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.

Differences in development, glycogen, and lipid content associated with cytochrome P450-mediated permethrin resistance in Culex pipiens quinquefasciatus (Diptera: Culicidae).

Insecticide resistance in populations of mosquitoes is an escalating problem that can directly affect disease prevalence. Determining the fitness associated with an insecticide resistance mechanism (allele) will provide for greater understanding of the evolution of resistance, and help inform effective vector management programs. Previously, a population cage experiment in which the alleles of two highly related strains of Culex pipiens quinquefasciatus (Say) SLAB (susceptible) and ISOP450 (permethrin resistant because of cytochrome P450-mediated detoxification) were placed in direct competition in the absence of insecticide revealed that the P450 resistance allele frequency decreased over time. In the current study, SLAB and ISOP450 development, mortality and energetic resources derived from glycogen, other sugars and lipids were measured to identify biological parameters that might explain the previously observed fitness cost. SLAB exhibited shorter egg-to-adult female development time and larger body size when reared in groups when compared with ISOP450. ISOP450 female adults provided 20% sugar water lived longer than 20% sugar water fed females of the SLAB strain. No significant differences in larval development time, larval mortality, pupal stage duration time, pupal mortality, longevity of male and female adults provided with distilled water and males provided sugar water were found between the strains. The caloric content from glycogen and lipids were significantly higher in SLAB relative to ISOP450 in adults. The slower female emergence time and smaller body size when reared in groups combined with lower energy reserves (glycogen and lipids) associated with the resistance allele (in ISOP450) are likely fitness costs associated with the resistance allele of P450-mediated detoxification.

Multiplicative interaction between the two major mechanisms of permethrin resistance, kdr and cytochrome P450-monooxygenase detoxification, in mosquitoes.

Epistasis is the nonadditive interaction between different loci which contribute to a phenotype. Epistasis between independent loci conferring insecticide resistance is important to investigate as this phenomenon can shape the rate that resistance evolves and can dictate the level of resistance in the field. The evolution of insecticide resistance in mosquitoes is a growing and world-wide problem. The two major mechanisms that confer resistance to permethrin in Culex mosquitoes are target site insensitivity (i.e. kdr) and enhanced detoxification by cytochrome P450 monooxygenases. Using three strains of mosquitoes, and crosses between these strains, we assessed the relative contribution of the two independent loci conferring permethrin resistance, individually and when present together. We found that for all genotype combinations tested, Culex pipiens quinquefasciatus exhibited multiplicative interactions between kdr and P450 detoxification, whether the resistance alleles were homozygous or heterozygous. These results provide a basis for further analysis of the evolution and maintenance of insecticide resistance in mosquitoes.