Gene_locus Report for: lepde-ACHE

The molecular and biochemical properties of two acetylcholinesterases (LdAChE1 and LdAChE2) from the Colorado potato beetle, Leptinotarsa decemlineata, were investigated in this study. Polyacrylamide gel electrophoresis in conjunction with western blotting with LdAChE1- or LdAChE2-specific antibodies suggested that LdAChE1 exists in a soluble form, whereas LdAChE2 exists in both soluble and amphiphilic forms with a glycophosphatidylinositol anchor. Both LdAChEs exist as homodimers with each monomer connected with a disulfide bond. LdAChE1 was the most highly expressed in the thorax followed by the head, leg, and abdomen, whereas LdAChE2 was the most highly expressed in the head, followed by the thorax, leg, and abdomen. The overall expression levels of LdAChE1, however, were higher than those of LdAChE2 in all examined tissues. Kinetic analysis using recombinant LdAChE1 and LdAChE2 showed that LdAChE2 has a 4.8-fold higher catalytic efficiency toward acetylthiocholine iodide compared to LdAChE1. LdAChE2 was more sensitive to organophosphorus and carbamate insecticides than LdAChE1. The addition of irreversibly phosphorylated LdAChE1 via paraoxon titration significantly reduced LdAChE2 inhibition by insecticides and glycoalkaloids, suggesting a sequestration role of soluble LdAChE1 in the chemical defense against xenobiotics. Taken together, LdAChE2 may be the main enzyme for synaptic transmission, thus serving as a toxicologically more relevant target, whereas the soluble LdAChE1 may function as a bioscavenger.

Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos-methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance-associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos-methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides.

Insecticide-resistant populations of Colorado potato beetle (CPB), with insensitive acetylcholinesterase (AChE) have recently been reported from commercial potato fields of Hamedan province in west of Iran. The objective of this study was to clarify the molecular mechanism of this insensitivity. The serine to glycine change at position 291 (S291G) in the AChE gene was found previously in an azinphosmethyl-resistant strain of CPB (AZ-R). PCR-RFLP assays were used to monitor the frequency of the S291G resistance mutation in resistant field populations of CPB, Aliabad, Bahar, Dehpiaz, and Yengijeh. The S291G mutation was detected in 66.6, 73.3, 53.3 and 26.6% of Bahar, Dehpiaz, Aliabad and Yengijeh populations, respectively. Moreover, only 25% of samples from the resistant field populations were homozygous for S291G mutation. There was no significant correlation between the mutation frequencies and resistance levels in the resistant populations, indicating that other mutations may contribute to this variation. PCR-SSCP method was used to find sequence variation in the AChE gene. Based on the published nucleotide sequence information on AChE gene (GenBank L41180.1), five primer pairs were designed to amplify specific PCR products of 306 bp (first fragment: codons 24-142), 370 bp (second fragment: codons 141-261), 403 bp (third fragment: codons 248-381), 335 bp (fourth fragment: codons 376-486), and 335 bp (fifth fragment: codons 488-598). No specific PCR product of desirable size for either the fourth and fifth fragments could be obtained. The DNA amplification products were subjected to SSCP analysis to identify the DNA sequence variations between the susceptible strain and resistant populations. Ninety-five beetles of susceptible strain (15 beetles) and four field populations (20 of each) were screened. Allelic differences were detected by distinctive electrophoretic patterns of each single strand. Nucleotide sequence variations in the different SSCP patterns were verified by direct DNA sequencing. Alignment of the AChE gene sequences with the sequencing results revealed 16 point mutations (V44G, E128D, R140G, I143F, T248S, F250S, G251C, I261M, S265I, S291G, G353R, L356R, E366K, S378E, S378R, E382D) from the field populations of CPB, which may contribute to the AChE insensitivity. Further analysis on the RNA level is necessary for clarification and validation of this contention.

The molecular and biochemical properties of two acetylcholinesterases (LdAChE1 and LdAChE2) from the Colorado potato beetle, Leptinotarsa decemlineata, were investigated in this study. Polyacrylamide gel electrophoresis in conjunction with western blotting with LdAChE1- or LdAChE2-specific antibodies suggested that LdAChE1 exists in a soluble form, whereas LdAChE2 exists in both soluble and amphiphilic forms with a glycophosphatidylinositol anchor. Both LdAChEs exist as homodimers with each monomer connected with a disulfide bond. LdAChE1 was the most highly expressed in the thorax followed by the head, leg, and abdomen, whereas LdAChE2 was the most highly expressed in the head, followed by the thorax, leg, and abdomen. The overall expression levels of LdAChE1, however, were higher than those of LdAChE2 in all examined tissues. Kinetic analysis using recombinant LdAChE1 and LdAChE2 showed that LdAChE2 has a 4.8-fold higher catalytic efficiency toward acetylthiocholine iodide compared to LdAChE1. LdAChE2 was more sensitive to organophosphorus and carbamate insecticides than LdAChE1. The addition of irreversibly phosphorylated LdAChE1 via paraoxon titration significantly reduced LdAChE2 inhibition by insecticides and glycoalkaloids, suggesting a sequestration role of soluble LdAChE1 in the chemical defense against xenobiotics. Taken together, LdAChE2 may be the main enzyme for synaptic transmission, thus serving as a toxicologically more relevant target, whereas the soluble LdAChE1 may function as a bioscavenger.

Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos-methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance-associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos-methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides.

ABSTRACT: BACKGROUND: Invasive pest species offers a unique opportunity to study the effects of genetic architecture, demography and selection on patterns of genetic variability. Invasive Colorado potato beetle (Leptinotarsa decemlineata) populations have experienced a rapid range expansion and intense selection by insecticides. By comparing native and invasive beetle populations, we studied the origins of organophosphate (OP) resistance-associated mutations in the acetylcholinesterase 2 (AChE2) gene, and the role of selection and demography on its genetic variability.
RESULTS: Analysis of three Mexican, two US and five European populations yielded a total of 49 haplotypes. Contrary to the expectations all genetic variability was associated with a point mutation linked to insecticide resistance (S291G), this mutation was found in 100% of Mexican, 95% of US and 71% of European beetle sequences analysed. Only two susceptible haplotypes, genetically very differentiated, were found, one in US and one in Europe. The genetic variability at the AChE2 gene was compared with two other genes not directly affected by insecticide selection, diapause protein 1 and juvenile hormone esterase. All three genes showed reduction in genetic variability indicative of a population bottleneck associated with the invasion.
CONCLUSIONS: Stochastic effects during invasion explain most of the observed patterns of genetic variability at the three genes investigated. The high frequency of the S291G mutation in the AChE2 gene among native populations suggests this mutation is the ancestral state and thus, either a pre-adaptation of the beetle for OP resistance or the AChE2 is not the major gene conferring OP resistance. The long historical association with host plant alkaloids together with recombination may have contributed to the high genetic variation at this locus. The genetic diversity in the AChE2 locus of the European beetles, in turn, strongly reflects founder effects followed by rapid invasion. Our results suggest that despite the long history of insecticide use in this species, demographic events together with pre-invasion history have been strongly influential in shaping the genetic diversity of the AChE2 gene in the invasive beetle populations.

Insecticide-resistant populations of Colorado potato beetle (CPB), with insensitive acetylcholinesterase (AChE) have recently been reported from commercial potato fields of Hamedan province in west of Iran. The objective of this study was to clarify the molecular mechanism of this insensitivity. The serine to glycine change at position 291 (S291G) in the AChE gene was found previously in an azinphosmethyl-resistant strain of CPB (AZ-R). PCR-RFLP assays were used to monitor the frequency of the S291G resistance mutation in resistant field populations of CPB, Aliabad, Bahar, Dehpiaz, and Yengijeh. The S291G mutation was detected in 66.6, 73.3, 53.3 and 26.6% of Bahar, Dehpiaz, Aliabad and Yengijeh populations, respectively. Moreover, only 25% of samples from the resistant field populations were homozygous for S291G mutation. There was no significant correlation between the mutation frequencies and resistance levels in the resistant populations, indicating that other mutations may contribute to this variation. PCR-SSCP method was used to find sequence variation in the AChE gene. Based on the published nucleotide sequence information on AChE gene (GenBank L41180.1), five primer pairs were designed to amplify specific PCR products of 306 bp (first fragment: codons 24-142), 370 bp (second fragment: codons 141-261), 403 bp (third fragment: codons 248-381), 335 bp (fourth fragment: codons 376-486), and 335 bp (fifth fragment: codons 488-598). No specific PCR product of desirable size for either the fourth and fifth fragments could be obtained. The DNA amplification products were subjected to SSCP analysis to identify the DNA sequence variations between the susceptible strain and resistant populations. Ninety-five beetles of susceptible strain (15 beetles) and four field populations (20 of each) were screened. Allelic differences were detected by distinctive electrophoretic patterns of each single strand. Nucleotide sequence variations in the different SSCP patterns were verified by direct DNA sequencing. Alignment of the AChE gene sequences with the sequencing results revealed 16 point mutations (V44G, E128D, R140G, I143F, T248S, F250S, G251C, I261M, S265I, S291G, G353R, L356R, E366K, S378E, S378R, E382D) from the field populations of CPB, which may contribute to the AChE insensitivity. Further analysis on the RNA level is necessary for clarification and validation of this contention.

A survey of resistance to five conventional insecticides was conducted in 2009 and 2010 for the first generation 4th-instar larvae of Leptinotarsa decemlineata from Urumqi, Changji, Qitai and Qapqal. Compared with the Tekes population, a reference susceptible population, the Changji and Qapqal populations exhibited very high to moderate levels of resistance to cyhalothrin and deltamethrin, moderate to high levels of resistance to carbosulfan and carbofuran, and low levels of resistance to azinphosmethyl. Moreover, the Urumqi and the Qitai populations reached a high and a moderate level of resistance to carbosulfan, respectively. Synergistic effects of triphenyl phosphate, diethylmeleate, and piperonyl butoxide on cyhalothrin and carbosulfan in Changji population revealed that cytochrome P450s were involved in the resistance to cyhalothrin but not carbosulfan. A modified bi-PASA was developed to simultaneously detect point mutations of S291G in the AChE and L1014F in the LdVssc1 genes. The former mutation resulted in the resistance to carbamates and the latter in the resistance to pyrethroids. The rates of homozygous and heterozygous resistant individuals to carbamates (S291G mutation) were 17.6% and 14.7%, 50.6% and 42.2%, 49.9% and 41.7%, 51.3% and 41.4%, and 44.8% and 47.4%; to pyrethroids (L1014F mutation) were 5.8% and 8.7%, 36.1% and 27.0%, 41.8% and 24.8%, 12.2% and 9.7%, and 7.9% and 10.6%, respectively, in samples from Tekes, Changji, Qapqal, Urumqi and Qitai. I392T point mutation in the AChE was detected by RT-PCR among 18 individuals from Changji, Qapqal, Urumqi and Qitai. These results demonstrated that point mutations of S291G in the AChE and L1014F in the LdVscc1 are responsible for, at least partially, the resistance to carbamates and pyrethroids in L. decemlineata in some field populations in northern Xinjiang Uygur autonomous region.

The Colorado potato beetle (CPB), Leptinotarsa decemlineata is an important economic pest of potato worldwide. Resistance to organophosphates and carbamates in CPB has been associated in some cases to point mutations in the acetylcholinesterase (AChE) gene Ldace2, an orthologue of Drosophila melanogaster Dmace2. In this paper we report cloning and sequencing of Ldace1, an orthologue of Anopheles gambiae Agace1 that was previously unknown in CPB. The Ldace1 coding enzyme contains all residues conserved in a functionally active AChE. Ldace1 is expressed at higher levels (between 2- and 11-fold) than Ldace2 in embryos, in the four larval instars and in adults. Specific interference of Ldace1 by means of dsRNA injection resulted in a reduction of AChE activity to an approximate 50% compared to control, whilst interference of Ldace2 reduced AChE activity to an approximate 85%. Analysis of zymograms of AChE activity after interference indicates that LdAChE1 is the enzyme predominantly responsible for the activity visualised. Interference of Ldace1 in CPB adults caused a significant increase in mortality (43%) as early as three days post-injection (p.i.), suggesting the essential role of Ldace1. Interference of Ldace2 also caused a significant increase in mortality (29%) compared to control, although at seven days p.i. The effect of the interference of Ldace1 on susceptibility to the organophosphate chlorpyrifos points out that LdAChE1 could be a main target for this insecticide. In the light of our results, studies associating resistance in CPB to mutations in Ldace2 should be reviewed, taking into consideration analysis of the Ldace1 gene.

BACKGROUND Pyrethroids and organophosphates are the most frequently used insecticides for Colorado potato beetle, Leptinotarsa decemlineata (Say), control in the Czech Republic. Based on molecular methods, organophosphate and pyrethroid resistance alleles have been detected in samples from three sites. The accuracy of restriction fragment length polymorphism (RFLP) and bidirectional polymerase chain reaction amplification of specific alleles (Bi-PASA) for detection of resistance alleles is compared. RESULTS: Leptinotarsa decemlineata from three sites showed higher frequencies of resistance alleles to organophosphates than to pyrethroids. The rates of occurrence of individuals homozygous resistant (RR) to pyrethroids ranged from 20.0 to 22.9%, while the rates of occurrence of individuals RR to organophosphates ranged from 52.9 to 66.7%. The incidences of individuals with resistance alleles to both organophosphates and pyrethroids ranged from 8.6 to 13.6%. No relationship was found between incidence of the pyrethroid resistance allele and site, while incidence of the organophosphate resistance allele differed significantly according to site. CONCLUSION: Both RFLP and Bi-PASA were suitable for detecting resistance alleles to pyrethroids, and in most cases also for detecting resistance alleles to organophosphates. In contrast to Bi-PASA, RFLP was also suitable for samples with lower DNA quality when testing for the resistance allele to pyrethroids. On the other hand, RFLP was not as accurate as Bi-PASA in detection of the organophosphate resistance allele.

The functional attributes of specific point mutations, R30K, S291G, and I392T, associated with full-length acetylcholinesterase (AChE) cDNAs of organophosphate (OP)- and carbamate-resistant Colorado potato beetles (CPB), were determined using site-directed mutagenesis and baculovirus expression. Enzymatic and inhibitory properties of altered recombinant acetylcholinesterases(rAChEs) were examined. S291G increased the hydrolysis of substrates with larger substituted alkyl groups (e.g., BTC vs ATC) and increased the inhibitory action of inhibitors with larger alkyl groups (e.g., paraoxon, DFP, and N-propyl carbofuran vs. azinphosmethyl-oxon and N-methyl carbofuran). R30K in conjunction with S291G enhanced the hydrolysis activity of larger substrates and the inhibitory action of larger inhibitors. I392T attenuated the effects of S291G in that the altered rAChE with both S291G and I392T elicited substrate specificity and inhibitory properties more similar to the susceptible form of AChE without mutations.

The BERTS strain of Colorado potato beetle (CPB) was found to be highly resistant to N-methyl carbofuran but relatively susceptible to azinphosmethyl. N-Methyl carbofuran resistance was found to correlate well with acetylcholinesterase (AChE) insensitivity. In becoming resistant to N-methyl carbofuran, the AChE of the BERTS strain became more sensitive to N-propyl carbofuran inhibition. This negative cross-insensitivity correlated well to the increased relative toxicity of the BERTS strain to N-propyl carbofuran compared to the susceptible SS strain. BERTS beetles were sorted into BERTS-R and BERTS-S substrains using their AChE activity profiles. Sequence comparisons of AChE cDNAs from the two substrains revealed the presence of the point mutation that results in the S291G substitution previously found in the AChE of the azinphosmethyl-resistant AZ-R strain of CPB. A novel mutation present only in BERTS-S CPB, however, resulted in an additional I392T substitution in the AChE and apparently reverses the resistance conferring properties of the S291G substitution.

Three DNA-based genotyping techniques, bi-directional PCR amplification of specific allele (bi-PASA), single-stranded conformational polymorphism (SSCP) and minisequencing, have been developed and compared for the detection of the S291G (insensitive acetylcholinesterase) and L1014F (insensitive sodium channel) mutations associated with azinphos-methyl and permethrin resistance, respectively, in the Colorado potato beetle (Leptinotarsa decemlineata). Extraction of genomic DNA from individual neonates that were hatched from previously collected egg masses is the most efficient and reliable means to obtain suitable templates in terms of convenience, economy, speed and DNA quality. Bi-PASA, employing two allele-specific primers, appears to be the most efficient and rapid genotyping method for the simultaneous detection of both resistant/susceptible homozygous (SS, RR) and heterozygous (SR) alleles. Its resolution, however, is strongly dependent on the quality of template genomic DNA. SSCP also allows unambiguous genotyping, including the detection of heterozygous alleles, and is less dependent on template DNA quality, but requires a longer processing time. Minisequencing is amenable to a 96-well microtiter plate format for the processing of a large number of samples and allows direct detection of resistant/susceptible homozygous alleles but is not as efficient as the PASA and SSCP in detecting heterozygous alleles. In considering the advantages and disadvantages of each technique, DNA-based genotyping is best employed in combinations, with the bi-PASA as the primary method and the SSCP and minisequencing as the secondary validating methods. These methods are rugged, rapid, cost-effective and capable of resolving SS, RR and SR individuals. The availability of such DNA-based genotyping techniques, using neonate genomic DNA as templates, will enable the precise monitoring of the resistant and susceptible allele frequencies, including those of heterozygote individuals, in field populations of L. decemlineata.

An A to G point mutation that results in a serine to glycine amino acid change (S291G) in the acetylcholinesterase (AChE, EC 3.1.1.7) gene was identified previously as associated with azinphosmethyl resistance in Colorado potato beetle due to target site insensitivity. To efficiently validate the detection process of the S291G mutation and base the DNA diagnostic method on direct determination of nucleic acid sequence, a single-stranded conformational polymorphism (SSCP) protocol and a minisequencing reaction were developed. SSCP protocols using a 163-bp DNA template that spans the mutation resulted in an easy, rapid, cheap, and rugged DNA-based diagnostic method, which was capable of separating azinphosmethyl-susceptible and - resistant beetles. For minisequencing, PCR-amplified and biotinylated DNA templates from both susceptible and resistant beetles, which contain the mutation site, were bound to streptavidin-coated microplate strips. Minisequencing was accomplished with a detection primer that annealed adjacent to the point mutation, digoxigenin-labeled dATP, or alternatively, digoxigenin-labeled dUTP and AmpliTaq polymerase. The sequencing reaction added a digoxigenin-labeled dATP only when matched to the biotinylated DNA template (dATP and 3'...GGTCA...5'). Digoxigenin-labeled DNA was detected using peroxidase-conjugated digoxigenin antibodies and quantitated as optical density (OD) at 450 nm in a microplate reader. The OD readings obtained with digoxigenin-labeled dATP in the presence of susceptible AChE DNA template was 0.319 +/- 0.05, which was significantly higher than that obtained in the presence of the azinphosmethyl-resistant template (0.031 +/- 0.018) (P < 0.001). These highly significant results agree well with the susceptibility of AChE from individual insects as judged by AChE inhibition by azinphosmethyl-oxon and further support the contention that A to G point mutation, which occurs only in AChE gene of azinphosmethyl-resistant beetles, is responsible for enzyme insensitivity. Compared with SSCP, the minisequencing reaction provides a direct means to validate this specific point mutation. Coupling minisequencing with the ease and durability of SSCP will allow us to determine the presence or absence of the S291G mutation in an efficient and unambiguous manner. As such, similar approaches could be used to validate point mutations in any resistant strain of insect.

A serine to glycine point mutation of the acetylcholinesterase (AChE, EC 3.1.1.7) gene was identified previously in an azinphos-methyl-resistant (AZ-R) strain of the Colorado potato beetle, Leptinotarsa decemlineata(Say). To validate this mutation, the polymerase chain reaction (PCR) amplification of specific alleles (PASA) was coupled to a functional assay for AChE in individual insects. The remaining activities of AChE after inhibition by 50 uM azinphos-methyl-oxon was 22.3% +/- 6.5 and 48.0% +/- 3.9 (mean SD,n= 13) for the beetles from the azinphos-methyl-susceptible (SS) and AZ-R strains, respectively. This pattern of AChE inhibition established that AChE from the AZ-R beetles were significantly less sensitive to azinphos-methyl-oxon inhibition than that from the SS beetles. Using a competitive PASA technique to diagnose the azinphos-methyl-resistance allele of the AChE gene in these insects, the resistance allele was found in all AZ-R beetles but was never found in the SS beetles. These results agree well with the level of susceptibility of the AChE in individual insects and support the contension that the serine to glycine mutation is responsible for the reduced sensitivity of the AChE to inhibition by azinphos-methyl-oxon in the AZ-R strain of beetles. The utilization of the PASA technique for resistance monitoring in Colorado potato beetle is also discussed.

A serine to glycine point mutation of acetylcholinesterase (AChE, EC 1.1.1.7) was identified in an azinphosmethyl-resistant strain of Colorado potato beetle [Leptinotarsa decemlineata (Say)]. The position of the mutation corresponds to Val 238 of the Torpedo AChE and represents the first amino acid residue to form the alpha-helix, alpha-E'1. The predicted secondary structure of the mutation-containing region of AChE suggested that the transition from the turn to the alpha-helix occurs sooner in the sequence when serine is replaced by glycine. Thus, conformational changes in the AChE due to the alpha-helix deformation were expected to impinge upon both the catalytic and the peripheral binding sites, resulting in the modification of the bindings of organophosphorus insecticides and other ligands to these sites. The mutation appeared to be associated with the fitness of the beetle. The intrinsic rate of increase of the azinphosmethyl-resistant (AZ-R) strain was relatively low when the beetles were reared on the Russet Burbank potato cultivar, but was relatively high when they were reared on the NDA 1725-1 potato cultivar. Because these two potato cultivars contain different amounts of steroidal glycoalkaloids (e.g., alpha-solanine and alpha-chaconine), the different fitness of the AZ-R strain on different potato cultivars may be partially attributed to the increased sensitivity of the azinphosmethyl-resistant form of AChE to the inhibition by alpha-solanine and reduced sensitivity to alpha-chaconine as previously reported.

A cDNA encoding acetylcholinesterase (AChE, EC 1.1.1.7) was cloned from a cDNA library constructed from an insecticide-susceptible strain of Colorado potato beetle, Leptinotarsa decemlineata (Say). The complete amino acid sequence of AChE deduced from the cDNA consisted of 29 residues for the putative signal peptide and 600 residues for the mature protein with a predicted molecular weight of 67,994. Northern blot analysis of poly(A) RNA showed an approx 13.1-kb transcript. The mature protein sequence had 57 and 61% of amino acid residues identical to those of Drosophila melanogaster and Anopheles stephensi, respectively, and produced a remarkably similar hydropathy profile when compared to those of the two dipterous species. The three residues (Ser, Glu and His) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds were completely conserved when compared to the other seven AChEs from a broad range of animal species reported to date. Other properties of the deduced protein of AChE, including molecular weight and amino acid composition, agreed well with those of a previously reported study on the purified AChE from the same insect species. All these features firmly established that the cloned cDNA encodes AChE in Colorado potato beetle.