Ddt

DDT, imidacloprid, malathion

Thiodicarb

Pyrethroids Pyrethroids

Permethrin

Pyrethroids

Pyrethroids

Deltamethrin

Me-parathion, carbaryl Chlorpyriphos

Feyereisen etal. (1989),

Carino etal. (1992) Sabourault etal. (2001) Liu and Scott (1996) Liu and Scott (1996) Kasai and Scott (2000) Shono etal. (2002) Kasai and Scott (2001b) Guzov etal. (1998)

Amichot etal. (1994) Waters etal. (1992) Amichot etal. (1994) Maitra etal. (2000) Bride etal. (1997) Maitra etal. (1996) Maitra etal. (2000) Le Goff et al. (2003) Daborn etal. (2001, 2002) Daborn etal. (2002) Daborn etal. (2002) Brandt etal. (2002) Brandt etal. (2002), Le Goff et al. (2003)

Rose etal. (1997)

Pittendrigh etal. (1997) Ranasingheand Hobbs (1998)

Zhu and Snodgrass (2003)

Nikou etal. (2003)

Kasai etal. (2000)

Shen etal. (2003)

Scharf etal. (2001) Scharf etal. (1999)

aP450 protein level increased. bPresumed CYP6G1 ortholog of D. simulans.

et al., 2001; see Table 6 for comparison of Rutgers and sbo). A comparison of the coding sequence of CYP6A1 between Rutgers and two susceptible strains showed no (sbo) or little (aabys) sequence variation. Five amino acid changes were noted in aabys, two at the far N-terminus and three at the far C-terminus, well outside the regions (SRS) thought to influence enzyme activity (Cohen et al., 1994). The lack of CYP6A1 sequence difference between

Rutgers and sbo indicated that if CYP6A1 was implicated in diazinon resistance in the Rutgers strain, it was through elevation of enzyme activity alone. Reconstitution of recombinant CYP6A1 expressed in E. coli with its redox partners (Sabourault et al., 2001) provided the 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

Table 6 Comparison of a susceptible and a resistant strain of the housefly

Rutgers

Diazinon contact toxicity: LC50 (mg/pint 4.4 167.8 jar)

Resistance ratio

Diazinon topical toxicity: LD50 (mg/fly) Resistance ratio P450 level (nmol/mg protein) Aldrin epoxidation (pmol/min/pmol P450)

CYP6A1 mRNA relative level CYP12A1 mRNA relative level CYP6A1 protein level (fmol/abdomen) Diazinon metabolized by CYP6A1 Oxidative cleavage (pmol/min/fly) Desulfuration to oxon (pmol/min/fly) OP oxon metabolized by mutant ali-

esterase (pmol/min/fly) NADPH-dep. diazinon metabolism by microsomes (pmol/min/abdomen)

aNAIDM susceptible strain.

favorable ratio (2.7) between oxidative ester cleavage and desulfuration (see Section 4.1.4.2.2 and Table 4).

The nature of the chromosome 2 trans-acting factor and of the mutation leading to resistance in the Rutgers strain has long 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 (3.312.4 cM). This region carries an ali-esterase gene (MdaE7) implicated as its Lucilia cuprina ortholog in organophosphorus insecticide resistance (Newcomb et al., 1997; Claudianos et al., 1999). A Gly137 to Asp mutation in this ali-esterase abolishes carboxylesterase activity towards model compounds such as methylthiobutyrate, and confers a measurable phosphotriester hydrolase activity towards an organophosphate ("P=O"), chlorfenvin-phos. Chromosome 2 of the Rutgers strain carries this Gly137 to Asp mutation, and low CYP6A1 protein levels are correlated with the presence of at least one wild-type (Gly137) allele of MdaE7. 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 wild-type 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. Nature seems to have found the optimal loss-of-function mutation (Gly137 to Asp) as the Rutgers haplotype has swept through global populations of the housefly (C. Claudianos, J. Brownlie, V. Taskin, M. Kence, R.J. Russell, J.G. Oakeshott, personal communication). The mutant ali-esterase probably helps clearing the activated form (P=O) of the insecticide (Sabourault et al., 2001). The negative regulation by the Gly137 allele and the diazinon resistance-enhancing effect of the Asp137 allele may explain the incomplete dominance of the diazinon resistance trait. Housefly strains that are susceptible or that are not known/shown to overexpress CYP6A1 predictably carry at least one Gly137 allele (Scott and Zhang, 2003). The LPR strain that overexpresses CYP6A1 (Carino et al., 1992) and has increased OP metabolism (Hatano and Scott, 1993), as well as another resistant strain (NG98) carry other alleles (Scott and Zhang, 2003) that have impaired ali-esterase activity, Trp251 to Ser or Leu (Campbell et al., 1998; Claudianos et al., 2001). Corroborating, but indirect evidence for the hypothesis is the predicted pleiotropic effect of a trans-acting regulator. Constitutive overexpression of CYP12A1 whose product metabolizes diazinon as well (Guzov et al., 1998) and GST-1 in the Rutgers strain are also controlled by a chromosome 2 factor, possibly the same as the one controlling CYP6A1 expression.

Although alternative hypotheses can be advanced, such as the fortuitous genetic closeness between the ali-esterase and a factor that increases the level of CYP6A1, a diazinon metabolizing enzyme, such hypotheses do not have the benefit of elegance. The mutant ali-esterase cannot account for the carbamate and JHA resistance so is not the sole factor of resistance seen in many housefly strains. Diazinon-resistant L. cuprina have provided evidence for Oppenoorth's mutant ali-esterase hypothesis (Newcomb et al., 1997), but the role of P450 in OP resistance cannot be ignored. 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 L. cuprina diazinon resistance has also been presented (Kotze, 1995; Kotze and Sales, 1995).

4.1.4.5.3. Cyp6a2 in Drosophila: overexpression and mutant enzyme Insecticide-resistant strains of D. melanogaster have been studied at the molecular genetic level and the Cyp6a2 gene has been implicated in the metabolic resistance of several of

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