Investigations of the constitutive overexpression of CYP6D1 in the permethrin resistantLPR strain of house fly (Musca domestica)

Abstract

House fly (Musca domestica) CYP6D1 is a cytochrome P450 involved in metabolism of xenobiotics. CYP6D1 is located on chromosome 1 and its expression is inducible in response to the prototypical P450 inducer phenobarbital (PB) in insecticide susceptible strains. Increased transcription of CYP6D1 confers resistance to permethrin in the LPR strain, and this trait maps to chromosomes 1 and 2. However, the constitutive overexpression of CYP6D1 in LPR is not further increased by PB and the non-responsiveness to PB maps to chromosome 2. It has been suggested that a single factor on chromosome 2 could be responsible for both the constitutive overexpression and lack of PB induction of CYP6D1 in LPR. We examined the PB inducibility of CYP6D1v1 promoter from LPR using dual luciferase reporter assays in Drosophila S2 cells and found the CYP6D1v1 promoter was able to mediate PB induction, similar to the CYP6D1v2 promoter from the insecticide susceptible CS strain. Therefore, variation in promoter sequences of CYP6D1v1 and v2 does not appear responsible for the lack of PB induction of CYP6D1v1 in LPR; this suggests an unidentified trans acting factor is responsible. HR96 has been implicated in having a role in PB induction in Drosophila melanogaster and M. domestica. Therefore, house fly HR96 cDNA was cloned and sequenced to examine if this trans acting factor is responsible for constitutive overexpression of CYP6D1v1 in LPR. Multiple HR96 alleles (v1-v10) were identified, but none were associated with resistance. Expression levels of HR96 were not different between LPR and CS. Thus, HR96 is not the trans acting factor responsible for the constitutive overexpression of CYP6D1 in LPR. The identity of this trans acting factor remains elusive.

Figures

Fig. 1

CYP6D1v1 promoter sequence from −365 to −267 (from LPR) is able to mediate PB induction. Promoter constructs are numbered relative to the transcription start site (TSS) at +1. Relative luciferase activity was estimated by normalizing each signal of each promoter construct to the mean of signals of pGL3-Basic vector in the same replicate. Bars represent the mean relative luciferase activity ± S.D. of three independent transfections. Gray bars represent the signal in the presence of PB and white bars represent the control. Double asterisks indicate a greater PB induction relative to the next shorter promoter construct (p<0.01, Student’s t-test).

Fig. 2

Alignment of D. melanogaster (Dm) and M. domestica (Md) HR96 deduced protein sequences. (A) Protein sequence alignment of the DNA binding domain (DBD). Sequences representing the DBD were underlined. Locations of two amino acid substitutions (E82V and G110D) identified in alleles v8–v10 from the LPR strain were denoted with asterisks above the alignment. (B) Protein sequence alignment of the ligand binding domain (LBD). Sequences representing the LBD were underlined.

Fig. 3

Relative quantitation of HR96 transcript level of the CS and LPR strains. Abdomens of ten three-day-old male adults of CS and LPR were used to measure HR96 transcript level relative to Actin transcript level using real-time RT-PCR with comparative CT method. Bars represent means of relative quantitation of HR96 (normalized by Actin transcript level) ± S.D. of three PCR reactions of three biological sample pools (n = 9). The HR96 transcript levels were not significantly different between CS and LPR (by Student’s t-test).