A. Cuany

Inducibility of the Drosophila melanogaster cytochrome P450 gene, CYP6A2, by phenobarbital in insecticide susceptible or resistant strains

The importance of cytochrome P450s in the biology of cells or organisms is clearly established. While numerous studies concern vertebrates, little is known about invertebrates cytochrome P450s. In this paper, we have focused on CYP6A2 gene expression in Drosophila melanogaster. We show the expression of this cytochrome P450 gene in the Canton(s) strain (wild type) to be under the control of phenobarbital. In adults treated with phenobarbital, this gene is transcribed in the midgut, the pericuticular fat bodies and the Malpighian tubules. The induction factor is 15. In the RDDTR strain of Drosophila melanogaster, which is resistant to the insecticide DDT, this gene is constitutively overexpressed in the same tissues (overexpression factor is 6 relative to untreated Canton(s) flies). Phenobarbital is not as effective on RDDTR (induction factor is 2.5 relative to untreated RDDTR flies) as on wild type strains.

Esterase metabolism and reduced penetration are causes of resistance to deltamethrin in Spodoptera exigua HUB (Noctuidea; lepidoptera)

Abstract Using deltamethrin in toxicological experiments we have shown that a guatemalian strain of Spodoptera exigua has an LC 40 at least 100 times that of a sensitive one. Delayed penetration and cleavage of deltamethrin at the ester bond are two mechanisms responsible for this resistance. Degradation of deltamethrin is 17 times higher in the resistant strain compared to the sensitive one. This degradation is inhibited by DEF and paraoxon, indicating that esterases are likely involved in the metabolism of this insecticide. In addition it has been shown that the resistant strain has an enhanced esterase activity toward chromogenic substrates, such as naphthyl acetate or methylumbelliferyl acetate, the level depending on the substrate used. It is likely that activity toward chromogenic substrates and the hydrolysis of deltamethrin are related.

Molecular Polymorphism of Head Acetylcholinesterase from Adult Houseflies (Musca domestica L.)

Abstract: Acetylcholinesterase (AChE) from housefly heads was purified by affinity chromatography. Three different native forms were separated by electrophoresis on poly‐acrylamide gradient gels; Two hydrophilic forms presented apparent molecular weights of 75,000 (AChE1) and 150,000 (AChE2). A third component (AChE3) had a migration that depended on the nature and concentration of detergents. In the presence of sodium deoxycholate in the gel, AChE3 showed an apparent molecular weight very close to that of AChE2. Among the three forms, AChE3 was the only one found in purified membranes. The relationships among the various forms were investigated using reduction with 2‐mercaptoethanol or proteolytic treatments. Such digestion converted purified AChE3 into AChE2 and AChE1, and reduction of AChE3 and AChE2 by 2‐mercaptoethanol gave AChE1, in both cases with a significant loss of activity. These data indicate that the three forms of purified AChE may be classified as an active hydrophilic monomeric unit (G1) plus hydrophilic and amphiphilic dimers. These two components were termed G2sand G2m, where “s” refers to soluble and “m” to membrane bound.).

Differential Sensitivity of Mosquito Taxa to Vegetable Tannins

The sensitivity of larval Culicidae to vegetable tannins was investigated in different taxa representative of the fauna from alpine hydrosystems (Aedes rusticus, Culex pipiens) and foreign noxious fauna (Aedes aegypti, A. albopictus). Bioassays reveal that tannic acid at concentrations of 0.1–6 mM is significantly more toxic for C. pipiens than for Aedes taxa, and A. aegypti is more sensitive than A. albopictus and A. rusticus. Comparison of the rank order of sensitivity among taxa with the associated levels of cytochrome P-450, esterase, and glutathione-S-transferase activities suggests that cytochrome P-450 and esterases may be involved in the detoxification of tannins. A possible involvement of these detoxifying enzymes is also revealed in vivo by the synergistic effects of S,S,S-tributyl phosphorotrithioate (esterase inhibitor) and piperonyl butoxide (P-450 inhibitor). The differential sensitivity to tannins among taxa is discussed in terms of ecological implications within mosquito communities from alpine hydrosystems, where the acquisition of tannins–detoxifying enzymatic systems may be considered as a key innovation.

Purification and partial characterization of glutathione S-transferases from insecticide-resistant and lindane-induced susceptible Spodoptera littoralis (Boisd.) larvae

Abstract Cytosolic GST activity was followed after a single topical application of a non-lethal dose of lindane to fourth instar larvae of a susceptible strain of Spodoptera littoralis (Boisd.). The induction reached its peak 8 h after treatment for 1-chloro-2,4-dinitrobenzene (CDNB) conjugating GSTs (1.5-fold), and 12 h after the treatment for 1,2-dichloro-4-nitrobenzene (DCNB) comjugating enzymes (1.3-fold). CDNB conjugating GST activity in an insecticide-resistant strain of S. littoralis was twice that measured in the non-induced suceptible larvae. No significant difference was observed with DCNB. GSTs of both larvae were purified by affinity chromatography using a glutathione-agarose column, which resulted in a 30-fold prufication and a yield of 50–70%. Purified GSTs were characterized using CDNB as the substrate. Apparent K m and V max values calculated for CDNB and reduced glutathione (GSH) were significantly different between the resistant strain and the susceptible one, whether induced by lindane or not. The lindane treatment modified only K m values for CDNB and GSH in susceptible larvae. Monodimensional SDS-PAGE of GSTs purified from both strains revealed a large band of 27 kDa apparent molecular weight.

Characterization of microsomal oxidative activities in a wild-type and in a DDT resistant strain of Drosophila melanogaster

Abstract Resistance of a laboratory selected DDT strain of Drosophila melanogaster (RalDDT R ) has been found to be monofactorial and correlated to an increased level of activity of the cytochrome P450-dependent mixed function oxidase (MFO). Both strains metabolize DDT and deltamethrin via MFO activity. However, the resistant strain does it more rapidly. The amount of DDT metabolites, including kelthane, bis-4-chlorophenyl acid, bis-4-chlorophenyl-ethanol, and 1,1-bis ( p -chlorophenyl)2,2-dichloroethane, is approximately 9-fold greater with RalDDT R microsomes than with the wild-type strain Raleigh (Ral). Production of deltamethrin metabolites is 2.7-fold higher within the resistant strain. As compared to insecticides, lauric acid and the two steroids used as substrates in this study present many more sites for MFO metabolic action. Lauric acid is hydroxylated on positions 11 and 12 by both strains, but the amount of metabolites formed is 10-fold higher with RalDDT R microsomes. The 2,22-dideoxyecdysone is converted to two polar metabolites when incubated with RalDDT R microsomal preparations. These unidentified metabolites are neither 2-deoxyecdysone nor ecdysone. Also reported for the first time is the metabolization of testosterone by insect microsomes, which gives 13 oxiderivatives formed at different rates, depending on the strains.

Mechanisms of resistance to organophosphates in Tetranychus urticae (Acari: Tetranychidae) from Greece.

We investigated the mechanisms conferring resistance to methyl-parathion (44-fold) and to methomyl (8-fold) in Tetranychus urticae from Greece by studying the effect of synergists on the resistance and the kinetic characteristics of various enzymes in a resistant strain (RLAB) and a susceptible reference strain (SAMB). It is shown that S,S,S-tributyl phosphorotrithioate, a synergist that inhibits esterases and glutathione S-transferases, and piperonyl butoxide, a synergist that inhibits cytochrome P450 mediated monooxygenases, did not affect the level of methyl-parathion or methomyl resistance in RLAB and that resistance ratios to both insecticides did not change significantly in the presence of either synergist. Isoelectric focusing of esterase allozymes on single mites revealed no differences in staining intensity and glutathione S-transferase activity was not significantly different in the two strains. The activity of two cytochrome P450 monooxygenase groups was compared. No significant difference of 7-ethoxyresorufin-O-diethylase activity was observed between strains that were two-fold higher in RLAB than in SAMB. The kinetic characteristics of acetylcholinesterase, the target enzyme of organophosphates and carbamates, revealed that acetylcholinesterase in RLAB was less sensitive to inhibition by paraoxon and methomyl in comparison with SAMB. I(50), the inhibitor concentration inducing 50% decrease of acetylcholinesterase activity was greater (119- and 50-fold with paraoxon and methomyl, respectively) and the bimolecular constant k(i) was lower (39- and 47-fold with paraoxon and methomyl, respectively) in RLAB compared to SAMB.

Analysis of methidathion resistance mechanisms in Phytoseiulus persimilis A.H

Abstract A resistant strain of Phytoseiulus persimilis selected by methidathion pressure for several years metabolizes the [ 14 C]methidathion faster than does the corresponding susceptible strain. The metabolism is for the main part glutathione dependent and gives the methidathion conjugate on glutathione as a first metabolite: S [5-methoxy-2-oxo-1,3,4-thiadiazol-3(2H)-yl]- l -glutathione. In addition, glutathione transferase with chlorodinitrobenzene as a substrate has a threefold lower K m in R strain than in S strain. Furthermore, this reaction is competitively inhibited by methidathion with a K i which is threefold lower in R than in S strain. These results indicated that in this strain of P. persimilis resistance is due to an elevated detoxication of methidathion by a glutathione transferase. Other parameters known to be able to induce resistance in arthropods have been compared in resistant and sensitive strains. Esterase and monooxygenase activity measured with chromogenic substrates are the same in the two strains as is the level of acetylcholinesterase and its inhibition by methidathion oxon. No difference between the two strains has been found in the penetration kinetics measured with [ 14 C]methidathion. These results indicated that glutathione transferase is the only mechanism which has been selected in P. persimilis , although other mechanisms are known to be involved in resistance to other insecticides in phytoseiid mites.

Purification and characterization of a carboxylesterase involved in malathion-specific resistance from Tribolium castaneum (Coleoptera: Tenebrionidae)

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.

Induction of cytochrome P450 activities in Drosophila melanogaster strains susceptible or resistant to insecticides.

We analysed Drosophila melanogaster cytochrome P450s (P450) through the measurements of four enzymatic activities: ethoxycoumarin-O-deethylase, ethoxyresorufin-O-deethylase, lauric acid hydroxylation, and testosterone hydroxylation. We did these measurements in two Drosophila strains: one is susceptible to insecticides (Cantons) and the other is resistant to insecticides by enhanced P450 activities (RDDTR). In addition, we also treated the flies with eight chemicals (beta-naphtoflavone, benzo-alpha-pyrene, 3-methylcholanthrene, phenobarbital, aminopyrine, rifampicin, prochloraz, and clofibrate) known to induces genes from the families CYP1, CYP2, CYP3, CYP4, and CYP6. Metabolisation of all the substrates by P450 from flies microsomes was observed. The chemicals had different effects on these activities, ranging from induction to inhibition. The effects of these chemicals varied with the strains as most of them were ineffective on the RDDTR strain. The results showed that P450-dependent activities are numerous in Drosophila. Regulation features of these activities are complex. The availability of mutant strains as RDDTR should allow fundamental studies of P450 in insects.