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

  1. Charles S. Wondji1,7,
  2. Helen Irving1,
  3. John Morgan1,
  4. Neil F. Lobo2,3,
  5. Frank H. Collins2,3,
  6. Richard H. Hunt4,
  7. Maureen Coetzee5,6,
  8. Janet Hemingway1 and
  9. Hilary Ranson1
  1. 1 Liverpool School of Tropical Medicine, Liverpool, L3 5QA, United Kingdom;
  2. 2 The Eck Family Institute for Global Health & Infectious Diseases, University of Notre Dame, Notre Dame, Indiana 46556, USA;
  3. 3 Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA;
  4. 4 School of Animal, Plant & Environmental Sciences, University of the Witwatersrand, Private Bag X3 Johannesburg, South Africa;
  5. 5 Medical Entomology and Vector Control, Division of Virology and Communicable Disease Surveillance, University of the Witwatersrand, Private Bag X3 Johannesburg, South Africa;
  6. 6 National Institute for Communicable Diseases (NICD), Johannesburg, Private Bag X4, Sandringham 2131, South Africa

    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 f2 = 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.

    Footnotes

    • ↵7 Corresponding author.

      ↵E-mail c.s.wondji{at}liverpool.ac.uk; fax 44-151-705-3369.

    • [Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GenBank (http://www.ncbi.nlm.gov/Genbank/) under accession nos. EU852639–EU852651.]

    • Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.087916.108.

      • Received October 14, 2008.
      • Accepted December 8, 2008.
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    1. Published in Advance February 5, 2009, doi: 10.1101/gr.087916.108 Genome Res. 19: 452-459 Copyright © 2009 by Cold Spring Harbor Laboratory Press

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