Immunological detection of cytochrome P450 from insecticide resistant and susceptible house flies ( Musca domestica)

Abstract

An antiserum was raised in a rabbit against a cytochrome P450, termed P450 lpr, purified from the pyrethroid resistant LPR strain of house fly. This polyclonal antiserum was specific for P450 lpr as judged by both rocket immunoelectrophoresis and immunoblotting. The antiserum was used to detect immunoreactive cytochrome P450s in insecticide susceptible and resistant house flies. Single immunoreactive bands of a slightly variable position were obtained when crude microsomes from six insecticide resistant house fly strains (LPR, Dairy, Kashiwagura, 3rd-Y, EPR, ASPR) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting, while immunoreactive bands were barely detectable in insecticide susceptible ( aabys, S+) strains. The immunoreactive cytochrome P450s were isolated from each of eight house fly strains by high-performance liquid chromatography. The isolated immunoreactive cytochrome P450s from all strains were immunologically identical to P450 lpr by fused rocket immunoelectrophoresis, and only those from the Kashiwagura and ASPR strains differed in chromatographic and/or electrophoretic properties from P450 lpr. Amounts of immunoreactive P450 were determined in house fly microsomes. Immunoreactive P450 represented 68% (specific content: 0.48 nmol/mg protein) of the total (0.72 nmol/mg) cytochrome P450 in LPR microsomes and 6.6% (0.011 nmol/mg) of the total (0.15 nmol/mg) cytochrome P450 in S+. Thus, immunoreactive P450 was constituitively expressed at 44-fold higher levels in insecticide resistant LPR flies compared to the susceptible S+ strain.