Background: Aggravation of insecticide resistance in malaria vectors is threatening the efforts to control malaria by reducing the efficacy of insecticide-based interventions hence needs to be closely monitored. This study investigated the intensity of insecticide resistance of two major malaria vectors An. funestus sensu stricto (s.s.) and An. gambiae sensu lato (s.l.) collected in southern Ghana and assessed the bio-efficacy of several long-lasting insecticidal nets (LLINs) against these mosquito populations. Methods: The insecticide susceptibility profiles of Anopheles funestus s.s. and Anopheles gambiae s.l. populations from Obuasi region (Atatam), southern Ghana were characterized and the bio-efficacy of some LLINs was assessed to determine the impact of insecticide resistance on the effectiveness of these tools. Furthermore, molecular markers associated with insecticide resistance in both species were characterized in the F₀ and F₁ populations using PCR and qPCR methods. Results: Anopheles funestus s.s. was the predominant species and was resistant to pyrethroids, organochlorine and carbamate insecticides, but fully susceptible to organophosphates. An. gambiae s.l. was resistant to all four insecticide classes. High intensity of resistance to 5 × and 10 × the discriminating concentration (DC) of pyrethroids was observed in both species inducing a considerable loss of efficacy of long-lasting insecticidal nets (LLINs). Temporal expression analysis revealed a massive 12-fold increase in expression of the CYP6P4a cytochrome P450 gene in An. funestus s.s., initially from a fold change of 41 (2014) to 500 (2021). For both species, the expression of candidate genes did not vary according to discriminating doses. An. gambiae s.l. exhibited high frequencies of target-site resistance including Vgsc-1014F (90%) and Ace-1 (50%) while these mutations were absent in An. funestus s.s. Conclusions: The multiple and high intensity of resistance observed in both malaria vectors highlights the need to implement resistance management strategies and the introduction of new insecticide chemistries.

Monitoring cases of insecticide resistance aggravation and the effect on the efficacy of control tools is crucial for successful malaria control. In this study, the resistance intensity of major malaria vectors from Uganda was characterised and its impact on the performance of various insecticide-treated nets elucidated. High intensity of resistance to the discriminating concentration (DC), 5× DC, and 10× DC of pyrethroids was observed in both Anopheles funestus and Anopheles gambiae in Mayuge and Busia leading to significant reduced performance of long-lasting insecticidal nets (LLINs) including the piperonyl butoxide (PBO)-based nets (Olyset Plus). Molecular analysis revealed significant over-expression of cytochrome P450 genes (CYP9K1 and CYP6P9a/b). However, the expression of these genes was not associated with resistance escalation as no difference was observed in the level of expression in mosquitoes resistant to 5× DC and 10× DC compared to 1× DC suggesting that other resistance mechanisms are involved. Such high intensity of pyrethroid resistance in Uganda could have terrible consequences on the effectiveness of insecticide-based interventions and urgent action should be taken to prevent the spread of super-resistance in malaria vectors.

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 sequencing of candidate resistance genes detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNPs were 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 SNPs in the P450 CYP9K1 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 reduced genetic diversity in Uganda underlying an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid change in CYP9K1 was fixed in Ugandan mosquitoes but not in other An. funestus populations. This study sheds further light on the evolution of metabolic resistance in a major malaria vector by implicating more genes and variants that can be used to design field‐applicable markers to better track resistance Africa‐wide.

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide‐based interventions. Here, we deciphered the role of a 6.5‐kb structural variation (SV) in driving cytochrome P450‐mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole‐genome pooled sequencing detected an intergenic 6.5‐kb SV between duplicated CYP6P9a/b P450s in pyrethroid‐resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5‐fold higher activity (p < .0001) for the SV− carrying fragment than the SV− free one. Quantitative real‐time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV− (1.7‐ to 2‐fold) and homozygotes SV−/SV− (4‐to 5‐fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV−/SV−; odds ratio [OR] = 2,079.4, p < .001). The 6.5‐kb SV is present at high frequency in southern Africa (80%–100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid‐treated nets (OR 27.7; p < .0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote‐resistant at the three loci (SV+/CYP6P9a_R/CYP6P9b_R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid‐based nets.

Cross-resistance to insecticides in multiple resistant malaria vectors is hampering resistance management. Understanding its underlying molecular basis is critical to implementation of suitable insecticide-based interventions. Here, we established that the tandemly duplicated cytochrome P450s, CYP6P9a/b are driving carbamate and pyrethroid cross-resistance in Southern African populations of the major malaria vector Anopheles funestus. Transcriptome sequencing revealed that cytochrome P450s are the most over-expressed genes in bendiocarb and permethrin-resistant An. funestus. The CYP6P9a and CYP6P9b genes are overexpressed in resistant An. funestus from Southern Africa (Malawi) versus susceptible An. funestus (Fold change (FC) is 53.4 and 17 respectively), while the CYP6P4a and CYP6P4b genes are overexpressed in resistant An. funestus in Ghana, West Africa, (FC is 41.1 and 17.2 respectively). Other up-regulated genes in resistant An. funestus include several additional cytochrome P450s (e.g. CYP9J5, CYP6P2, CYP6P5), glutathione-S transferases, ATP-binding cassette transporters, digestive enzymes, microRNA and transcription factors (FC<7). Targeted enrichment sequencing strongly linked a known major pyrethroid resistance locus (rp1) to carbamate resistance centering around CYP6P9a/b. In bendiocarb resistant An. funestus, this locus exhibits a reduced nucleotide diversity, significant p-values when comparing allele frequencies, and the most non-synonymous substitutions. Recombinant enzyme metabolism assays showed that both CYP6P9a/b metabolize carbamates. Transgenic expression of CYP6P9a/b in Drosophila melanogaster revealed that flies expressing both genes were significantly more resistant to carbamates than controls. Furthermore, a strong correlation was observed between carbamate resistance and CYP6P9a genotypes with homozygote resistant An. funestus (CYP6P9a and the 6.5kb enhancer structural variant) exhibiting a greater ability to withstand bendiocarb/propoxur exposure than homozygote CYP6P9a_susceptible (e.g Odds ratio = 20.8, P<0.0001 for bendiocarb) and heterozygotes (OR = 9.7, P<0.0001). Double homozygote resistant genotype (RR/RR) were even more able to survive than any other genotype combination showing an additive effect. This study highlights the risk that pyrethroid resistance escalation poses to the efficacy of other classes of insecticides. Available metabolic resistance DNA-based diagnostic assays should be used by control programs to monitor cross-resistance between insecticides before implementing new interventions.

(1) Background: Malaria remains a global public health problem. Unfortunately, the resistance of malaria vectors to commonly used insecticides threatens disease control and elimination efforts. Field mosquitoes have been shown to survive upon exposure to high insecticide concentrations. The molecular mechanisms driving this pronounced resistance remain poorly understood. Here, we elucidated the pattern of resistance escalation in the main malaria vector Anopheles gambiae in a pesticide-driven agricultural hotspot in Cameroon and its impact on vector control tools; (2) Methods: Larval stages and indoor blood-fed female mosquitoes (F₀) were collected in Mangoum in May and November and forced to lay eggs; the emerged mosquitoes were used for WHO tube, synergist and cone tests. Molecular identification was performed using SINE PCR, whereas TaqMan-based PCR was used for genotyping of L1014F/S and N1575Y kdr and the G119S-ACE1 resistance markers. The transcription profile of candidate resistance genes was performed using qRT-PCR methods. Characterization of the breeding water and soil from Mangoum was achieved using the HPLC technique; (3) Results: An. gambiae s.s. was the only species in Mangoum with 4.10% infection with Plasmodium. These mosquitoes were resistant to all the four classes of insecticides with mortality rates <7% for pyrethroids and DDT and <54% for carbamates and organophophates. This population also exhibited high resistance intensity to pyrethroids (permethrin, alpha-cypermethrin and deltamethrin) after exposure to 5× and 10× discriminating doses. Synergist assays with PBO revealed only a partial recovery of susceptibility to permethrin, alpha-cypermethrin and deltamethrin. Only PBO-based nets (Olyset plus and permaNet 3.0) and Royal Guard showed an optimal efficacy. A high amount of alpha-cypermethrin was detected in breeding sites (5.16-fold LOD) suggesting ongoing selection from agricultural pesticides. The 1014F-kdr allele was fixed (100%) whereas the 1575Y-kdr (37.5%) and the 119S Ace-1^R (51.1%) were moderately present. Elevated expression of P450s, respectively, in permethrin and deltamethrin resistant mosquitoes [CYP6M2 (10 and 34-fold), CYP6Z1(17 and 29-fold), CYP6Z2 (13 and 65-fold), CYP9K1 (13 and 87-fold)] supports their role in the observed resistance besides other mechanisms including chemosensory genes as SAP1 (28 and 13-fold), SAP2 (5 and 5-fold), SAP3 (24 and 8-fold) and cuticular genes as CYP4G16 (6 and 8-fold) and CYP4G17 (5 and 27-fold). However, these candidate genes were not associated with resistance escalation as the expression levels did not differ significantly between 1×, 5× and 10× surviving mosquitoes; (4) Conclusions: Intensive and multiple resistance is being selected in malaria vectors from a pesticide-based agricultural hotspot of Cameroon leading to loss in the efficacy of pyrethroid-only nets. Further studies are needed to decipher the molecular basis underlying such resistance escalation to better assess its impact on control interventions.