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- Title
The duplicated P450s CYP6P9a/b drive carbamates and pyrethroids cross-resistance in the major African malaria vector Anopheles funestus.
- Authors
Mugenzi, Leon M. J.; A. Tekoh, Theofelix; S. Ibrahim, Sulaiman; Muhammad, Abdullahi; Kouamo, Mersimine; Wondji, Murielle J.; Irving, Helen; Hearn, Jack; Wondji, Charles S.
- Abstract
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. Author summary: Insecticide-based vector control measures are extensively used in the fight against malaria. Unfortunately, insecticides are in danger of losing their potency due to the development of resistance in vector species populations. The worst-case scenario is when a single resistance mechanism confers resistance to many insecticide classes, thereby hindering resistance management. The application of appropriate insecticide-based interventions depends critically on understanding the underlying molecular basis of insecticide resistance. Here, we performed gene expression profiling (RNAseq) and whole genome sequencing (PoolSeq) of multiple resistant An. funestus collected from Ghana, Uganda, Malawi, laboratory-resistant (FUMOZ), and susceptible (FANG) strains. We found overexpression of metabolic cytochrome P450s CYP6P9a/b genes in Malawi and CYP6P4a/b genes in Ghana and also detected signatures of a strong selection of some regions of the genome around these genes. In vitro expression of CYP6P9a/b P450s showed metabolic activities towards carbamates and transgenic flies overexpressing resistant alleles of both genes were significantly more resistant to carbamates. Furthermore, phenotype/genotype association studies revealed a strong correlation between carbamate resistance and CYP6P9a/b-resistant genotypes. Altogether, this study demonstrated that P450-based pyrethroid resistance also confers resistance to carbamate insecticides.
- Subjects
GHANA; MALAWI; INSECTICIDE resistance; PYRETHROIDS; ATP-binding cassette transporters; CARBAMATES; GENE expression profiling; ANOPHELES
- Publication
PLoS Genetics, 2023, Vol 18, Issue 3, p1
- ISSN
1553-7390
- Publication type
Article
- DOI
10.1371/journal.pgen.1010678