Global spread and genetic variants of the two CYP9M10 haplotype forms associated with insecticide resistance in Culex quinquefasciatus Say.

Insecticide resistance develops as a genetic factor (allele) conferring lower susceptibility to insecticides proliferates within a target insect population under strong positive selection. Intriguingly, a resistance allele pre-existing in a population often bears a series of further adaptive allelic variants through new mutations. This phenomenon occasionally results in replacement of the predominating resistance allele by fitter new derivatives, and consequently, development of greater resistance at the population level. The overexpression of the cytochrome P450 gene CYP9M10 is associated with pyrethroid resistance in the southern house mosquito Culex quinquefasciatus. Previously, we have found two genealogically related overexpressing CYP9M10 haplotypes, which differ in gene copy number (duplicated and non-duplicated). The duplicated haplotype was derived from the non-duplicated overproducer probably recently. In the present study, we investigated allelic series of CYP9M10 involved in three C. quinquefasciatus laboratory colonies recently collected from three different localities. Duplicated and non-duplicated overproducing haplotypes coexisted in African and Asian colonies indicating a global distribution of both haplotype lineages. The duplicated haplotypes both in the Asian and African colonies were associated with higher expression levels and stronger resistance than non-duplicated overproducing haplotypes. There were slight variation in expression level among the non-duplicated overproducing haplotypes. The nucleotide sequences in coding and upstream regions among members of this group also showed a little diversity. Non-duplicated overproducing haplotypes with relatively higher expression were genealogically closer to the duplicated haplotypes than the other non-duplicated overproducing haplotypes, suggesting multiple cis-acting mutations before duplication.

Use of isogenic strains indicates CYP9M10 is linked to permethrin resistance in Culex pipiens quinquefasciatus.

Previous studies on a strain of Culex pipiens quinquefasciatus from Saudi Arabia indicated permethrin resistance was a result of cytochrome P450 mediated detoxification and kdr. The P450 detoxification was found to be larval specific and associated with a fitness cost in certain environments. The P450 responsible for resistance (and the fitness cost) has not been identified, but recently two candidate P450s (CYP4H34 and CYP9M10) have been found. We measured cytochrome P450 and cytochrome b5 content as well as the expression levels of CYP4H34 and CYP9M10 in a susceptible (SLAB) and two isogenic strains (isolated by repeated backcrossing and selection) of mosquito (ISOP450 and ISOJPAL) resistant to permethrin. Cytochrome P450 protein levels of the resistant strains were significantly higher (1.5-fold) than SLAB, but were not significantly different from one another. Expression of CYP4H34 in the larvae and adults of the resistant (ISOP450 and ISOJPAL) and susceptible (SLAB) strains were not statistically different. CYP9M10 was found to be significantly over-expressed in larvae of both permethrin-resistant isogenic strains (1800-fold in ISOP450 and 870-fold in ISOJPAL) when compared to SLAB. Partial sequence analysis of CYP9M10 revealed eight polymorphic sites that distinguished the susceptible allele from the resistant allele. We conclude that CYP9M10 is linked to permethrin resistance in these strains of C. p. quinquefasciatus, and is likely to be the P450 gene responsible for resistance in these strains.