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====== CYP6D1, the LPR strain, and pyrethroid resistance in the house fly ======



The LPR strain of the house fly is highly resistant to pyrethroids with a phenoxybenzyl moiety. This permethrin-selected strain has several resistance mechanisms with important contributions of the genes pen (for reduced penetration) and kdr (for target site resistance)(Liu and Scott, 1995) and with P450-based detoxification as a major contributor. 

An abundant form of P450 (P450Lpr) was purified from abdomens of adult LPR flies, and immunological data indicated that P450Lpr represents 67% of the P450 in microsomes from LPR flies, a 10-fold (Wheelock and Scott, 1990) increase over the reference susceptible strain. The P450Lpr gene, CYP6D1 (Tomita and Scott, 1995) is located on chromosome 1 of the house fly (Liu et al., 1995), and is constitutively overexpressed by about 10-fold in the LPR strain. This overexpression is not caused by gene amplification, but by increased transcription. It has been claimed that increased transcription of CYP6D1 causes insecticide resistance (Liu and Scott, 1998), but transgenic expression of CYP6D1 in Drosophila (Korytko et al., 2000a) has not been reported to confer resistance, and heterologously expressed CYP6D1 (Smith and Scott, 1997) has not been reported to metabolize pyrethroids. Instead, the evidence for the role of CYP6D1 in pyrethroid resistance is based on the inhibition of microsomal deltamethrin and cypermethrin metabolism by anti-P450Lpr antibodies (Wheelock and Scott, 1992b, Korytko and Scott, 1998). Deltamethrin is metabolized preferentially at the gem-dimethyl group on the acid moiety (Wheelock and Scott, 1992b) whereas cypermethrin is mainly hydroxylated at the 4' position on the alcohol moiety, at an extremely low rate (Zhang and Scott, 1996a). Whether overexpression or point mutations of CYP6D1 or both are involved in pyrethroid resistance in the LPR strain is yet unknown. Indeed, the CYP6D1 gene sequence from 5 strains shows a high polymorphism, with 57 variable sites in the coding region alone, of which 12 are non-silent (Tomita et al., 1995). Six amino acid changes are specific to the LPR strain (CYP6D1v1) when compared to pyrethroid-susceptible strains and several of these mutations appear to align with SRS3. 
The 170-fold piperonyl butoxide-suppressible resistance is conferred by a combination of the resistant chromosomes 1 and 2 from the parent LPR strain in the homozygous condition (Liu and Scott, 1995). There is no substantial resistance or CYP6D1 overproduction conferred by isolated LPR chromosomes 1 or 2, or by their subsequent combination (Liu and Scott, 1995, Liu and Scott, 1996). Thus, in the LPR strain, P450-mediated resistance requires both copies of the LPR chromosomes 1 and 2. The resistance and CYP6D1 overexpression linked to chromosome 1 are dominant, whereas the contributions of chromosome 2 are mostly recessive (Liu and Scott, 1996, Liu and Scott, 1997a). These data suggest a unique combination in the LPR strain of chromosome 2 trans-acting factor(s) with at least a matched cis-factor on chromosome 1. A key sequence difference between the 5'UTR of the CYP6D1v1 allele of LPR and of susceptible strains is the presence of a 15 bp insert that interrupts a binding site of the transcriptional repressor mdGfi-1, and reduces the amount of mdGfi-1 binding to the   CYP6D1 promoter in electrophoretic mobility shift assays (Gao and Scott, 2006). This strongly suggests that the 15-bp insertion is the major cis-mutation on chromosome 1. This insert is also found in a permethrin-resistant strain NG98 from Georgia, USA that carries a virtually identical CYP6D1v1 haplotype (Seifert and Scott, 2002).
However, in a strain from neighboring Alabama with high permethrin resistance (ALHF), chromosomes 1 and 2 play little role but chromosome 5 plays a major piperonyl butoxide-suppressible role (Liu and Yue, 2001). Two P450 genes located on chromosome 5, CYP6A36 and CYP6A5v2 (a probable recent duplicate of CYP6A5) are constitutively overexpressed in that strain (Zhu and Liu, 2008; Zhu et al., 2008a). A gene closely linked to CYP6D1 on chromosome 1 codes for a similar (78% identity) P450, CYP6D3 (Kasai and Scott, 2001a). CYP6D3 is 12-fold overexpressed in adult flies of the LPR strain, but it is also expressed in larvae (Kasai and Scott, 2001b), as opposed to CYP6D1 which has an adult-specific pattern of expression. CYP6D1 is overexpressed by about 2.4 fold in the Japanese strain YPER (Kamiya et al., 2001; Shono et al., 2002). This strain does not carry the CYP6D1v1 allele, and chromosome 2 has a major role in this permethrin-resistant strain. CYP6D3v2 and CYP6A24 are also overexpressed in Japanese strains (Kamiya et al., 2001).

Pyrethroid resistance in the house fly appears thus to involve multiple mutations playing a role in the contribution of several P450s to the multifactorial resistance.