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.

The sibling species Drosophila melanogaster and Drosophila simulans differ in the expression profile of glutathione S-transferases.

Two major forms of glutathione S-transferase are known in Drosophila melanogaster: GST D and GST 2. In the present paper we report the existence of a third major form of glutathione S-transferase in Drosophila simulans. Induction with phenobarbital revealed a different regulation of GST between these species. Despite the fact that these two species are closely related, there was a difference in the expression profile of the enzyme implicated in the detoxification system, suggesting variations in capacity to suit their environment.

Comparative ability to detoxify alder leaf litter in field larval mosquito collections.

The larvicidal effects of polyphenols from dietary alder leaf litter were investigated in different field collections of three detritivorous Aedes taxa (Ae. detritus, Ae. cataphylla, Ae. rusticus) and compared to the cytochrome P450 monooxygenase, glutathione S-transferase, and esterase activities. Larvae from polyphenol-rich habitats had a higher tolerance for polyphenols and higher midgut cytochrome P450 and esterase activities than larvae from polyphenol-poor habitats. Furthermore, the role of P450 enzymes in the mechanism of resistance to alder polyphenols was suggested by the synergistic effect in vivo of piperonyl butoxide in the resistant Ae. rusticus. This confirms the importance of polyphenols to larval mosquito performance, and provides evidence for the importance of specific detoxification mechanisms for tolerance to dietary polyphenols. Arch.

Tissue-specific induction and inactivation of cytochrome P450 catalysing lauric acid hydroxylation in the sea bass, Dicentrarchus labrax.

Microsomal cytochrome P450-dependent lauric acid hydroxylase activities were characterized in liver, kidney, and intestinal mucosa of the sea bass (Dicentrarchus labrax). Microsomes from these organs generated (omega-1)-hydroxylauric acid and a mixture of positional isomers including (omega)-, (omega-2)-, (omega-3)- and (omega-4)-hydroxylauric acids, which were identified by RP-HPLC and GC-MS analysis. Peroxisome proliferators, such as clofibrate and especially di(2-ethylhexyl) phthalate, increased kidney microsomal lauric acid hydroxylase activities. The synthesis of 11-hydroxylauric acid was enhanced 5.3-fold in kidney microsomes. Liver microsomal lauric acid hydroxylase activities were weakly affected and no significant induction was found in small intestine microsomes from clofibrate or di(2-ethylhexyl) phthalate-treated fish. The differences in lauric acid metabolisation and the tissue-specific induction by peroxisome proliferators suggest the involvement of several P450s in this reaction. Incubations of liver and kidney microsomes with lauric acid analogues (11- or 10-dodecynoic acids) resulted in a time- and concentration-dependent loss of lauric acid hydroxylase activities. The induction of these activities in fish by phthalates, which are widely-distributed environmental pollutants, may be taken into consideration for the development of new biomarkers.

Pyrethroid resistance in Culex quinquefasciatus from west Africa.

Pyrethroid resistance was investigated in thirty-three samples of Culex quinquefasciatus Say from twenty-five cities in Côte d'Ivoire and Burkina Faso. Permethrin resistance ratios at LC50 ranged from 9.5- to 82-fold in Côte d'Ivoire and from 17- to 49-fold in Burkina Faso. For deltamethrin, resistance ratios were lower and ranged from nine to thirty-eight in both countries. A strain was selected with permethrin to investigate resistance mechanisms. After forty-two generations of selection, permethrin resistance level reached 3750-fold, but deltamethrin resistance remained unexpectedly unchanged. This indicated that a specific mechanism was involved in permethrin resistance. Synergist assays and biochemical tests indicated that resistance was partly due to P450-dependent oxidases. A target site insensitivity (kdr) was also involved, associated with DDT cross resistance and a dramatic loss of permethrin knockdown effect on adults. This resistance should be taken into consideration when planning the use of pyrethroid-impregnated materials in urban areas, as Culex is by far the main source of nuisance. Any failure in nuisance control due to resistance is likely to demotivate people in using impregnated materials.

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.

Cytochrome P-450 field insecticide tolerance and development of laboratory resistance in grape vine populations of Drosophila melanogaster (Diptera: Drosophilidae).

Studies were conducted between 1993 and 1996 using 3 natural grape vine populations, 1 susceptible laboratory strain, and 1 resistant selected strain of Drosophila melanogaster L. In vitro monooxygenase activity (ethoxycoumarine-O-deethylation) (ECOD) was recorded from microsomal fractions of all strains. Results varied over a 6-fold range between susceptible laboratory Canton and resistant selected RDDT strains and over a 2-fold range between the Canton strain and natural populations of flies. Few significant variations of ECOD activity were detected among the natural populations despite many insecticide treatments, but activities were significantly correlated with toxicological tolerance to 5 of the 15 insecticides (deltamethrin, fipronil, chlorpyriphos ethyl, DDT, and diazinon). Moreover, immunoblotting responses of microsomal protein encoded by Cyp6A2 showed that the levels of expression were quantitatively correlated with toxicological tolerance to almost the same group of insecticides (deltamethrin, fipronil, chlorpyriphos ethyl, DDT, fenvalerate, and fenthion). However, the level of CYP6A2 expression in some natural strains (still weakly resistant) was almost comparable with one of the resistant strains. In vivo monooxygenase activity recorded in individual abdomens of flies showed that frequency distributions of ECOD activity in natural populations overlapped those of the resistant and laboratory strains, which were much narrower. Substantial and fast frequency changes (of the narrowness) that obtained in laboratory were related to either the time of rearing of 1 of the natural populations or selecting this population with an insecticide that has a toxicology correlated with both of the monooxygenase signs measured. Perspectives on using the CYP6A2 expression and ECOD activity for detecting a resistance mechanism by cytochrome P450 in field populations are discussed.

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.

A microfluorometric method for measuring ethoxycoumarin-O-deethylase activity on individual Drosophila melanogaster abdomens: interest for screening resistance in insect populations.

We developed a method for measuring ethoxycoumarin deethylase (ECOD) activity using a single Drosophila abdomen. The activities obtained were well correlated with the classic method from microsomes (r = 0.902). This new method, performed in microtitration plates, was at least six times more sensitive compared to the conventional cuvet fluorometric one. Moreover, it was possible among a large number of insects to differentiate those with low or high ECOD activities. This improved procedure has been checked upon crosses between resistant strain (with high ECOD activity) and susceptible strain (with low ECOD activity). The results demonstrate the possible separation of resistant phenotypes and emphasize the importance of this approach in assessing the spreading of insecticide resistance in natural populations of insects.

Fate of a terminal olefin with Drosophila microsomes and its inhibitory effects on some P-450 dependent activities.

In vitro bioassays were used to analyze the metabolism of the 11-dodecenoic acid (11-DDNA) by microsomes prepared from Drosophila melanogaster RalDDTR strain. 11-DDNA is metabolized to 11,12-epoxylauric acid (epoxyLA) in a NADPH-dependent way. The microsomal production of epoxyLA reaches a plateau very quickly, suggesting the occurrence of an enzyme inactivation process. After incubation of microsomes with (1-14C)11-DDNA, three proteins of Mr approximately 50 kDa were labeled. 11-DDNA inhibits the microsomal metabolism of lauric acid and 7-ethoxycoumarin in a time and NADPH-dependent process. An inhibition of metabolites generated from DDT and testosterone was also obtained but at higher concentrations. These results are discussed according to the fact that RalDDTR is an insecticide resistant strain characterized as a high metabolizer of the insecticide DDT and also of lauric acid, testosterone, and ethoxycoumarin.

Molecular polymorphism of head acetylcholinesterase from adult houseflies (Musca domestica L.).

Acetylcholinesterase (AChE) from housefly heads was purified by affinity chromatography. Three different native forms were separated by electrophoresis on polyacrylamide 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 G2s and G2m, where "s" refers to soluble and "m" to membrane bound.

Identification and affinity labeling of very high affinity binding sites for the phenylalkylamine series of Ca+ channel blockers in the Drosophila nervous system.

The interaction of putative Ca2+ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-[3H]D888 and (+/-)-[3H]verapamil. These ligands recognize a single class (Kd = 0.1-0.4 nM; Bmax = 1600-1800 fmol/mg of protein) of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a Kd value as exceptionally low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca2+ channel blockers as well as bepridil inhibited (-)-[3H]D888 binding in a competitive way with Kd values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor Kd = 0.24 nM), was used in photoaffinity experiments. A protein of Mr 135,000 +/- 5,000 was specifically labeled after ultraviolet irradiation.

Single-mosquito test to determine genotypes with an acetylcholinesterase insensitive to inhibition to propoxur insecticide.

A sensitive technique allowing to identify the three genotypes (AceSS, AceRR and AceRS) of the Ace gene existing in natural populations of Culex pipiens in southern France is described. The technique is based on the comparison of AChE (acetylcholinesterase) activity in 3 equal aliquots taken from the homogenate of a single mosquito (a) in absence of inhibitor (RA), (b) in presence of eserine that inhibits the AChE encoded by AceS and AceR alleles (RI) and (c) in presence of a concentration of propoxur inhibiting the AChE coded by the AceS allele but not by the AceR allele (RG). The mosquito tested is AceSS when RG = RI, AceRR when RG = RA and AceRS when RI less than RG less than RA.