ugandan Anopheles gambiae population to insecticides using WHO procedures and assessed the potential restoration of susceptibility in the hybrid line Mayuge/KISUMU in an insecticide-free environment for eighteen (18) generations. Results: This An gambiae population exhibited a very high intensity of resistance to permethrin, deltamethrin, and alphacypermethrin with a consistent loss of efficacy of all long-lasting insecticidal nets (LLINs) tested including PBO-based and new generation nets Interceptor G2 (IG2) and Royal guard. Molecular analysis revealed a fixation of the L1014S-kdr mutation together with the overexpression of some P450 metabolic genes (CYP6Z1, CYP9K1, CYP6P1, 3 & 4) besides the cuticular resistance-related genes (CYP4G16) and sensorial appendage proteins (SAP1, SAP2, and SAP3) but no GSTe2 overexpression. In the absence of selection pressure, the mortality rate after exposure to insecticides increased significantly over generations, and restoration of susceptibility was observed for most of the insecticides in less than 10 generations. Accordingly, a significant reduction in the frequency of KdrE was observed after 13 generations coupled with reduced expression of most metabolic resistance genes. Conclusions: The results of this study show that the high intensity of pyrethroid resistance observed in An gambiae from Uganda associated with the loss of efficacy of LLINs could compromise vector control efforts. The study also highlights that an early rotation of insecticides could help manage resistance to insecticides by restoring the susceptibility. However, the persistence of Kdr mutation together with overexpression of some metabolic genes after many generations in the absence of selection pressure indicates the potential implication of modifiers alleviating the cost of resistance which needs to be further investigated. [ABSTRACT FROM AUTHOR]

Background: Cameroon is considering the implementation of indoor residual spraying (IRS) as a complementary measure to control malaria in the context of high pyrethroid resistance in major malaria vectors. Non-pyrethroid insecticide classes such as organophosphates and carbamates may be utilized in IRS due to widespread pyrethroid resistance. However, the success of this strategy depends on good knowledge of the resistance status of malaria vectors to carbamates and organophosphates. Here, we assessed the susceptibility profile of Anopheles gambiae sensu lato with respect to carbamates and organophosphate and the distribution of the molecular mechanism underlying resistance to these insecticides. Methods: Anopheles gambiae s.l. mosquitoes were collected from nine settings across the country and bio-assayed with bendiocarb, propoxur and pirimiphos-methyl. The Ace-1 target-site G119S mutation was genotyped using a TaqMan assay. To investigate the polymorphism in the Ace-1 gene, a region of 924 base pairs in a sequence of the gene was amplified from both live and dead females of An. gambiae exposed to bendiocarb. Results: Pirimiphos-methyl induced full mortality in An. gambiae s.l. from all study sites, whereas for carbamates, resistance was observed in four localities, with the lowest mortality rate recorded in Mangoum (17.78 ± 5.02% for bendiocarb and 18.61 ± 3.86% for propoxur) in the southern part of Cameroon. Anopheles coluzzii was found to be the predominant species in the northern tropical part of the country where it is sympatric with Anopheles arabiensis. In the localities situated in southern equatorial regions, this species was predominant in urban settings, while An. gambiae was the most abundant species in rural areas. The G119S Ace-1 target-site mutation was detected only in An. gambiae and only in the sites located in southern Cameroon. Phylogenetic analyses showed a clustering according to the phenotype. Conclusion: The occurrence of the Ace-1 target-site substitution G119S in An. gambiae s.l. populations highlights the challenge associated with the impending deployment of IRS in Cameroon using carbamates or organophosphates. It is therefore important to think about a resistance management plan including the use of other insecticide classes such as neonicotinoids or pyrrole to guarantee the implementation of IRS in Cameroon. [ABSTRACT FROM AUTHOR]