Isolation and sequence analysis of P450 genes from a pyrethroid resistant colony of the major malaria vector Anopheles funestus

Abstract

Pyrethroid resistance has been demonstrated in populations of Anopheles funestus from South Africa and southern Mozambique. Resistance is associated with elevated P450 monooxygenase enzymes. In this study, degenerate primers based on conserved regions of Anopheles gambiae P450 CYP4, 6 and 9 families were used to amplify genomic and cDNA templates from A. funestus. A total of 12 CYP4, 12 CYP6 and 7 CYP9 partial genes have been isolated and sequenced. BLAST results revealed that A. funestus P450s generally have a high sequence identity to A. gambiae with above 75% identity at the amino acid level. The exception is CYP9J14. The A. gambiae P450 showing highest identity to CYP9J14 exhibits only 55% identity suggesting that CYP9J14 may have arisen from a recent duplication event. Molecular phylogenetic analysis based on amino acid sequences also supported this hypothesis. Intron positions, but not size, were highly conserved between the two species. The high level of orthology that exists in the P450 gene families of these two species may facilitate the prediction of individual P450 protein function.

• Anopheles funestus
• pyrethroid resistance
• P450
• orthologous genes
• gene duplication
• protein function

Acknowledgements

We thank Ms Humbulani Mafumo for technical assistance and Dr Nelson for naming the P450 genes. This work received financial support from UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) to LLK and a Welcome Trust Research Developmental Award to BDB and the National Health Laboratory Service.