Jean-Marc Bride

Drosophila acetylcholinesterase: Mechanisms of resistance to organophosphates

Quantitative and qualitative changes of acetylcholinesterase can affect the sensitivity of insects to insecticides. First, the amount of acetylcholinesterase in the central nervous system is important in Drosophila melanogaster, flies which overexpress the enzyme are more resistant than wild-type flies. On the contrary, flies which express low levels of acetylcholinesterase are more susceptible. An overproduction of acetylcholinesterase outside the central nervous system also protects against organophosphate poisoning, that is, flies producing a soluble acetylcholinesterase, secreted in the haemolymph, are resistant to organophosphates. Second, resistance can also result from a qualitative modification of acetylcholinesterase. Four mutations have been identified in resistant strains: Phe115 to Ser, Ileu199 to Val, Gly303 to Ala and Phe368 to Tyr. Each of these mutations led to a different pattern of resistance and combinations between these mutations led to highly resistant enzymes.

Biochemical characterization of the esterases A1 and B1 associated with organophosphate resistance in the Culex pipiens L. Complex

Abstract Two esterases, A1 and B1, displaying a high activity in organophosphate (OP) resistant Culex pipiens L. from southern France and in C. quinquefasciatus Say from California, respectively, have been analyzed. Both enzymes are shown to be soluble and to constitute a large proportion of the proteins (1–3% for esterase A1 and 6–12% for esterase B1). The size of native esterase A1 was estimated between 118 and 134 kDa, that of esterase B1 67 kDa. Upon SDS denaturation, esterase B1 leads a single polypeptide of 67 kDa which suggests that it is a monomeric protein; esterase A1 leads also a single polypeptide of 60 kDa suggesting a homodimeric structure of the protein. These observations are discussed with regards to esterase E4 of Myzus persicae Sultz.

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.).

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.

Drosophila melanogaster acetylcholinesterase: Identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes

AceIJ29 and AcIJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23° C for AceIJ29 or above 25° C for AceIJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser374 to Phe in AceIJ29 and Pro75 to Leu in AceIJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp248 by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.

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.

Very high conservation between Cyp6a2 from Drosophila melanogaster and its ortholog Cyp6a26 from D-simulans

Although Drosophila simulans is closely related to D. melanogaster, very few cytochrome P450 genes have been studied in this species until now. As Cyp6a2 from D. melanogaster is a major gene implicated in the detoxification of xenobiotic molecules, we decided to look for its ortholog in D. simulans. The isolated gene, Cyp6a26, presents structural characteristics very similar to those of Cyp6a2: an identical size of 1 590‐bp comprising two exons separated by a 69‐bp intron and a nucleotide sequence homology of 95%. Many putative transcriptionally important motifs were identified in the upstream DNAs of the two genes but only 16 elements are in common positions. Treatment of flies with phenobarbital leads to an increased production of Cyp6a26 mRNAs. The expression of Cyp6a26 mRNAs varies following developmental stages in the same manner as Cyp6a2. Immunohistochemistry experiments of phenobarbital‐treated adult drosophila show that the spatial expression pattern of the two proteins is also conserved between the two species. All these data argue in favor of the conservation of the function of these homologous genes between the two Drosophila species.

Insect Acetylcholinesterase and Resistance to Insecticides

Extensive utilization of pesticides against insects provides us with a good model for studying the adaptation of an eukaryotic genome submitted to a strong selective pressure. Since the early 1950s, organophosphates and carbamates have been widely used to control insect pests around the world. These insecticides are hemisubstrates that inactivate acetylcholinesterase by phosphorylating or carbamylating the active serine (Aldridge, 1950). A mechanism for insect resistance to insecticides consists of the alteration of acetylcholinesterase which is less sensitive to organophosphates and carbamates. In 1964, Smissaert first described a resistant acarina carrying a modified acetylcholinesterase. Since this report, altered acetylcholinesterases have been detected in several resistant insect species such as aphids, colorado potato beetle or mosquitoes (review in Fournier and Mutero, 1994). Resistance is very variable, from 2 to 200, 000 fold depending on the species or on the strain, suggesting that several mutations may be responsible for resistance of the enzyme (Pralavorio and Fournier, 1992).