Jean-Baptiste Bergé

Voltage‐dependent Na+ channels in pyrethroid‐resistant Culex pipiens L mosquitoes

In some insect species, knockdown resistance (kdr) to pyrethroids and DDT is linked to point mutations in the sequence of the para-type voltage-dependent sodium channel gene. The effects of pyrethroids were assayed on six Culex pipiens strains: two were susceptible to pyrethroids and the four others displayed various levels of resistance, but, in each case, a kdr-type mechanism was strongly suggested. Degenerate primers were designed on the basis of the corresponding sequences of the para orthologous gene reported from several orders of insects. These primers were used to amplify the region of the sodium channel gene which includes the positions where the kdr and super-kdr mutations have been found in Musca domestica. As expected, the amplified fragment was highly homologous to the para sequences. The super-kdr-like mutation (methionine to threonine at position 918 of the M domestica para sequence) was never detected in any strain. In contrast, the same kdr mutation (leucine to phenylalanine at position 1014) was present in some Culex pyrethroid-resistant samples. An alternative substitution of the same leucine to a serine was detected in one strain slightly resistant to pyrethroids but highly resistant to DDT. These data have allowed us to design a PCR-based diagnostic test on genomic DNA to determine the presence or the absence of the kdr allele in single C pipiens collected in several countries. The validity of this test was checked by comparing the frequency of the resistance allele and the toxicological data.

Acetylcholinesterases from Musca domestica and Drosophila melanogaster Brain Are Linked to Membranes by a Glycophospholipid Anchor Sensitive to an Endogenous Phospholipase

Abstract: The sensitivity of acetylcholinesterases (AChEs) from Musca domestica and from Drosophila melanogaster to the phosphatidylinositol‐specific phospholipase C from Bacillus cereus and to the glycosylphosphatidylinositol‐specific phospholipase C from Trypanosoma brucei was investigated. B. cereus phospholipase C solubilizes membrane‐bound AChE, and both phospholipases convert amphiphilic AChEs into hydrophilic forms of the enzyme. The Upases uncover an immunological determinant that is found on other glycosylphosphatidylinositol‐anchored membrane proteins after the same treatment. This immunological determinant is also present on the native hydrophilic form of AChE. The polypeptide bearing the active site of the membrane‐bound enzyme migrates faster during sodium dodecyl sulfate‐polyacrylamide gel electrophoresis than the same polypeptide from the soluble enzyme. We conclude that AChE from insect brain is attached to membranes via a glycophospholipid anchor. This anchor is covalently linked to the polypeptide bearing the active esterase site of the enzyme and can be cleaved by an endogenous lipase.

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.

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.).

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.

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.

Target modification as a molecular mechanism of pyrethroid resistance in Drosophila melanogaster

Abstract “Knock down” resistance (kdr) to the pyrethroid deltamethrin was investigated using susceptible (Tubingen) and resistant (TubingenDDT) strains of Drosophila melanogaster. Toxicological and pharmacological data show that resistance involves a modification of the affinity of the insecticide for its receptor site on the voltage-dependent sodium channel. Genetic studies indicate that the kdr factor is linked to the second chromosome where one sodium channel gene, sch, is located. Cloning and sequencing the alleles of this gene from both strains revealed a single substitution that may be responsible for the loss of toxicity of deltamethrin in the resistant strain.