Recent studies have revealed that non-Cyclorrhapha insects possess two acetylcholinesterases (AChEs): Drosophila Ace-orthologous (o-Ace) and -paralogous (p-Ace) forms. In these insects, p-Ace is considered as a neural target of insecticides because mutations conferring insecticide insensitivity have been consistently identified in equivalent forms from various insects. To clarify the functional differentiation between the two AChE isoforms, we characterized the expression of two AChE genes in nerve cell-rich and -poor tissues of German cockroaches. The two AChE genes were expressed at comparable levels in both the nerve cord and head. Two AChE isoforms with mutually discriminatory fenitroxon sensitivities were exhibited in the nerve cord, indicating that o-Ace and p-Ace function in the neurons. pAce was mainly expressed in the nerve cord and head, while o-Ace was ubiquitously expressed at a comparable level. Moreover, o-Ace transcripts as well as AChE activity were detected in the fat body where no nervous tissue is observed. No AChE activity was detected in the hemolymph. These results show that p-Ace is better as an insecticidal target because of its abundant transcripts in nervous tissue. In addition, the roles of o-Ace, which is present ubiquitously without secretion into the hemolymph in insects, are discussed.
Several amino acid substitutions causing insensitivity have been found in the active site of Ace-paralogous acetylcholinesterase (AP-AChE); Gly119Ser (Culex pipiens, Anopheles gambiae), Ala201Ser (Aphis gossypii), Phe290Val (Nephotettix cincticeps), Ser331Phe (Myzus persicae, A. gossypii), Phe331Cys (Tetranychus urticae) and Phe331Trp (Cx. tritaeniorhynchus). To confirm the responsibility of these substitutions to the insensitivity, the six substitutions were introduced into the AP-AChE cDNA of Cx. tritaeniorhynchus resistant strain (Toyama), and their biochemical properties were examined by using a baculovirus-insect cell system. The substitution Gly119Ser gave the enzyme a high level of insensitivity to carbamate insecticides but a slight insensitivity to organophosphates. On the other hand, an amino acid substitution Phe331Trp located in the acyl pocket induced a very high level of insensitivity to organophosphates and ten times lower insensitivity to carbamates. The amino acid replacement appear to render the acyl pocket less hydrophobic and smaller, and then alter the accessibility of the substrates and inhibitors to this site.
Mechanisms of resistance to pirimicarb were surveyed in Iranian populations of the peach-potato aphid, Myzus persicae. Insecticide bioassays were carried out using topical application on three different populations collected from Karaj and Rasht in the Northern provinces. Results of the bioassays indicated that Rasht and Karaj-S populations were susceptible to pirimicarb. A population collected from the college greenhouse (Karaj-R) was resistant to pirimicarb. LD 50 (Ͼ100 ng/insect) of pirimicarb was not calculated for the Karaj-R population due to limited solubility of this insecticide. The activity and PAGE patterns of esterase in resistant (Karaj-R) and susceptible populations showed that one mechanism of resistance to pirimicarb was esterase-based resistance. Esterase activity of the Karaj-R population was 3.25-fold higher than the susceptible population (Karaj-S), and one of the two esterase bands with higher activity in the Karaj-R population showed strong susceptibility to pirimicarb. The acetylcholinesterase (AChE) of the Karaj-R population had lower affinity to artificial substrates, acetylthiocholine, propionylthiocholine and butyrylthiocholine, than Rasht and Karaj-S populations. The I 50 of pirimicarb for Karaj-R, Karaj-S and Rasht populations was 1.89ϫ 10 Ϫ5 M, 1.37ϫ10 Ϫ8 M and 1.37ϫ10 Ϫ8 M, respectively. These results suggest that AChE of the Karaj-R population is insensitive to pirimicarb. Ratios of AChE insensitivity of the Karaj-R population to the Karaj-S population were 1,384.6, 11.8 and 2.7 for pirimicarb, monocrotophos and aldicarb, respectively; however, the ratios were 0.32, 0.15 and 0.10 for carbofuran, propoxure and MTMC, respectively, showing negatively correlated sensitivity to pirimicarbinsensitive AChE.
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