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===== CYP6A1 and diazinon resistance in the house fly Rutgers strain =====


[[cyp6a1|CYP6A1]] is the first insect P450 cDNA to have been cloned. The gene is phenobarbital-inducible and is constitutively overexpressed in the multiresistant Rutgers strain (Feyereisen et al., 1989). A survey of 15 house fly strains (Cariño et al., 1992) showed that CYP6A1 is constitutively overexpressed at various degrees in eight resistant strains, but not in all resistant strains - notably R-Fc known to possess a P450-based resistance mechanism. Thus the first survey with a P450 molecular probe confirmed the results of the first survey of house fly strains with marker P450 activities (aldrin epoxidation and naphthalene hydroxylation)(Schonbrod et al., 1968): there is no simple relationship between resistance and a molecular marker, here the level of expression of a single P450 gene. That different P450 genes would be involved in different cases of insecticide resistance was a sobering observation (Cariño et al., 1992), even before the total number of P450 genes in an insect genome was known. 

The constitutive overexpression of CYP6A1 in larvae and in adults is linked to a semi-dominant factor on chromosome 2 (Cariño et al., 1994), but the CYP6A1 gene maps to chromosome 5 (Cohen et al., 1994), implying the existence of a chromosome 2 trans-acting factor(s) differentially regulating CYP6A1 expression in the two strains (Cariño et al., 1994). The gene copy number and the coding sequence of CYP6A1 are identical between Rutgers and a standard susceptible strain (sbo) (Cariño et al., 1994; Cohen et al., 1994). Competitive ELISA using purified recombinant CYP6A1 protein as standard showed that the elevated mRNA levels are indeed translated into elevated protein levels (Sabourault et al., 2001). Reconstitution of recombinant CYP6A1 expressed in E.coli with its redox partners (Sabourault et al., 2001) provided conclusive evidence for its role in diazinon resistance, as CYP6A1 metabolizes the insecticide with a high turnover (18.7 pmol/pmol CYP6A1/min), and a favorable ratio (2.7) between oxidative ester cleavage and desulfuration.

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The nature of the chromosome 2 trans-acting factor and of the mutation leading to resistance in the Rutgers strain has remained enigmatic despite considerable circumstantial evidence for a major resistance factor on chromosome 2 (Plapp, 1984). Diazinon resistance and high CYP6A1 protein levels could not be separated by recombination in the short distance between the ar and car genes (3.3-12.4 cM). This region carries an ali-esterase gene (MdαE7). A Gly137 to Asp mutation in this ali-esterase abolishes carboxylesterase activity towards model compounds such as methylthiobutyrate, and confers a low but measurable phosphotriester hydrolase activity towards an organophosphate ("P=O"), chlorfenvinphos, in both the sheep blowfly //Lucilia cuprina// LcαE7 and //Musca domestica// MdαE7 enzymes (Newcomb et al., 1997, Claudianos et al., 1999). Chromosome 2 of the Rutgers strain carries this MdαE7 Gly137 to Asp mutation, and low CYP6A1 protein levels are correlated with the presence of at least one wildtype (Gly137) allele of MdαE7. Recombination in the ar-car region could not dissociate diazinon susceptibility, low CYP6A1 protein level and the presence of a Gly137 allele of the ali-esterase (Sabourault et al., 2001). It was therefore hypothesized that the wildtype ali-esterase metabolizes an (unknown) endogenous substrate into a negative regulator of CYP6A1 transcription. Removal of this regulator (by loss-of-function of the ali-esterase) would increase CYP6A1 production and hence, diazinon metabolism. House fly strains that are susceptible or that are not known to overexpress CYP6A1 predictably carry at least one wild type Gly137 allele (Scott and Zhang, 2003). The LPR strain that overexpresses CYP6A1 (Cariño et al., 1992) and has increased OP metabolism (Hatano and Scott, 1993), as well as other resistant strains, carry other alleles of MdαE7 (Scott and Zhang, 2003; Gacar and Taskin, 2009). These alleles, Trp251 to Ser or Leu, also have impaired ali-esterase activity in //Musca domestica// (Claudianos et al., 1999; Gacar and Taskin, 2009). The hypothesis that the wild type allele of MdαE7 is a trans-acting negative regulator causing low expression of CYP6A1 in susceptible strains has implications. The pleiotropic effect of trans regulation is compatible with constitutive overexpression of CYP12A1 (whose product metabolizes diazinon as well, Guzov et al., 1998) and of GST-1 that are both controlled in the Rutgers strain by a chromosome 2 factor, possibly the same as the one controlling CYP6A1 expression.

There are alternative explanations, for instance very close linkage of the ali-esterase MdαE7 with an uncharacterized trans-acting factor, or interference of small inversions with genetic crosses and recombination in that region. The fragmented current assembly of the house fly genome is a hindrance to further research in this (historically important) case of resistance.

The diazinon resistance (Rop-1) and malathion resistance (Rmal) in //Lucilia cuprina//, are linked to the Gly137Asp and Trp251Leu mutations in LcαE7 (Newcomb et al., 1997). The Trp251Leu mutation enhances hydrolysis of dimethylorganophosphates and of the malathion carboxylesters, while the Gly137Asp mutation enhances preferentially the hydrolysis of diethylorganophosphates but virtually abolishes malathion carboxylesterase activity (Devonshire et al., 2003). In view of these data, could the Gly137Asp mutation alone be responsible for diazinon resistance in the blowfly ? The elegant calculations of Devonshire et al. (2003) show that despite the very low activity of the Gly137Asp mutant esterase to hydrolyze the oxon form of the pesticide (kcat ~ 0.05 min-1), the 10-20 fold resistance to diazinon and parathion could indeed be accounted by LcαE7. Those calculations, based on the oxon form do not take into account the necessary P450-dependent desulfuration of the parent OP to produce the oxon, that is invariably associated with ester cleavage (see above), so that P450 may still play a role in the blowfly. Indeed, the Q strain of the sheep blowfly is more resistant to parathion than to paraoxon (Hughes and Devonshire, 1982) and indirect evidence for a P450 involvement in //Lucilia cuprina// diazinon resistance has also been presented (Kotze, 1995, Kotze and Sales, 1995). When transgenically expressed in Drosophila (Daborn et al., 2012), the Lucilia Gly137Asp mutant esterase confers about 7-fold resistance to diazinon, which is significant but lower than the house fly Rutgers resistance. 
 
In the Rutgers strain of the house fly, the situation is different. The mutant ali-esterase cannot account for the P450-dependent carbamate and JHA resistance that are linked to Chromosome 2. Furthermore, the higher resistance to diazinon than in //Lucilia cuprina// (120x vs 10x) requires  a more efficient clearance of the oxon. The contribution of CYP6A1 alone accounts for over 5 times more than the mutant ali-esterase to the timely removal of the toxic form. Lethality of the MdαE7 null (Sabourault et al., 2001) suggests that the Gly137Asp mutation is the optimal loss-of-function mutation as the Rutgers haplotype has swept through global populations of the house fly (Claudianos, 1999; Gacar and Taskin, 2009). The low phosphotriester hydrolase activity of the mutant ali-esterase probably helps clearing the activated form (P=O) of the insecticide (Sabourault et al., 2001). The endogenous function of the wild type MdαE7 gene remains to be elucidated.