Insecticide-resistant House Fly Strains Research Articles

A Cluster of Cytochrome P450 Genes of the CYP6 Family in the House Fly

A cluster of genes of the CYP6 family was found in a series of overlapping lambda DASH clones from a genomic library of the house fly, Musca domestica. Four complete genes, CYP6A3, CYP6A4, CYP6A5, and CYP6C1, and fragments of two other genes, CYP6A6 and CYP6C2, were closely linked on a 24-kb segment of DNA. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified segments of two of the genes showed that the cluster is localized on chromosome V of the house fly. Each gene contained a short intron of 57 to 125 bp interrupting a conserved Glu codon, as in the previously described CYP6A1 gene. The gene fragment CYP6A6 consisted only of the coding region downstream from this intron, i.e., about one-third of the complete P450. The gene fragment CYP6C2 was missing a short amino-terminal part of the coding region, and may represent the two last exons of a larger gene. Gene duplication and chromosomal inversion events may explain the origin of this cluster. The P450 proteins deduced from the nucleotide sequences shared 39-71% amino acid identity with each other. This low identity and the lack of evidence of recent gene conversion events suggested that this cluster may be evolutionarily ancient and that homologous clusters may be found in other holometabolous insects. Evidence for transcription of the genes and for correct splicing of the introns was obtained by northern blotting and reverse transcription polymerase chain reaction (RT-PCR) experiments. No overexpression was observed in any of three insecticide-resistant house fly strains. RT-PCR and sequencing also revealed the existence of other genes or alleles closely related to the members of this cluster.

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

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.

Studies on the effect of phenobarbital on γ-glutamyl transferase activity in various housefly strains

The effect of pretreatment with phenobarbital (PB) on γ-glutamyl transferase (GGT) activity in an insecticide-susceptible housefly strain (CSMA) and three insecticide-resistant housefly strains (Fc, Rutgers, and Hirokawa) was investigated. Pretreatment with PB had little if any effect on GGT activity in the susceptible and resistant housefly strains, indicating that not all the enzymes involved in mercapturic acid biosynthesis in the housefly are inducible.

Studies on γ-glutamyl transferase in the housefly

Variation in γ-glutamyl transferase activity between developmental stages, body regions, sexes and strains of the housefly were determined. Enzyme activity increases during the early larval stages, decreases in the pupal stage and increases again in the adult housefly. The female has slightly higher γ-glutamyl transferase activity than the male, depending on age, and enzyme activity of body regions was found to decrease in the order: abdomen > head > thorax. No correlation was found between γ-glutamyl transferase activity and the resistance mechanism involving glutathione S- transferase in several susceptible and insecticide-resistant housefly strains.