**5. Role of AChE in Colorado potato beetle resistance to carbamates and organophosphates**

Organophosphates and carbamates are neural toxins, characterized by high toxicity and a quick action. They act inhibitory on acetylcholinesterase (AChE), during neural transmis‐ sion. Organophosphates phosphorylate enzymes, whereas carbamates form an enzymeinhibitor complex. The forming of this complex is not irreversible but the reactivation rate is 105 –106 slower when compared to a similar reaction in case of ACh and AChE. The enzyme cannot disintegrate new ACh. Although organophosphates and carbamates have a similar mode of action, there are also some pronounced differences between them, mainly as a result of different binding site in the active center and due to different geometries of nucleophilic attack [71].

Acetylcholinesterase (AChE; EC 3.1.1.7) is a key enzyme in the nervous system (55), terminat‐ ing nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Carboxylesterases belong to a multifunctional carboxylesterase/cholinesterase superfamily (CCE). They are ubiquitous in most living organisms, including animals, insects, plants, and microbes. CCEs, regarding their physiological and biochemical functions could be divided in three groups: dietary/detoxification, hormone/semiochemical processing, and neurodevelop‐ mental [72]. AChE is the major target for organophosphate and carbamate insecticides, which inhibit enzyme activity. Such inhibition of AChE causes excessive excitement in nerves, a blockage of neurotransmission, and the death of insects. Insensitivity of AChE to organophos‐ phates and carbamates is one of the important mechanisms for insecticide resistance. Changes in AChE lead to an efficient mechanism of resistance to OP and carbamate insecticides and that is site insensitivity at the target enzyme, acetylcholinesterase (AChE) [73, 74].

For testing the resistance to organophosphates and carbamates, caused by altered AChE, the determination of kinetic constants, especially the Michaelis-Menten constant (Km) is of great importance. It measures the affinity of the enzyme to its substrate [42].

The first case of AChE with decreased susceptibility to pesticides was described by Smissaert in 1961 [75]. Ioannidis et al. [76] first characterized a field population of the carbofuran-resistant CPB. The resistance was determined to be autosomal and monofactorial, leading to a decrease in AChE sensitivity to carbofuran inhibition. A study of a crude enzyme preparation [77] showed that AChE from the AZ-R strain had a 2.4-fold reduction of affinity to acetylthiocholine (ATCh) compared with AChE from a susceptible (SS) strain.

As previously recorded, altered acetylcholine esterase plays a critical role in resistance to organophosphates and carbamates [76, 78, 79]. The measurement of AChE activity is com‐ monly used as a biomarker of exposure to different pesticides [80]. Kinetic analysis of AChE was used to explain the resistance of some insect strains and the selectivity of some organo‐ phosphate and carbamate insecticides. A lot of different studies have described CPB resistance to OPs and CBs [46, 77, 81, 82]. Azinphosmethyl resistance has been reported in CPB; high level of resistance (136-fold) in a nearly isogenic CPB strain (AZ-R) was due to multiple resistance mechanisms, including reduced penetration, enhanced xenobiotic metabolism, and target site insensitivity [83]. Russel et al. [84] suggest that interspecific comparisons of bioassay and biochemical data suggest two major patterns of resistance to OPs/CBs resulting from an insensitive AChE: Pattern I resistance, which is generally more effective for carbamates, and Pattern II resistance, at least as effective for organophosphates as it is for carbamates and may even be specific to organophosphates in some cases.

The role of acetylcholinesterase (AChE) as the primary mechanism for removing the excitatory neurotransmitter, acetylcholine (ACh), from cholinergic synapses and its role as the target site for organophosphate and carbamate inhibitors and accumulation of ACh that results from the inhibition of AChE leads to the prolonged stimulation and, in many cases, the desensitization of the ACh receptors, eventually to severe neurological disruption, and ultimately to death [85]. Since AChE causes death, irreversible inhibitors have been developed as insecticides: organophosphates and carbamates. They have similar properties to acetylcholine but are hemisubstrates ultimately leading to irreversible inhibition of the enzyme. This inhibition leads to an accumulation of acetylcholine in the synapses (active site of the enzyme is therefore occupied and incapable of hydrolyzing its normal substrate) which in turn leaves the acetyl‐ choline receptors permanently open, resulting in the death of the insect [75].

sion. Organophosphates phosphorylate enzymes, whereas carbamates form an enzymeinhibitor complex. The forming of this complex is not irreversible but the reactivation rate

Acetylcholinesterase (AChE; EC 3.1.1.7) is a key enzyme in the nervous system (55), terminat‐ ing nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Carboxylesterases belong to a multifunctional carboxylesterase/cholinesterase superfamily (CCE). They are ubiquitous in most living organisms, including animals, insects, plants, and microbes. CCEs, regarding their physiological and biochemical functions could be divided in three groups: dietary/detoxification, hormone/semiochemical processing, and neurodevelop‐ mental [72]. AChE is the major target for organophosphate and carbamate insecticides, which inhibit enzyme activity. Such inhibition of AChE causes excessive excitement in nerves, a blockage of neurotransmission, and the death of insects. Insensitivity of AChE to organophos‐ phates and carbamates is one of the important mechanisms for insecticide resistance. Changes in AChE lead to an efficient mechanism of resistance to OP and carbamate insecticides and

that is site insensitivity at the target enzyme, acetylcholinesterase (AChE) [73, 74].

importance. It measures the affinity of the enzyme to its substrate [42].

(ATCh) compared with AChE from a susceptible (SS) strain.

even be specific to organophosphates in some cases.

For testing the resistance to organophosphates and carbamates, caused by altered AChE, the determination of kinetic constants, especially the Michaelis-Menten constant (Km) is of great

The first case of AChE with decreased susceptibility to pesticides was described by Smissaert in 1961 [75]. Ioannidis et al. [76] first characterized a field population of the carbofuran-resistant CPB. The resistance was determined to be autosomal and monofactorial, leading to a decrease in AChE sensitivity to carbofuran inhibition. A study of a crude enzyme preparation [77] showed that AChE from the AZ-R strain had a 2.4-fold reduction of affinity to acetylthiocholine

As previously recorded, altered acetylcholine esterase plays a critical role in resistance to organophosphates and carbamates [76, 78, 79]. The measurement of AChE activity is com‐ monly used as a biomarker of exposure to different pesticides [80]. Kinetic analysis of AChE was used to explain the resistance of some insect strains and the selectivity of some organo‐ phosphate and carbamate insecticides. A lot of different studies have described CPB resistance to OPs and CBs [46, 77, 81, 82]. Azinphosmethyl resistance has been reported in CPB; high level of resistance (136-fold) in a nearly isogenic CPB strain (AZ-R) was due to multiple resistance mechanisms, including reduced penetration, enhanced xenobiotic metabolism, and target site insensitivity [83]. Russel et al. [84] suggest that interspecific comparisons of bioassay and biochemical data suggest two major patterns of resistance to OPs/CBs resulting from an insensitive AChE: Pattern I resistance, which is generally more effective for carbamates, and Pattern II resistance, at least as effective for organophosphates as it is for carbamates and may

–106 slower when compared to a similar reaction in case of ACh and AChE. The enzyme cannot disintegrate new ACh. Although organophosphates and carbamates have a similar mode of action, there are also some pronounced differences between them, mainly as a result of different binding site in the active center and due to different geometries of

is 105

28 Insecticides Resistance

nucleophilic attack [71].

CPB populations resistant to azinphosmethyl contained two mutations in the AChE (S291G and R30K), which made the enzyme less sensitive to azinphosmethyl and carbofuran [73, 86]. In the strain resistant to carbofuran, the presence of two mutations (I392T and S291G) did not result in resistance, but the presence of just one (S291G) conferred high resistance to carbofuran and medium resistance to azinphosmethyl [73]. Compared to the susceptible strain, due to altered acetylcholine esterase, strain resistance to azinphosmethyl had a reduced substrate affinity for ATCh and azinphosmethyl oxon [87]. Modifications in acetylcholine esterase, resulting in resistance, may be selective. It was found that while one resistant strain was highly insensitive to arylcarbamates, another strain with the same affected enzyme was highly insensitive to organophosphates, but not arylcarbamates. Such changes in AChE made yet another resistant strain more sensitive to α-chaconine, a glycoalkaloid present in potatoes and an inhibitor of AChE. Additionally, modified AChE also had increased sensitivity to tomatine, which is also glycoalkaloid present in tomatoes [78].

Zhu and Clark [77] demonstrated that the less bulky substrates, such as ATCh, interact poorly with AChE from the AZ-R strain than AChE from the SS strain of CPB. Such structure–activity relationships may be an indication that a similar alteration in amino acid residues has taken place in the acyl pocket size in AChE from the AZ-R strain and has resulted in the altered substrate and inhibitor profile.

The target site of organophosphate (OP) and carbamate insecticides is the same; they inhibit the activity of AChE. The function of acetylcholinesterase (AChE) is degradation of acetyl‐ choline (ACh – neurotransmitter) in the insect cholinergic synapses. Mutations in the AChEencoding locus have been shown to confer target site insensitivity to organophosphate and carbamate insecticides, leading to modification of AChE (MACE). A range of other amino acid substitutions in AChE confer insecticide resistance, and these mutations typically reside near to or within the active site of the enzyme. Such AChE mutations, associated with insecticide resistance, mostly known as Ace in Drosophila, have also been observed in other species, including *L. decemlineata*. Based on bioassays and the literature, modified/insensitive AChE confers two major patterns of resistance to OPs/carbamates [84]. Pattern I resistance is characterized by significantly higher resistance ratios (RR) (much greater reduction in the sensitivity of AChE at the biochemical level) to carbamates than to organophosphate insecti‐ cides. Pattern II resistance is characterized by resistance ratios (and/or reductions in the sensitivity of AChE) that are approximately equivalent for both carbamates and OPs. There are also a few species for which an insensitive AChE has been reported and for which molecular data have been collected, but for which the resistance profiles for both OPs and carbamates have not been reported. For CPB, both patterns were registered.

In a few cases of each pattern, gene sequencing has identified the molecular nature of the alteration leading to the lowered sensitivity to inhibitors. Although it is not possible yet to relate with full confidence the mechanism by which these structural changes alter sensitivity to inhibitors. Pattern I mutations may involve changes in the active site, such as a common Gly^Ser mutation in the oxyan–ion hole, whereas Pattern II changes may result in a constriction of the cleft leading to an active site that limits the access of inhibitors and, presumably, of ACh itself. Insensitivity to inhibitors may be accompanied by a reduced ability to hydrolyze ACh. Whether this is always deleterious to the organism is unclear since it is generally considered that, as in vertebrates, AChE is present in insects at a level considerably in excess of that needed for basic neurological functions under normal physiological conditions.

Biochemical studies using an affinity-purified AChE from the SS strain established that the AChE associated with CPB possessed typical characteristics of other AChEs and consists of two different molecular forms: the major form (92%) was a hydrophilic dimer, whereas the minor form (8%) was an amphiphilic dimer. Both molecular forms had virtually identical molecular weights and isoelectric points. Amino acid analysis indicates that the mole percen‐ tages of amino acids of the AChE from CPB were highly comparable to those previously reported for AChE from Drosophila [77].

According to Zhu and Clark [77], affinity (Km) and hydrolyzing efficiency (Vmax) of AChE purified from a near-isogenic azinphosmethyl-resistant (AZ-R) strain of CPB to selected substrates, including acethylthiocholine, acetyl-(5-methyl) thiocholine, and propionythiocho‐ line, were lower than those of AChE purified from a susceptible (SS) strain. AChE from the SS strain was significantly inhibited by higher amounts of acethylthiocholine and acetyl-(fjmethyl) thiocholine, whereas AChE from the AZ-R strain was activated by higher amounts of all four substrates examined.

Finally, it is important to notice results on toxicological tests and measuring activity of AChE of CPB populations in Serbia, resistant to OPs and carbamates [88]. The order of resistance levels for OPs and carbamates was completely opposite. Experiments showed that acetylcho‐ linesterase (AChE) activity of CPB was very pronounced and easily measured. At a constant AChE concentration, increasing the substrate concentration will cause a positive, linear, and dependent increase in the reaction. The same applies in the reaction with constant substrate concentration and increased enzyme concentrations. AChE activity is significantly affected not only by location, but also by substrate concentration (acetylthiocholine iodide ATChI). Considering that ATChI (substrate) in increased concentrations inhibits normal AChE activity, it can be concluded that altered AChE affected the change in the population order. The total AChE activity is in correlation with the determined resistance to carbamates.
