Extensive utilization of pesticides against insects provides us with a good model for studying the adaptation of a eukaryotic genome to a strong selective pressure. One meanism ofresistance is the alteration ofacetyichoinesterase (EC 3.1.1.7), the molecular target for organophosphates and carbamates. Here, we report the sequence analysis of the Ace gene in several resistant field strains of Drosophila melanogaster. This analysis resulted in the identification of five point mutations associated with reduced sensitivities to insecticides. In some cases, several of these mutations were found to be combined in the same protein, leading to different resistance patterns. Our results suggest that recombination between resistant alleles preexisting in natural populations is a mechanism by which insects rapidly adapt to new selective pressures.Although insecticide resistance is an important agricultural problem, this phenomenon also provides a good model for studying adaptation of eukaryotes to a toxic environment. Resistance to insecticides results from three main mechanisms: reduction in the insecticide penetration; increased metabolization of the insecticide by esterases, mixedfunction oxidases, or glutathione transferases; and modification of the insecticide target.

Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane (DDT) resistance in the RDDT R strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT R strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2b position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.Keywords: cytochrome P450; mutation; insecticide; resistance; structure.Many cytochrome P450 enzymes are known to be essential for the protection of organisms against xenobiotics. In insects, the involvement of cytochrome P450 enzymes in plant toxin or insecticide resistance has already been suggested or demonstrated [1][2][3][4][5][6][7], although high resistance levels to insecticides still remain unexplained. To date, only three of the cytochrome P450 enzymes linked to resistance have been shown to be able to metabolize insecticides. Two were cloned from the house fly: CYP6A1 metabolizes aldrin, heptachlor [8], terpenoids [9] and diazinon [10] and CYP12A1 metabolizes aldrin, heptachlor, diazinon and azinphosmethyl [11]. The third is CYP6A2 from Drosophila melanogaster. Baculovirus-directed production of wild-type CYP6A2 showed metabolism of cyclodiene and organophosphorous insecticides, but 1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane (DDT) metabolism could not be detected [12]. In addition, sequence polymorphism of CYP6A1 and CYP6D1 has been documented in the house fly, but there is no link between these instances of polymorphism and insecticide resistance [7,13,14]. These results are in contrast with known instances of cytochrome P450 polymorphisms in humans, which are well known to affect the metabolism of drugs [15,16] and even pesticides [17]. In fact, only two examples of pesticide...

ABSTRACï Resistance mechanisms of a strain (MSE) of Culex pipiens L., collected in sauthern France in 1979 and highiy resistant to chlorpynfos. were investigated by comparing the resistance characteristics to vanous organophasphates and carbamates in the absence or presence of synergists and determining the biochemical characteristics of four enzymes (esterases. glutathione-S-transferases, mixed function oxidases, and acetylcholinesterase) compareci with a susceptible strain and a chlorpyrifas-resistant strain (S54) collected in the same area in 1960 and 1974, respectively. Cblorpyrifos resistance in S54 was due to a detoxifying esterase as previously dexribed, whereas resistance in MSE was associateci with an acetylcholinesterase insensitive to the inhibition by chlorpyrifoxon and some carbamates (propoxur, carbosulfan. and carbaryl), and with an increase of oxidative metabolism.

Target site insensitivity and metabolic resistance mediated by esterases have been previously suggested to be involved in resistance to malathion in a field-derived strain (W) of Ceratitis capitata. In the present study, we have obtained the coding sequence for acetylcholinesterase (AChE) gene (Ccace) of C. capitata. An allele of Ccace carrying only a point mutation Gly328Ala (Torpedo numbering) adjacent to the glutamate of the catalytic triad was found in individuals of the W strain. Adult flies homozygotes for this mutant allele showed reduced AChE activity and less sensitivity to inhibition by malaoxon, showing that target site insensitivity is one of the factors of malathion resistance. In addition, all individuals from the resistant W strain showed reduced aliesterase activity, which has been associated with specific malathion resistance in higher Diptera. However, the alphaE7 gene (CcalphaE7), sequenced in susceptible and resistant individuals, did not carry any of the mutations associated with organophosphorus insecticide resistance in other Diptera. Another esterase mechanism, perhaps a carboxylesterase selective for malathion, in addition to mutant AChE, thus contributes to malathion resistance in C. capitata.