Role of Carboxylesterases (ALiE) Regarding Resistance to Insecticides: Case Study of Colorado Potato Beetle (Leptinotarsa decemlineata Say)

Abstract

Colorado potato beetle is one of the most important pests because of rapid and strongly developed resistance to insecticides. Resistant insects' populations may detoxify or degrade the toxin faster than susceptible insects, or quickly rid their bodies of the toxic molecules. Resistant populations may possess higher levels or more efficient forms of these enzymes. Insecticide metabolic destruction inside the target organism is a common defensive mechanism, decreasing the duration and intensity of the exposure of the target site, lowering the probability of a lethal outcome. Three major mechanisms of metabolic transformation of insecticides underlie the vast majority of examples of biotransformation-based resistance: (i) oxidation; (ii) ester hydrolysis; and (iii) glutathione conjugation. Pyrethrins, pyreth-roids, organophosphates, carbamates and other insecticides are degraded by hydrolysis. Insecticide detoxification primarily unfolds through molecule hydrolysis on different sites, thereby splitting ester, carboxyl-ester, amide and other chemical bonds. The most important hydrolytic enzymes are phosphoric triesters and carboxylesterases (ALiE esterases). Structural mutations in mutant carboxylesterases have now been widely described showing metabolic resistance to organophosphate and pyrethroid insecticides and relatively few cases of resistance to carbamates role. Carboxylesterases role in Colorado potato beetle resistance was confirmed by many authors.

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Chapter 7
Role of Carboxylesterases (ALiE) Regarding Resistance
to Insecticides: Case Study of Colorado Potato Beetle
(Leptinotarsa decemlineata Say)
Sladjan Stankovic and Miroslav Kostic
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/66254
Provisional chapter
Role of Carboxylesterases (ALiE) Regarding Resistance to
Insecticides: Case Study of Colorado Potato Beetle
(Leptinotarsa decemlineata Say)
Sladjan Stankovic and Miroslav Kostic
Additional information is available at the end of the chapter
Abstract
Colorado potato beetle is one of the most important pests because of rapid and
strongly developed resistance to insecticides. Resistant insects’ populations may
detoxify or degrade the toxin faster than susceptible insects, or quickly rid their
bodies of the toxic molecules. Resistant populations may possess higher levels or
more ecient forms of these enzymes. Insecticide metabolic destruction inside the
target organism is a common defensive mechanism, decreasing the duration and
intensity of the exposure of the target site, lowering the probability of a lethal
outcome. Three major mechanisms of metabolic transformation of insecticides
underlie the vast majority of examples of biotransformation-based resistance: (i)
oxidation; (ii) ester hydrolysis; and (iii) glutathione conjugation. Pyrethrins, pyreth-
roids, organophosphates, carbamates and other insecticides are degraded by
hydrolysis. Insecticide detoxication primarily unfolds through molecule hydrolysis
on dierent sites, thereby spliing ester, carboxyl-ester, amide and other chemical
bonds. The most important hydrolytic enzymes are phosphoric triesters and
carboxylesterases (ALiE esterases). Structural mutations in mutant carboxylesterases
have now been widely described showing metabolic resistance to organophosphate
and pyrethroid insecticides and relatively few cases of resistance to carbamates role.
Carboxylesterases role in Colorado potato beetle resistance was conrmed by many
authors.
Keywords: insecticide resistance, metabolism, hydrolysis, carboxylesterase, ALiE
esterase
© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction
Worldwide, Leptinotarsa decemlineata (Say.)—Colorado potato beetle (CPB) is one of the most
important pests of potatoes, an insect extremely dicult to control due to rapid and strongly
developed resistance against insecticides [1–4]. None of the control techniques, during the long-
lasting history, developed against this pest has provided long-term protection for potato crops.
CPB still remains a major threat to potato production because of its resistance to all major groups
of insecticides [5–18]. Numerous alternative control strategies for Colorado potato beetle were
investigated in the last few decades [19–31].
In insects, generally, the factors that lead to resistance are (1) morphological, (2) physiological
and biochemical, and (3) behavioral [32]. Regarding CPB’s resistance to insecticides, the most
important, investigated, and best described are the metabolic changes. Populations of resistant
insects may detoxify or degrade the toxin faster than susceptible insects or quickly rid their
bodies of the toxic molecules. This type of resistance is the common mechanism and often
presents the greatest challenge. Resistant populations may possess higher levels or more
ecient forms of these enzymes. In addition to being more ecient, these enzyme systems
also may have a broad spectrum of activity. The metabolic destruction of an insecticide inside
the target organism is a common defensive mechanism that leads to a decrease in the duration
and intensity of the exposure of the target site and thereby lowers the probability of a lethal
outcome. Three major mechanisms of metabolic transformation of insecticides underlie the
vast majority of examples of biotransformation-based resistance: (i) oxidation; (ii) ester
hydrolysis; and (iii) glutathione conjugation.
In the rst stage, the metabolism of insecticides is manifested through many reactions, most
important of which are oxidation, reduction, and hydrolysis. In the second stage, conjugates
are formed, which are practically nontoxic [33]. Selective toxicity of insecticides mostly comes
from the balance of the reactions included in activation and in detoxication. Pyrethrins,
pyrethroids, organophosphates (OP), carbamates, and other insecticides are degraded by
hydrolysis. This is the basis for the selective eect of insecticides and for insects’ resistance
mechanisms. Insecticide detoxication primarily unfolds through molecule hydrolysis on
dierent sites, thereby breaking ester, carboxylester, amide, and other chemical bonds [32, 34].
The most important hydrolytic enzymes are phosphoric triesters and carboxylesterases (ALiE
esterases, nonspecic, or B-esterases).
Esterase-related insect resistance is based on the following:
•Increase in the total amount of esterase—by altering regulatory genes or regulatory loci
combined with structural genes, which results in change in enzyme synthesis in the
organism or amplication of genes responsible for DNA methylation.
•Change in their activity—by altering structural genes that directly determine the nature of
enzymes [1].
The majority of widely used insecticides are esters. This includes virtually all carbamate and
OPs, most pyrethroids, and others. In almost all the cases, the hydrolysis of the ester group
Insect Physiology and Ecology160

leads to a signicant decrease in or elimination of toxicity. Consequently, esterase activity often
plays a key role in determining the comparative responses and resistance to current insecti-
cides [1].
In insects, the primary groups of esterases of interest hydrolyze esters of carboxylic acids, and
they are, therefore, termed carboxylesterases. The topic of their nature and signicance in
insecticide toxicology and resistance has been reviewed by dierent researchers [35–38]. A
useful functional method with special relevance for insecticides was developed based on the
ability of the esterase to either hydrolyze OPs (type A) or to become inhibited by them (type
B). There are relatively a few cases of high-level esterase-mediated metabolic resistance to
carbamates. Structural mutations in mutant carboxylesterases have now been described from
four insect species, showing metabolic resistance to OP insecticides [39]. The role of esterase
in CPB resistance was conrmed by many authors [4, 40–44].
2. Hydrolytic metabolic pathways
Insecticide detoxication is primarily performed by hydrolysis of molecules at dierent sites.
Hydrolysis means spliing of molecules by adding water. This chemical reaction splits
dierent chemical bonds, such as ester bonds (with phosphoric, carbamine, chrysanthemum,
and other acids), carboxyl ester, amide, and other bonds [32–34].
Hydrolysis of such bonds is done enzymatically and nonenzymatically. Besides hydrolases
(esterases, phosphatases, carboxylesterases, and amidases), in spliing some of the mentioned
chemical bonds there are also some other enzymes involved, such as mixed function oxidases
(MFO) and glutathione S transferases (GST) [33]. Hydrolytically, pyrethrins and pyrethroids,
organophosphates, carbamates, and some other insecticides can decompose faster or more
slowly. This type of metabolism often makes a basis for a selective mode of action of insecticides
and a mechanism of insect resistance to insecticides [34].
2.1. Hydrolytic enzymes
Hydrolases are widely spread in diverse plant and animal tissues and dierent parts of cells.
In vertebrates, they can be located in blood plasma, and they are able to aack a large number
of xenobiotic esters, but natural substrates of these esterases are unknown. In mammals, A-
type esterases (with –SH group) can be found in the serum, and they are associated with the
lipoprotein fraction. Some hydrolases, especially B-type hydrolases (with –OH group), are
bound to membranes in the microsomal fraction. Up to 7% of all microsomal proteins is
membrane-bound esterases. Such esterases can also be found in the serum and pancreatic
uid. Hydrolases can be present in the cellular uid [32].
Most of these enzymes are not puried. Hence, it is often unknown if the investigated
hydrolytic reactions are catalyzed by one enzyme of weak specicity (for example, one enzyme
with both esterase and amidase function) or by a mixture of two or more enzymes with higher
specicities. Unlike oxidases and transferases, hydrolases do not require any coenzymes;
however, from time to time, they require cations for activation [32].
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There are still some diculties in naming hydrolases and their specicities for substrates. The
same bonds, especially in phosphates, can be aacked not only by hydrolases but also by other
enzymes (MFO and transferases). In the broadest sense, one can identify phosphatases,
carboxyl esterases, carboxyl amidases, and epoxide hydrolases [41]. Cyclic phenyl saligenin
phosphate, S,S,S-tributyl phosphorotrithioate (DEF), and profenofos are stated as hydrolase
inhibitors [45].
2.2. Hydrolysis of phosphorus esters
Enzymes that catalyze a hydrolytic aack on phosphorous esters or anhydride bonds are
marked as hydrolases of phosphoric triesters or phospho-triesterases. In insects, these enzymes
are not puried, so they have not been adequately compared with puried mammalian
hydrolases. These hydrolases do not aack phosphoro-trithioates in any scope. The hydrolase
reaction is activated with 1 mM Mn2+ and Co2+ [46].
In several cases, the activity of these enzymes is higher in resistant insect species. Phosphate
triester hydrolysis results in forming an anion metabolite that is a weak AChE inhibitor,
nally resulting in detoxication of the starting compound. There are two types of phosphate-
ester bonds: an anhydride bond (P—O; P—S; P—C; P—N; and others) and an alkyl-ester bond
(R—O—P). There is also an alkyl–nitrogen bond (R—N—P). These bonds are split not only
by hydrolases but also by MFO and G-S transferases. Enzymatic hydrolysis is found in
mammals, insects, plants, and microorganisms [34].
Some of these bonds can be split nonenzymatically, for example, when it comes to chlor-
pyrifos oxon in trout, hydrolysis is not stimulated by Ca2+ and EDTA does not deactivate
this reaction. None of the known inhibitors has aected the hydrolysis. It is also known
that trichlorfon can be nonenzymatically transformed into dichlorvos in some plant and an-
imal species [34]. Hydrolases aack oxo forms of phosphates, such as paraoxon, diazoxon,
malaoxon (Figure 1), and dichlorvos [46].
Figure 1. Dierent hydrolases and their specicities for substrates [47].
Insect Physiology and Ecology162

Hydrolases (A-esterases) split not only phosphate-ester bonds such as P—O and P—S but also
anhydride bonds such as P—O—P, P—F, P—C. Metabolic spliing of the P—S─aryl bond is
done at P–S, hydrolytically for thiolate esters and oxidatively for thiolothionates. Fonofos is
hydrolyzed after oxidative desulphurization takes place. In some mammals, trichlorfon is
hydrolytically metabolized by spliing the P—C bond, and in some others (rabbits), there is a
dierent kind of metabolic reactions. Omethoate (thiolate) is also hydrolytically metabolized
in rats by spliing of the P—S bond. However, spliing of the P—S bond in the P—S alkyl
structure is not hydrolytic, while spliing of the S—C bond most probably is. The former can
result in a P–OH product and the laer in a P—SH product. The ester bond in malathion is
split into small scale by breaking the P—S bond (about 1%) and the S—C bond (about 0.5%)
by a liver homogenate. Spliing of alkyl-ester bonds in organophosphates is done not only
hydrolytically but also oxidatively and through a group transfer. Hydrolysis, for example,
splits the ethyl-phosphorous bond in paraoxon. This reaction is conducted in a soluble liver
fraction in mammals and in vivo in insects. In rats, there is also hydrolytic O-demethylation
of omethoate [34].
2.3. Hydrolysis of carboxylic esters
Carboxyl esterases are also called ALiE esterases or B-esterases. The enzyme that catalyzes the
spliing of the carboxyl ester bond in organophosphates is referred to as EC. 3.1.1.1. [34, 38].
It is widely spread in mammals (in the liver, kidneys, lungs, spleen, small intestine, and uid)
[34] and in insects, of both susceptible and resistant species. A puried carboxyl esterase in
insects (ALiE) has the molecular weight of 16,000 Da [46].
After investigating the specicities of carboxylesterases in the liver of rats, it has been ascer-
tained that nonphosphorous mono- and di-carboxyl esters serve as substrates. The α-carboxyl
ester bond in malathion is hydrolyzed to form malathion α-monoacid. Similarly, the homoge-
nate of the housey gives α-monoacid more than β-isomer (α/β ratio is 3.5–5.0). However,
esterases taken from the horse liver and rat liver microsomes primarily produce malathion
β-monoacid (α/β ratio is 0.1). Malathion di-acid in rats does not emerge in vitro, but in vivo,
under the inuence of an unknown factor [35, 39].
Organophosphates of dierent structures inhibit carboxyl esterases or the metabolism of
malathion and other insecticides from this group in vivo [34]. EPN oxygen analogue and n-
propyl-paraoxon inhibit these enzymes and show a strong eect (I50 about 10−8 M) [46]. These
compounds are also strong synergists for malathion and acethion in houseies and mites. Tri-
o-cresyl phosphate (TOCP), which is not an insecticide, is also tested as a carboxylesterase
inhibitor. Compounds of such type are weak esterase inhibitors in vitro, but strong inhibitors
of malathion metabolism in vivo. TOCP is in vivo transformed into saligenin cyclic-o-tolyl
phosphate, marked as M-1, which selectively inhibits carboxyl esterases (pI50 for the mouse
plasma esterase amounts to 7.2). TOCP increases malathion toxicity four times, and M-1 100
times. Other triaryl phosphates with a 0-alkyl group can be activated metabolically in a similar
way. Moreover, increased toxicity of malathion for mice is also exhibited by chlorothion
(insecticide) and S,S,S-tributyl-phosphoro-trithioate (DEF) (defoliant), inhibiting its metabo-
lism in vivo. In resistant insect species, DEF also synergizes paraoxon, azinphos-methyl,
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carbamates, and DDT and with EPN it synergizes dicrotophos, dimethoate, and phorate in
Anthonomus grandis [34].
Several cases have shown that the increased carboxylesterase activity is a mechanism of insect
resistance to malathion. In general, the activity of these enzymes susceptibility to insects is low.
Dierences between the enzymes in susceptible and resistant insects are quantitative, and in
Tetranychus urticae, they are also qualitative [46].
In the liver of mammals and products of several insect species, there are enzymes that
hydrolyze pyrethroids. It is not excluded that carboxylesterase that hydrolyzes (+)-trans-
resmethrin is the same one that hydrolyzes malathion. The enzymes in some insects, such as
Oncopeltus fesciatus and Trichoplusia ni, degrade (+)-cis compounds, whereas isomer specicity
is less pronounced in the enzymatic products from Musca domestica and Blaella germanica.
Relative hydrolysis speed is much higher in mammals than in insects, which is most likely the
basis of the selective toxicity of pyrethroids [34].
Figure 2. Hydrolysis of dierent OPs pesticides by OPH + CbE enzymes [48].
Insect Physiology and Ecology164

Lile is known not only about the nature of esterases that hydrolase carbamates but also about
biochemistry of these processes. Enzymes in the plasma of rabbits, sheep, and pigs are more
ecient in carbaryl hydrolysis than enzymes in other mammals. In insects, perhaps, it is about
a nonspecic esterase, aromatic esterase, primarily responsible for carbamate hydrolysis [34].
In some organophosphates (malathion, acethion, and phenthoate), the presence of the carboxyl
ester group makes these compounds susceptible to hydrolysis at that site. In malathion,
hydrolysis splits one carbethoxy group, forming nontoxic monoacid (Figure 2). The anionic
charge of the carboxyl group shifts near phosphorous so that the electrophilicity is reduced by
the eect of eld. Hence, malathion-α-monoacid is inactive. The metabolism of malathion in
insects and mammals is quantitatively similar, but qualitatively quite dierent. Due to its much
faster metabolism in mammals, they are usually less susceptible to malathion than insects.
Moreover, such type of malathion metabolism predominates in mammals. The presence of the
carboxyl ester group in organophosphates, however, does not always lead to reduced toxicity.
Mevinphos is much more toxic than it can be expected from its structure that includes the
carbethoxy group. In detoxication of mevinphos, the carboxyl esterase plays no or a very
lile role, and this bond is hydrolyzed nonenzymatically [32, 34, 46].
Some arthropod species resistant to malathion have a much higher carboxylesterase activity
than susceptible species. The carboxylesterase activity is, at least in part, responsible for
resistance to organophosphorous insecticides in several insect and mite species (mosquitoes,
houseies, cicadas, and mites) [36, 40–43].
Carbamates can also be hydrolyzed by esterases (Figure 3). The isolated carbamic acid is
immediately hydrolyzed into CO2 and methyl or dimethyl-amine. The carbamate ester bond is
quite stable in plants and insects, but easy to hydrolyze in the majority of animal species [32, 34].
Figure 3. Hydrolytic pathway for carbaryl [49].
Besides, oxidative metabolites with a still intact carbamate ester bond are also subjected to
hydrolysis by the same or perhaps dierent enzymes. In many cases, it is impossible to
determine whether oxidation happens before or after hydrolysis [34]. N-substituted carba-
mates, the products of oxidative N-dealkylation are hydrolytically more unstable than N-
methyl carbamates (about 100 times as much).
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The rate of enzymatic hydrolysis depends on carbamate structure and the type of organism.
Therefore, rats hydrolyze about 25% carbaryl, 33% propoxur, and 75% maxacarbate or isolan.
Although most mammals hydrolyze carbamate ester bonds easily, these compounds are
resistant in monkeys and pigs. On the other hand, many insects have diculties in hydrolyzing
carbamates, whereas hydrolysis is the main pathway to carbamate decomposing in B. german‐
ica. The hydrolysis of carbaryl has been the most studied carbamate hydrolysis. It occurs in a
large percentage in many mammals, in a small number of insect species (B. germanica, for
example), and in a very few plants [34].
Pyrethrins and pyrethroids are quite dierently hydrolyzed in living organisms. The
dierences are considerably due to the compound’s structure and the type of organism. The
hydrolysis is catalyzed by some kind of carboxylesterases. The basic metabolic pathway in
pyrethroids (such as permethrin) is hydrolytic spliing of the ester bond, but oxidation is also
important. Hydrolysis is, however, irrelevant and lile important in the metabolism of
pyrethrins (pyrethrin I, cynarin I, etc.) and related to older pyrethroids (allethrin, tralomethrin,
tetramethrin, barthrin, etc.) [32, 34].
In pyrethroids with cyclopropane acid, the stereochemistry of 1,3-bond of this ring strongly
aects the metabolism of these compounds. When the substituent is transposed to the carboxyl
group at C-1, spliing of the ester bond is easier than when it is cis-positioned. The adding of
a –CN group to α-carbon of 3-phenoxybenzyl alcohol decreases the susceptibility of molecules
to hydrolytic (and oxidative) decomposition. Insects normally hydrolyze pyrethroids more
slowly but split the ester bond of trans-isomers faster than mammals. However, there is an
exception. The larvae of Chrysopa spp., which have unexceptionally high levels of esterase,
hydrolyze cis-isomers of permethrin and cypermethrin faster than trans-isomers. This level of
esterase activity is undoubtedly the main factor of resistance of these species to pyrethroids
[50]. Like in mammals and in insects, the main primary metabolic process of pyrethroids
(permethrin, deltamethrin, cypermethrin, etc.) in plants (beans, coon, etc.) is the spliing of
the ester bond. Fenvalerate acts similarly in dierent plants (tomatoes, tobacco, leuce,
cabbage, etc.). In all plants, the –CN group disappears [32, 34, 50].
Permethrin, a compound without the –CN group, is hydrolyzed in mammals primarily by
spliing of the ester bond, during which the trans-isomer is about 100 times more susceptible
(rats and mice). In goats and cows, about 30% metabolites of permethrin has the preserved
ester bond (both cis- and trans-isomer). Spliing of the permethrin ester bond is more dicult
in sh. Insects (cockroaches, houseies, cabbage moth, and caterpillars) tear permethrin into
acid and alcohol part, among others, which form conjugates with glucose and amino acids. In
all three species, trans-permethrin is metabolized more easily. In vivo and in vitro results are
similar. The metabolism of permethrin (Figure 4) is, in its basis, similar in other insect species
and some mites species [34, 50]. The main pathway in the metabolism of deltamethrin,
cypermethrin, and cyhalothrin in mammals (mice and rats) is the spliing of the ester bond,
primarily by carboxylesterases, whereby trans-isomers are more susceptible. These com-
pounds are basically similarly metabolized in insects [45, 50]. Generally speaking, esterases
play a main role in the metabolism of pyrethroids in caterpillars and oxidases in houseies. In
Spodoptera lioralis and T. ni, for example, prophenophos inhibits the hydrolytic decomposi-
Insect Physiology and Ecology166

tion of trans-permethin for 65%, and cis-cypermethrin for more than 90%, thereby increasing
the toxicity of the former four times and the laer 20 times (T. ni), i.e., three times for both
compounds (S. lioralis).
Figure 4. Esterase-mediated hydrolysis of the pyrethroid bifenthrin [51].
Phenyl-saligenin, a cyclic phosphate, increases the toxicity of trans-permethrin in Chrysopa
carnea 68 times. Other compounds also undergo hydrolysis. For example, dinobuton (and
similar compounds) is initially hydrolytically activated into dinoseb. Acaricide cycloprate is
hydrolyzed into cyclopropane acid that afterward binds with carnitine, which eventually has
a lethal eect [34, 50].
2.4. Hydrolysis of carboxamides
Amide bonds are relatively similar to ester bonds. Breaking of these bonds is catalyzed by
carboxamidases. Carboxamidases are actually carboxylesterases capable of selecting amides
as substrates [32]. In vertebrates, these enzymes are located only in the liver, and they are
primarily related to the microsome fraction. Dierent divalent cations and nucleoids do not
aect the enzymatic activity. The oxidative derivative of dimethoates is not hydrolyzed by
carboxyamides but inhibits this enzyme. Dimethoate of amidases from dierent sources diers
in susceptibility to EPN-induced inhibition in vivo. Hence, housey and Oncopeltus fasciatus
amidases are not susceptible to EPN, while mammal amidases are highly susceptible. On the
whole, these enzymes are susceptible to organophosphates as inhibitors, including profenofos,
TOCP, DEF, etc. [34].
Breaking the amide bond among organophosphates is primarily determined for dimethoate,
dicrotophos, and vamidothion [32]. When compared to other pathways, spliing of the amide
bond of dimethoates by hydrolysis amounts to 27.3% in houseies, 2.5% in rat liver, and 0.0%
of rice leaves in vitro. Spliing of the S─C bond in dimethoate is hydrolytic. Vamidothion is
similarly metabolized. It has been determined that spliing of the amide bond of dimethoates
and related insecticides is important for their selective toxicity. Besides, the amide bond of
dimethoate is also hydrolyzed nonenzymatically on the leaf surface after oxidative desulphu-
rization. However, the amide bond of phosphamidon is not split in plants and animals.
The metabolism of benzoyl phenyl urea (BPU) of derivatives is primarily conducted by the
hydrolysis of amide bonds. In a large number of insect species (Tribolium castaneum, S.
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lioralis, Spodoptera exigua, etc.), it has been recorded that these compounds (diubenzuron,
chloruazuron, diubenzuron, etc.) degrade rapidly. For example, diubenzuron is rapidly
eliminated from insects (t½ about 7 h), and chloruazuron slowly (t½ > 100 h). Adding
hydrolase inhibitors (DEF and related compounds) in food prolongs the retention time (t½
from 7 to 18 h for diubenzuron) and increases the toxicity of diubenzuron for T. castaneum
and S. lioralis. The main metabolites in BPU hydrolysis are chloroaniline, chlorpheniramine
urea, and polar metabolites. The activity of these enzymes in hydrolysis of BPU is completely
inhibited by DEF or prophenophos in the concentration of about 10−5 M [32]. Therefore, DEF
and prophenophos express a synergetic activity with diubenzuron in S. exigua that has
developed resistance to them, ranging from 3.7 (to DEF) to 5.2 times to prophenophos [34,
45].
2.5. Hydrolysis of epoxides
Epoxide hydrolases (or epoxide hydrases, epoxide hydratases, EH), discovered in 1968, take
part in the metabolism of epoxides. These enzymes (EH) are widely spread among mammals
and insects. They are located in microsomes (MEH), solution (CEH), or other parts of mammal
liver cells and dierent insect tissues [32, 34, 44, 46]. pH optimum for the EH activity is most
often in the alkaline range, and in insects, it ranges from 7.9 to 9.0 [46]. They show a pronounced
specicity for substrates, and there is a great variation among the dierent species. The
mechanism of hydration is not clear enough. Perhaps it includes a nucleophilic aack of the
OH group on oxirane carbon [46]. The EH activity from liver microsomes does not depend on
NADPH or CO2 and this enzyme is not aected by BDO-type synergists [34]. A certain number
of EH inhibitors in insects include sesamex, piperonyl butoxide, a Cecropia hormone, and some
organophosphates, which are partially in contrast to these enzymes from the mammal liver.
Nevertheless, this enzyme is inhibited for about 80% with the same concentration of Cu2+ ions
[46]. Specic EH inhibitors in mammals are phenyl-glycidoles. S,S-enantiomers are stronger
inhibitors (I50 1.6 × 10−6 M) than R,R-isomers. These compounds are also substrates for EH [44].
EHs catalyze the spliing of the epoxide ring of dierent insecticides and other compounds.
Thereby, they form certain trans-diols that are less toxic, so these enzymes are included in
epoxide detoxication processes. These processes are most studied in cyclodienes. Spliing of
the epoxide ring of some cyclodienes has been shown in many insect and mammal species. In
vitro, dieldrin and heptachlor epoxide are quite stable in this form of degradation. In vivo,
however, especially in mammals, the main metabolite of dieldrin is trans-diol, which indicates
the spliing of the epoxide ring [34]. Epoxides of various alkenes and arenas are enzymatically
hydrolyzed, thus forming trans-di-hydro diols [46].
3. Determination of esterase activity in Colorado potato beetle
In our research, activity of Colorado potato beetle ALiE/Carboxylesterase was determined,
using spectrophotometry at a wavelength of 585 μm, rst described by Gomori [52]. Average
enzyme was prepared out of 40 CBP fourth-instar larvae, using 40 ml of phosphate buer
Insect Physiology and Ecology168

(0.02 M, pH7). Incubation of the average enzyme of Colorado potato beetle with dierent
inhibitors (PBO, DEF, eserine sulfate (ES)) proved that decomposition of 1-naphtyl acetate (1-
NA) to 1-naphtyl (1-N) is directly related to the activity of esterase. Incubation of an average
enzyme with PBO gave, in all cases, much lower reduction in activity, than in the incubation
with DEF or with eserine sulfate (ES). This indicates that the formation of 1-naphtyl does not
occur due to the activity of oxidase or a glutathione transferase (GST), but due to the activity
of esterases.
Activity of ALiE at varying concentrations of the substrate 1-NA showed that the increasing
the concentration of the substrate aects the increase in 1-N amount, and this dependence is
linear. Statistical analysis has obtained high-correlation coecient (0.9279) and a very small
statistical error (SE = 0.1601), indicating a high dependence of the examined parameters. The
value of the Michaelis constant (Km) was low (6.664 × 10−3). Km values for the activity of most
enzymes typically range from 10−10 to 10−2 M dm−3, which indicates quite high specicity of the
tested enzyme for substrate.
3.1. Determination of the calibration line for 1-naphthol
In the experimental conditions, for the ALiE enzyme, 1-naphthylacetate (1-NA) is commonly
used as a suitable substrate. Enzyme decomposes substrate 1-NA into 1 naphthol (1-N). In
order to be able to determine the amount of generated 1-N, it is necessary to determine the
calibration line. For these assays, it is also necessary to adjust the conditions of the experiment
when examining the functioning of the enzyme. Tests were carried out in the visible region of
wavelengths of light, for dierent concentrations of 1-N. Very low concentrations of 1-N does
not give the expressed absorbance maximum. With increasing concentration, the maximum
distinguishes more clearly, and most notably at higher concentrations. For the spectropho-
tometer device UV-VIS Perkin-Elmer 130, determined peak is at a wavelength of 585 nm and
all subsequent determinations of 1-N were carried out at a wavelength of 585 nm.
Concentration (μM) Absorbance per replication Average absorbance
1 2 3
0.5999 0.205 0.201 0.233 0.213
1.1998 0.216 0.274 0.220 0.237
2.3995 0.219 0.234 0.233 0.229
4.7991 0.290 0.276 0.270 0.279
9.5981 0.323 0.349 0.358 0.343
19.1963 0.467 0.511 0.538 0.505
38.3925 0.642 0.643 0.610 0.632
76.7851 0.890 0.883 0.810 0.861
153.5702 0.822 0.822 0.815 0.820
Table 1. Data for the calibration line for 1-naphthol.
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The initial concentration contained 22.14 mg/l of 1-N. Since 1 M solution of the compound is
containing 144.17 gl−1, this concentration expressed as a molar solution amounted to
153.57 μM of the compound. Absorbance values for the nine dierent concentration of 1-N are
shown in Table 1 and Chart 1. In order to avoid the eect of these concentrations of the eld
in which the readings are unreliable, for the calculation of the regression line only concentra-
tions from of 76.8 to 4.8 μM were used.
Chart 1. Data for the calibration line for 1 naphtol.
Statistical regression analysis calculated the following regression line for 1-N:
Y = 0.2918 + 0.0078 × X, as a basis for the calculation of the 1-N amounts for the appropriate
absorbance. Statistical data analysis found out that the regression is linear and directly
dependent. The correlation coecient is 0.98, which indicates a high dependence of investi-
gated parameters.
3.2. Determination of presence and activities of ALiE
Average enzyme was prepared out of 40 Colorado potato beetle fourth-instar larvae (L4), from
the locality of Dobanovci (Belgrade, Serbia), using 40 ml of phosphate buer (0.02 M, pH7).
Experimental conditions in terms of the average amount of enzyme in the required amount of
the reaction mixture and the temperature were constant, but the substrate concentration was
varied. It is found that the activity of Colorado potato beetle ALiE exists, since in all the
examined substrate concentrations in the experimental conditions there is a degradation of the
NA-1 1-N due to the enzyme (ALiE) activity (Chart 2).
Increasing the concentration of the substrate aects the increase in creation of 1-N, and this
dependence is linear. Statistical analysis shows high correlation coecient and a very small
statistical error (as shown in Table 2), which indicates the high dependency of investigated
parameters.
Insect Physiology and Ecology170

Chart 2. Activity of ALiE at varying concentrations of the substrate 1-NA.
The value of the Michaelis constant (Km) is small. These values of Km for most of the enzymes
activity typically range from 10−2 to 10−10 M dm−3, indicating quite high specicity of the
investigated enzyme regarding substrate.
Correlation coecient Statistical error Regression line Km (Mdm−3)
0.9279 0.1601 Y = 0.2849 + 0.00189 × X6.664 × 10−3
Table 2. ALiE activity at varying substrate (1-NA) concentrations—statistical parameters.
3.3. Determining the type of enzyme activity using inhibitors
For experiments with inhibitors, we used the average enzyme from Colorado potato beetle of
the fourth-grade larvae (L4), population Dobanovci, which contains not only a complex of
enzymes but also a variety of other compounds that can react with inhibitors. PBO is a typical
oxidase inhibitor and therefore is often used as an insecticide synergist, which are subject to
oxidative detoxication. DEF is a specic inhibitor of ALiE esterases, while the ES is specic
cholinesterase inhibitor.
Pre-incubation of the enzyme with PBO showed minimal reduction in activity in the degra-
dation of 1-NA. Statistical analysis showed that there are still signicant dierences in the
results for the variant, which applies only to the enzyme and variant combinations of enzymes
and piperonyl butoxide.
Pre-incubation of the enzyme with DEF resulted in signicantly decreased activity of the
enzyme. Dierences in the enzyme activity without inhibitor and pre-incubation with DEF are
very signicant. There are very signicant dierences between variants, such as the enzyme
pre-incubated with the PBO and variants when pre-incubated with DEF.
The greatest reduction in the enzyme activity was obtained when the enzyme is pre-incubated
with eserine sulphate (ES). Statistical analysis showed that there are very signicant dierences
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between the basic enzyme activity and its activity after pre-incubation with ES. Duncan’s test
proved that all the variants belong to dierent groups.
Dierent concentrations of the CPB average enzyme (ALiE) from population Dobanovci,
depending on the increase in the concentration of the enzyme, produce increasing amounts of
1 N, at a constant amount of substrate (1 NA). Similar instances happened with variable
concentrations of substrates in the presence of a constant amount of average enzyme. Since
the reactions are specic to this group of enzymes, these results indicate their distinguished
activity in potato beetle. Incubating the average enzyme with dierent inhibitors has been
proven in the case of Colorado potato beetle that degradation of 1-NA into 1-N comes under
the inuence of esterase. Incubation of average enzyme with PBO gave in all cases much lower
lower reduction in activity, than the impairment of enzyme activities incubated with DEF or
eserine sulphate (ES), which indicates that the creation of 1-N does not come due to the eect
of oxidase or glutathione transferase (GST), but due to the eect of esterase (Table 3).
Treatment Absorbance per replication Average Median Variance Standard deviation Duncan test
12345
Enzyme 0.82 0.88 0.82 0.82 0.90 0.848 0.82 1.52−3 0.039 a
Enzyme + PBO 0.80 0.80 0.78 0.80 0.84 0.804 0.80 4.80−4 0.022 b
Enzyme + DEF 0.72 0.70 0.70 0.75 0.72 0.718 0.72 4.20−4 0.020 c
Enzyme + ES 0.66 0.65 0.69 0.66 0.67 0.665 0.66 1.75−4 0.013 d
LSD0.05 = 0.06.
LSD0.01 = 0.08.
Table 3. Statistical parameters of the inhibitor eect on the activity of CPB larvae ALiE.
4. Conclusion
Resistant insect populations may detoxify or degrade the toxin faster than susceptible insects,
or quickly rid their bodies of the toxic molecules. Resistant populations may possess higher
levels or more ecient forms of these enzymes. The metabolic destruction of an insecticide
inside the target organism is a common defensive mechanism. Three major mechanisms of
metabolic transformation of insecticides underlie the vast majority of examples of biotrans-
formation-based resistance: (i) oxidation; (ii) ester hydrolysis; and (iii) glutathione conjugation.
Pyrethrins, pyrethroids, organophosphates, carbamates, and other insecticides are degraded
by hydrolysis. The most important hydrolytic enzymes are phosphoric triesters and carboxy-
lesterases (ALiE esterases, nonspecic, or B-esterases). Structural mutations in mutant
carboxylesterases have now been widely described showing metabolic resistance to organo-
phosphate and pyrethroid insecticides. There are relatively few cases of high-level esterase-
mediated metabolic resistance to carbamates. The role of carboxylesterases in Colorado potato
beetle’s resistance to insecticides was conrmed by many authors. CPB is resistant to all major
Insect Physiology and Ecology172

groups of insecticides, including organophosphates and carbamates. Insecticide resistance
presence and level are measurable [53]. ALiE’s role in the emergence of resistance to organo-
phosphorus and other insecticides in insects, especially in Colorado potato beetle, is investi-
gated. In most of the insect species in which the testing was performed, the dependence of the
increase in the activity of the enzyme matched with the increase of the insecticide resistance.
Probably, the primary role of this enzyme is its importance for the absorption of organophos-
phorus insecticides, which becomes nontoxic, and then to gradually decompose to nontoxic
components. Increased concentrations of the average CPB enzymes produced increasing
amounts of 1-naphthol (1-N) at a constant amount of substrate (1-NA). Similar results
happened with variable concentrations of substrate (1-NA) in the presence of a constant
amount of average enzyme. Since the reactions are specic to this group of enzymes, these
results indicate their distinguished activity in Colorado potato beetle.
Acknowledgements
Work was supported by Ministry of Education and Science, Republic of Serbia, Grant No. III
46008. We would like to acknowledge Dr. Anton Zabel for his visionary work and also thank
Ivana Stanimirovic and Dr. Slavica Colic.
Author details
Sladjan Stankovic1* and Miroslav Kostic2
*Address all correspondence to: sstankovic@ipn.bg.ac.rs
1 Institute for Science Application in Agriculture, Belgrade, Serbia
2 Institute for Medicinal Plant Research “Dr Josif Pancic”, Belgrade, Serbia
References
[1] Kostic M., Stankovic S., Kuzevski J. (2015): Role of AChE in Colorado potato beetle
(Leptinotarsa decemlineata Say) resistance to carbamates and organophosphates. In:
Insecticides Resistance (Trdan S., ed.), InTech, Rijeka, Croatia, 19–40. hp://dx.doi/
10.5772/61460.
[2] Alyokhin A., Chen Y.H., Udalov M., Benkovskaya G., Lindström L. (2013): Evolutionary
considerations in potato pest management. In: Insect Pests of Potato, 543–571.
[3] Stanković S., Kostić M., Sivčev I., Janković S., Kljajić P., Todorović G., Jevđović R. (2012):
Resistance of Colorado potato beetle (Coleoptera: Chrysomelidae) to neonicotinoids,
Role of Carboxylesterases (ALiE) Regarding Resistance to Insecticides: Case Study of Colorado Potato Beetle...
http://dx.doi.org/10.5772/66254
173

pyrethroids and nereistoxins in Serbia. Romanian Biotechnological Leers, 17(5), 7599–
7609.
[4] Zhao J.Z., Bishop B.A., Graus E.J. (2000): Inheritance and synergism of resistance to
imidacloprid in the Colorado potato beetle (Coleoptera: Chrysomelidae). Journal of
Economic Entomology, 93, 1508–1514.
[5] Stanković S., Zabel A., Kostić M., Manojlović B., Rajković S. (2004): Colorado potato
beetle (Leptinotarsa decemlineata Say) resistance to organophosphates and carbamates
in Serbia. Journal of Pest Science, 77, 11–15.
[6] Zabel A., Stankovic S., Kostic M., Sivcev I., Kuzevski J., Kostic I., Krnjajic S. (2016):
Acetylcholinesterase [AChE] activity of Colorado potato beetle populations in Serbia
resistant to carbamates and organophosphates. Romanian Biotechnological Leers (in
press).
[7] Alyokhin A., Baker M., Mota-Sanchez D., Dively G., Graus E. (2008): Colorado potato
beetle resistance to insecticides. American Journal of Potato Research, 85(6), 395–413.
[8] Stanković S., Zabel A., Kostić M., Šestović M. (2004): Comparative analysis of Colorado
potato beetle (Leptinotarsa decemlineata Say) resistance monitoring methods. Pesticides,
18(3), 159–175.
[9] Zabel A., Rajkovic S., Manojlovic B., Stankovic S., Kostic M. (2000): Nenicotinoids in
the control of Leptinotarsa decemlineata Say. 2nd Balkan Symposium on Vegetables and
Potatoes. Thessaloniki, Greece. Book of Abstracts.
[10] Zabel A., Rajkovic S., Manojlovic B., Stankovic S., Veljkovic I. (2000): New pesticides in
potato protection against the Colorado potato beetle (Leptinotarsa decemlineata Say.) and
late blight (Phytophtora infestans Mont. de Bary) on potato. 2nd Balkan Symposium on
Vegetables and Potatoes. Thessaloniki, Greece. Book of Abstracts.
[11] Stanković S., Kostić M., Sivčev I. (2003): Colorado potato beetle (Leptinotarsa decemli‐
neata Say) resistance levels to endosulfan in Serbia. Plant Protection, 54(1–4), 105–113.
[12] Alyokhin A., Ferro D. (1999): Relative tness of Colorado potato beetle (Coleoptera:
Chrysomelidae) resistant and susceptible to the Bacillus thuringiensis Cry3A toxin.
Journal of Economic Entomology, 92, 510–515.
[13] Jiang W.H., Wang Z.T., Xiong M.-H. (2010): Insecticide resistance status of Colorado
potato beetle (Coleoptera: Chrysomelidae) adults in northern Xinjiang Uygur autono-
mous region. Journal of Economic Entomology, 103(4), 1365–1371.
[14] Szendrei S., Graus E., Byrne A., Ziegler A. (2012): Resistance to neonicotinoid
insecticides in eld populations of the Colorado potato beetle (Coleoptera: Chrysome-
lidae). Pest Management Science, 68, 941–946.
[15] Wȩgorek P., Zamojska J., Mrówczyński M. (2011): Susceptibility level of the Colorado
potato beetle (Leptinotarsa decemlineata Say) to chlorpyrifos and acetamiprid in poland
Insect Physiology and Ecology174

and resistance mechanisms of the pest to chlorpyrifos. Journal of Plant Protection
Research, 51(3), 279–284.
[16] Alyokhin A., Dively G., Paerson M., Mahoney M., Rogers D., Wollam J. (2006):
Susceptibility of imidacloprid-resistant Colorado potato beetles to non-neonicotinoid
insecticides in the laboratory and eld trials. American Journal of Potato Research,
83,485–494.
[17] Alyokhin A., Dively G., Paerson M., Castaldo C., Rogers D., Mahoney M., Wollam J.
(2007): Resistance and cross-resistance to imidacloprid and thiamethoxam in the
Colorado potato beetle. Pest Management Science, 63, 32–41.
[18] Zhou Z., Zhaoxu Z., Jinhuan P., Wenchao G, Zhong N., Tian Y., Xiaa G., Wua J. (2012):
Evaluation of the resistance of transgenic potato plants expressing various levels of
Cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say) in the labora-
tory and eld. Pest Management Science, 68, 1595–1604.
[19] Zabel A., Manojlović B., Rajković S., Stanković S., Kostić M. (2002): Eect of Neem
extract on Lymantria dispar L. (Lepidoptera: Lymantridae) and Leptinotarsa decemlineata
Say. (Coleoptera: Chrysomelidae). Journal of Pest Science, 75, 19–26.
[20] Ascher K.R.S., Meisner J., Klein M., Rejesus R.S., Obra J.B., Zabel A., Kostic M.,
Manojlovic B., Stankovic S., Tomasi O., Sekulovic D., Beimain S.R., Golob P., Andan
H.F., Atarigiya J., Chare F.A., Can P., Gupta P., Siddiqui M.R., Joseph S., Shivpuri A.,
Gupta R.B.L. (2000): Abstracts of presentations on selected topics at The XlVth Inter-
national plant protection congress (IPPC) 2. NEEM (Azadirachta indica). Phytoparasitica,
28(1), 87–90.
[21] Gökçe A, Whalon M.E., Çam M.E., Yanar Y., Demirtaş İ., Gören N. (2006): Plant extract
contact toxicities to various developmental stages of Colorado potato beetles (Coleop-
tera: Chrysomelidae), Annals of Applied Biology, 149: 197–202.
[22] Safaei Khorram M., Taher Nasabi N., Jafarnia S., Khosroshahi S. (2011): The toxicity of
selected monoterpene hydrocarbons as single compounds and mixtures against
dierent developmental stages of Colorado potato beetle, Leptinotarsa decemlineata say
(Coleoptera: Chrysomelidae). Journal of Entomology, 8(5), 404–416.
[23] Trdan S., Vidrih M., Andjus L., Laznik Z. (2009): Activity of four entomopathogenic
nematode species against dierent developmental stages of Colorado potato beetle,
Leptinotarsa decemlineata, (Coleoptera, Chrysomelidae). Helminthologia, 46(1), 14–20.
[24] Kostić M., Zabel A., Popov V., Ristić M., Stanković S., Manojlović B., Rajković S. (2001):
Eects of essential oils of some species of the genus Tanacetum on potato leaf arac‐
tiveness to larvae of the Colorado potato beetle. Food in the 21st Century. Book of
Abstracts, 136–137.
[25] Kostić M., Kostić I., Marković T., Jevđović R., Stanković S., Todorović G., Nedić N.
(2012): Disruption of aractant properties of potato foliage on Leptinotarsa decemlineata
Role of Carboxylesterases (ALiE) Regarding Resistance to Insecticides: Case Study of Colorado Potato Beetle...
http://dx.doi.org/10.5772/66254
175

Say by the use of Salvia ocinalis L. essential oil. Proceedings of the 7th Conference on
Medicinal and Aromatic Plants of Southeast European Countries, Serbia, 351–356.
[26] Zabel A., Manojlovic B., Stanković S., Thomasi O., Kostić M.(2000): The ecacy of
essential oil of Myristica fragrans Hout to Leptinotarsa decemlineata Say. and Lymantria
dispar L. Plant Protection, 233–234, 211–220.
[27] Stević T., Kostić M., Stanković S., Ristić M., Soković M. (2003): Eect of thujone to
bioagents. Macedonian Pharmaceutical Bulletin. Book of Abstracts, 49(1,2), 165.
[28] Kostic M., Dražić S., Zabel A., Manojlovic B., Rajkovic S., Stankovic S. (2002): Disturb-
ance of potato araction for Colorado potato beetle larvae by essential oils of some
species of the genus Tanacetum. Archive of Agricultural Sciences, 221–222, 87–99.
[29] Kostic M., Stanković S., Ristic M., Jevdjovic R., Rajkovic S. (2003): Eect of essential oils
of genus Tanacetum plants to the aractiveness of potato foliage for adults of Colorado
potato beetle. Medicinal Raw Materials, 23, 69–82.
[30] Kostic M., Stevic T., Stankovic S., Soković M., Ristic M. (2003): Eect of camphor on
biological Agenses. VI Conference of Plant Protection. Abstracts, 2003, Number 112.
[31] Kostić M., Dražić S., Popović Z., Stanković S., Sivčev I., Živanović T. (2007): Develop-
mental and feeding alternations in Leptinotarsa decemlineata Say. (Coleoptera: Chryso-
melidae) caused by Salvia ocinalis L. (Lamiaceae) essential oil. Biotechnology and
Biotechnological Equipment, 21, 4.
[32] Hassall K.A. (1990): Biochemistry and Uses of Pesticides. Second Edition, VCH, New
York.
[33] Šestović M., Peric I. (1995): Fundamentals of the metabolism of insecticides: 1. Enzymes
that catalyze the oxidation processes of the rst phase. Pesticides, 10, 7–23.
[34] Šestović M., Perić I., Vukša P. (1995): Fundamentals of the metabolism of insecticides:
3. Reductive and hydrolytic processes of the rst phase. Pesticides, 10, 181–196.
[35] Oakesho J.G., Claudianos C., Russell R.J., Robin G.C. (1999): Carboxyl/cholinesterases:
a case study of the evolution of a successful multigene family. BioEssays, 21, 1031–1042.
[36] Wheelock C.E., Shan G., Oea J.A. (2005): Overview of carboxylesterases and their role
in metabolism of insecticides. Journal of Pesticide Science, 30, 75–83.
[37] Russell R., Claudianos C., Campbell P.M., Horne I., Sutherland T.D., Oakesho J.G.
(2004): Two major classes of target site insensitivity mutations confer resistance to
organophosphate and carbamate insecticides. Pesticide Biochemistry and Physiology,
79, 84–93.
[38] Hollingwort R.M., Dong, K. (2008): The Biochemical and Molecular Genetic Basis of
Resistance to Pesticides in Arthropods. In: Global Pesticide Resistance in Anthropods
(eds M.E. Whalon, D. Mota-Sanchez and R.M. Hollingworth), pp. 40–50. CABI Pub-
lishing, U.K.
Insect Physiology and Ecology176

[39] Oakesho J.G., Claudianos C., Campbell P.M., Newcomb R.D., Russell R.J. (2005):
Biochemical genetics and genomics of insect esterases. In: Comprehensive Molecular
Insect Science-Pharmacology (Gilbert L.I., Latrou K., Gill S.S. eds.), vol. 5, Elsevier,
Oxford, 309–381.
[40] Anspaugh D.D., Kennedy G.G., Roe R.M. (1995): Purication and characterization of a
resistance-associated esterase from the Colorado potato beetle, Leptinotarsa decemlineata
(Say.). Pesticide Biochemistry and Physiology, 53, 84–96.
[41] Argentine A., Zhu K.Y., Lee S.H., Clark J.M. (1994): Biochemical mechanisms of
azinphosmethyl resistance in isogenic strains of Colorado potato beetle. Pesticide
Biochemistry and Physiology, 48 63–78.
[42] Zabel A. (1991): Esterase activity and resistance to organophosphate insecticides in
potato beetle (Leptinotarsa decemlineata Say.). Faculty of Agriculture, Novi Sad.
[43] Feng Gong L., Fu K.-Y., Li Q., Guo W.-C., Tursun A., Li G.-Q. (2014): Identication of
carboxylesterase genes and their expression proles in the Colorado potato beetle
Leptinotarsa decemlineata treated with pronil and cyhalothrin. Pesticide Biochemistry
and Physiology, doi: 10.1016/j.pestbp.2014.12.015.
[44] Argentine J.A., Clark J.M. (1990): Selection for abamectin resistance in Colorado potato
beetle (Coleoptera: Chrysomelidae). Pestic. Sci., 28: 17–24. doi: 10.1002/ps.2780280104.
[45] Ishaaya I. (1990): Benzoylphenyl ureas and other selective control agents—mechanism
and application. In: Pesticides and Alternatives (Casida J. E. ed.), Elsevier, Amsterdam,
365–376.
[46] Dauterman W.C., Hodgson E. (1978): Detoxication mechanisms in insects. In: Biochem-
istry of Insects (Rockstein M. ed.), Acad. Press, NY, 541–577.
[47] Dauterman W.C. (1971): Biological and non-biological modications of organophos-
phorous compounds. Bulletin of the World Health Organization, 44, 133.
[48] da Silva N.A., Birolli W.C., Seleghim M.R., Porto A.L. (2013): Biodegradation of the
organophosphate pesticide profenofos by marine fungi. In: Applied Bioremediation—
Active and Passive Approaches (Patil Y. ed.), InTech, doi: 10.5772/56372.
[49] Dorough H.W., Ballard S.K. (1982): Degradation of pesticides by animals. In: Biode-
gradation of Pesticides (Matsumura F., Murti C.R.K. eds.), Plenum Press, N.Y., 3–20.
[50] Ishaaya I., Casida J.E. (1983): Pyrethroid detoxication and synergism in insects. In:
Pesticide Chemistry: Human Welfare and the Environment (Miyamoto J., Kearney P.C.
eds.), vol. 3, Pergamon Press, N.Y., 307–310.
[51] Wheelock C.E., Miller J.L., Miller M.J., Phillips B.M., Huntley S.A., Gee S.J., Tjeerdema
R.S., Hammock B.D. (2006): Use of carboxylesterase activity to remove pyrethroid-
associated toxicity to Ceriodaphnia dubia and Hyalella azteca in toxicity identication
evaluations. Environmental Toxicology and Chemistry, 25(4), 973–984.
Role of Carboxylesterases (ALiE) Regarding Resistance to Insecticides: Case Study of Colorado Potato Beetle...
http://dx.doi.org/10.5772/66254
177

[52] Gomori G. (1953): Human esterases. Journal of Laboratory and Clinical Medicine, 42,
445–453.
[53] Miyata, T. (1983): Detection and monitoring methods for resistance in arthropod based
on biochemical characteristics. In: Pest Resistance to Pesticides (Georghiou G.P., Saito
T. eds.), Plenum Press, New York, 99–117.
Insect Physiology and Ecology178