Transcription profiling of a recently colonised pyrethroid resistant Anopheles gambiae strain from Ghana

Abstract

Background: Mosquito resistance to the pyrethroid insecticides used to treat bednets threatens the sustainability of malaria control in sub-Saharan Africa. While the impact of target site insensitivity alleles is being widely discussed the implications of insecticide detoxification--though equally important--remains elusive. The successful development of new tools for malaria intervention and management requires a comprehensive understanding of insecticide resistance, including metabolic resistance mechanisms. Although three enzyme families (cytochrome P450s, glutathione S-transferases and carboxylesterases) have been widely associated with insecticide detoxification the role of individual enzymes is largely unknown.

Results: Here, constitutive expression patterns of genes putatively involved in conferring pyrethroid resistance was investigated in a recently colonised pyrethroid resistant Anopheles gambiae strain from Odumasy, Southern Ghana. RNA from the resistant strain and a standard laboratory susceptible strain, of both sexes was extracted, reverse transcribed and labelled with either Cy3- or Cy5-dye. Labelled cDNA was co-hybridised to the detox chip, a custom-made microarray containing over 230 A. gambiae gene fragments predominantly from enzyme families associated with insecticide resistance. After hybridisation, Cy3- and Cy5-signal intensities were measured and compared gene by gene. In both females and males of the resistant strain the cytochrome P450s CYP6Z2 and CYP6M2 are highly over-expressed along with a member of the superoxide dismutase (SOD) gene family.

Conclusion: These genes differ from those found up-regulated in East African strains of pyrethroid resistant A. gambiae and constitute a novel set of candidate genes implicated in insecticide detoxification. These data suggest that metabolic resistance may have multiple origins in A. gambiae, which has strong implications for the management of resistance.

Figure 1

Microarray analysis comparing the permethrin resistant Odumasy strain with the standard susceptible Kisumu strain. Data are shown for the differences in expression levels between the permethrin resistant Odumasy strain and the standard susceptible Kisumu strain for both females (A) and males (B). Each dot represents the mean estimates, fold (M) and p-value (adjusted for multiple testing), for one gene from the complete microarray experiment. Names are shown for genes that are at least two-fold differentially expressed. Underlined names show genes that are common to both the female-female and male-male comparison. Horizontal line represents the level of significance α = 0.001, and vertical lines indicate two-fold change threshold.

Figure 2

Microarray analysis comparing females with males within each strain. Data are shown for the gene expression levels of susceptible Kisumu females vs. males (A) and the permethrin resistant Odumasy females vs. males (B). Each dot represents the mean estimates, fold (M) and p-value (adjusted for multiple testing), for one gene from the complete microarray experiment. Names are shown for genes that are at least two-fold differentially expressed. Underlined names show genes that are common to both the Kisumu female-male and the Odumasy female-male comparison. Horizontal line represents the level of significance α = 0.001, and vertical lines indicate two-fold change threshold. *CYP6Z1 was spotted on the detox chip as both cDNA and 70-mer oligonucleotide probe [16].

Figure 3

Semi-quantitative RT-PCR validation of selected microarray data. Gene expression levels of CYP307A1 and CYP6M2 between females and males of the susceptible Kisumu strain were compared. (A) Peak intensities of amplified cDNA products of the two target mRNAs were measured and normalised by the internal control, the ribosomal S7 product. Primers used to amplify cDNA are shown in Table 1. (B) Measurements were taken from four biological replicates for each sex (15 one-day adult mosquitoes per replicates). Dots represent normalised peak intensities for each replicate. Horizontal bars show mean values. CYP307A1 shows a 3.4-fold over-expression in males (one-sided t-test, p < 0.001) whereas CYP6M2 was 1.1-fold over-expressed in males but statistically not significant (one-sided t-test, p = 0.3).