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. 2009 Mar;19(3):452-9.
doi: 10.1101/gr.087916.108. Epub 2009 Feb 5.

Two duplicated P450 genes are associated with pyrethroid resistance in Anopheles funestus, a major malaria vector

Charles S Wondji  1 , Helen Irving, John Morgan, Neil F Lobo, Frank H Collins, Richard H Hunt, Maureen Coetzee, Janet Hemingway, Hilary Ranson
Affiliations

Two duplicated P450 genes are associated with pyrethroid resistance in Anopheles funestus, a major malaria vector

Charles S Wondji et al. Genome Res. 2009 Mar.

Abstract

Pyrethroid resistance in Anopheles funestus is a potential obstacle to malaria control in Africa. Tools are needed to detect resistance in field populations. We have been using a positional cloning approach to identify the major genes conferring pyrethroid resistance in this vector. A quantitative trait locus (QTL) named rp1 explains 87% of the genetic variance in pyrethroid susceptibility in two families from reciprocal crosses between susceptible and resistant strains. Two additional QTLs of minor effect, rp2 and rp3, were also detected. We sequenced a 120-kb BAC clone spanning the rp1 QTL and identified 14 protein-coding genes and one putative pseudogene. Ten of the 14 genes encoded cytochrome P450s, and expression analysis indicated that four of these P450s were differentially expressed between susceptible and resistant strains. Furthermore, two of these genes, CYP6P9 and CYP6P4, which are 25 and 51 times overexpressed in resistant females, are tandemly duplicated in the BAC clone as well as in laboratory and field samples, suggesting that P450 gene duplication could contribute to pyrethroid resistance in An. funestus. Single nucleotide polymorphisms (SNPs) were identified within CYP6P9 and CYP6P4, and genotyping of the progeny of the genetic crosses revealed a maximum penetrance value f(2) = 1, confirming that these SNPs are valid resistance markers in the laboratory strains. This serves as proof of principle that a DNA-based diagnostic test could be designed to trace metabolic resistance in field populations. This will be a major advance for insecticide resistance management in malaria vectors, which requires the early detection of resistance alleles.

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Figures

Figure 1.
Figure 1.
Plot of LOD scores associated with pyrethroid resistance in Family 1 (A) and in Family 10 (B) using F6 progeny. The y-axis indicates LOD ratio scores, and the x-axis indicates chromosome positions. (Solid lines) LOD estimated by composite interval mapping, (dashed lines) LOD estimated by interval mapping. The straight line along the top of each graph represents the threshold value for LOD as determined by permutations. Names of markers are listed around QTL locations.
Figure 2.
Figure 2.
Schematic representation of gene organization of BAC 29F01 containing the rp1 QTL in An. funestus (top) compared with the organization of orthologous genes in An. gambiae (bottom). (Vertical arrows) Intergenic regions; (horizontal arrows) the 5′–3′orientation of each gene; (asterisks) duplicated genes.
Figure 3.
Figure 3.
Comparison of the patterns of gene expression of four candidate genes CYP6P9 (A), CYP6P4 (B), CYP6AA4 (C), and CYP6P1 (D) between the resistant strain FUMOZ and the susceptible FANG in different life stages. The normalized expression ratio of each gene against the SP7 gene is represented on the primary vertical axis, while the secondary vertical axis represents the fold change of each gene between FUMOZ and FANG. Significant differential expressions are indicated by asterisks: (***) P < 0.001, (**) P < 0.01.
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