A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

Although cytochrome P450 (CYP450) enzymes are frequently up-regulated in mosquitoes resistant to insecticides, no regulatory motifs driving these expression differences with relevance to wild populations have been identified. Transposable elements (TEs) are often enriched upstream of those CYP450s involved in insecticide resistance, leading to the assumption that they contribute regulatory motifs that directly underlie the resistance phenotype. A partial CuRE1 (Culex Repetitive Element 1) transposable element is found directly upstream of CYP9M10, a cytochrome P450 implicated previously in larval resistance to permethrin in the ISOP450 strain of Culex quinquefasciatus, but is absent from the equivalent genomic region of a susceptible strain. Via expression of CYP9M10 in Escherichia coli we have now

demonstrated time- and NADPH-dependant permethrin metabolism, prerequisites for confirmation of a role in metabolic resistance, and through qPCR shown that CYP9M10 is >20-fold over-expressed in ISOP450 compared to a susceptible strain. In a fluorescent reporter assay the region upstream of CYP9M10 from ISOP450 drove 10� expression compared to the equivalent region (lacking CuRE1) from

the susceptible strain. Close correspondence with the gene expression fold-change implicates the

upstream region including CuRE1 as a cis-regulatory element involved in resistance. Only a single CuRE1

bearing allele, identical to the CuRE1 bearing allele in the resistant strain, is found throughout Sub-

Saharan Africa, in contrast to the diversity encountered in non-CuRE1 alleles. This suggests a single

origin and subsequent spread due to selective advantage. CuRE1 is detectable using a simple diagnostic.

When applied to C. quinquefasciatus larvae from Ghana we have demonstrated a significant association

with permethrin resistance in multiple field sites (mean Odds Ratio ¼ 3.86) suggesting this marker has

relevance to natural populations of vector mosquitoes. However, when CuRE1 was excised from the allele

used in the reporter assay through fusion PCR, expression was unaffected, indicating that the TE has no

direct role in resistance and hence that CuRE1 is acting only as a marker of an as yet unidentified

regulatory motif in the association analysis. This suggests that a re-evaluation of the assumption that TEs

contribute regulatory motifs involved in gene expression may be necessary