Madeleine Pralavorio

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.

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.

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.

Drosophila Acetylcholinesterase: Analysis of Structure and Sensitivity to Insecticides by In Vitro Mutagenesis and Expression

Mutations in Drosophila hold promise for studying effects of protein alterations on development, neurophysiology and behavior of the organism. For that purpose, several mutations which affect acetylcholinesterase (AChE) were isolated (Greenspan et al.,1980; Morton and Singh, 1984; Pralavorio and Fournier, 1992). These mutations are either lethal, temperature-sensitive or modify the catalytic properties of the enzyme. Lethal and conditional mutations were obtained by mutagenesis experiments and mutations affecting the catalytic properties of the enzyme were found in insects resistant to insecticides which have an altered AChE less sensitive to inhibition by organophosphate and carbamate compounds.

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).