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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
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
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 ecient 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 detoxication primarily unfolds through molecule hydrolysis
on dierent sites, thereby spliing 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 conrmed by many
Keywords: insecticide resistance, metabolism, hydrolysis, carboxylesterase, ALiE
© 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.
Worldwide, Leptinotarsa decemlineata (Say.)—Colorado potato beetle (CPB) is one of the most
important pests of potatoes, an insect extremely dicult 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 . 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
ecient forms of these enzymes. In addition to being more ecient, 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 . Selective toxicity of insecticides mostly comes
from the balance of the reactions included in activation and in detoxication. Pyrethrins,
pyrethroids, organophosphates (OP), carbamates, and other insecticides are degraded by
hydrolysis. This is the basis for the selective eect of insecticides and for insects’ resistance
mechanisms. Insecticide detoxication primarily unfolds through molecule hydrolysis on
dierent sites, thereby breaking ester, carboxylester, amide, and other chemical bonds [32, 34].
The most important hydrolytic enzymes are phosphoric triesters and carboxylesterases (ALiE
esterases, nonspecic, 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 amplication of genes responsible for DNA methylation.
•Change in their activity—by altering structural genes that directly determine the nature of
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 signicant decrease in or elimination of toxicity. Consequently, esterase activity often
plays a key role in determining the comparative responses and resistance to current insecti-
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 signicance in
insecticide toxicology and resistance has been reviewed by dierent 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 . The role of esterase
in CPB resistance was conrmed by many authors [4, 40–44].
2. Hydrolytic metabolic pathways
Insecticide detoxication is primarily performed by hydrolysis of molecules at dierent sites.
Hydrolysis means spliing of molecules by adding water. This chemical reaction splits
dierent 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 spliing some of the mentioned
chemical bonds there are also some other enzymes involved, such as mixed function oxidases
(MFO) and glutathione S transferases (GST) . 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 .
2.1. Hydrolytic enzymes
Hydrolases are widely spread in diverse plant and animal tissues and dierent parts of cells.
In vertebrates, they can be located in blood plasma, and they are able to aack 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 .
Most of these enzymes are not puried. Hence, it is often unknown if the investigated
hydrolytic reactions are catalyzed by one enzyme of weak specicity (for example, one enzyme
with both esterase and amidase function) or by a mixture of two or more enzymes with higher
specicities. Unlike oxidases and transferases, hydrolases do not require any coenzymes;
however, from time to time, they require cations for activation .
Role of Carboxylesterases (ALiE) Regarding Resistance to Insecticides: Case Study of Colorado Potato Beetle...
There are still some diculties in naming hydrolases and their specicities for substrates. The
same bonds, especially in phosphates, can be aacked 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 . Cyclic phenyl saligenin
phosphate, S,S,S-tributyl phosphorotrithioate (DEF), and profenofos are stated as hydrolase
2.2. Hydrolysis of phosphorus esters
Enzymes that catalyze a hydrolytic aack on phosphorous esters or anhydride bonds are
marked as hydrolases of phosphoric triesters or phospho-triesterases. In insects, these enzymes
are not puried, so they have not been adequately compared with puried mammalian
hydrolases. These hydrolases do not aack phosphoro-trithioates in any scope. The hydrolase
reaction is activated with 1 mM Mn2+ and Co2+ .
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 detoxication 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 .
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 aected the hydrolysis. It is also known
that trichlorfon can be nonenzymatically transformed into dichlorvos in some plant and an-
imal species . Hydrolases aack oxo forms of phosphates, such as paraoxon, diazoxon,
malaoxon (Figure 1), and dichlorvos .
Figure 1. Dierent hydrolases and their specicities for substrates .
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 spliing 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 spliing the P—C bond, and in some others (rabbits), there is a
dierent kind of metabolic reactions. Omethoate (thiolate) is also hydrolytically metabolized
in rats by spliing of the P—S bond. However, spliing of the P—S bond in the P—S alkyl
structure is not hydrolytic, while spliing of the S—C bond most probably is. The former can
result in a P–OH product and the laer 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. Spliing 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 .
2.3. Hydrolysis of carboxylic esters
Carboxyl esterases are also called ALiE esterases or B-esterases. The enzyme that catalyzes the
spliing of the carboxyl ester bond in organophosphates is referred to as EC. 188.8.131.52. [34, 38].
It is widely spread in mammals (in the liver, kidneys, lungs, spleen, small intestine, and uid)
 and in insects, of both susceptible and resistant species. A puried carboxyl esterase in
insects (ALiE) has the molecular weight of 16,000 Da .
After investigating the specicities 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 housey 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 inuence of an unknown factor [35, 39].
Organophosphates of dierent structures inhibit carboxyl esterases or the metabolism of
malathion and other insecticides from this group in vivo . EPN oxygen analogue and n-
propyl-paraoxon inhibit these enzymes and show a strong eect (I50 about 10−8 M) . These
compounds are also strong synergists for malathion and acethion in houseies 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,
Role of Carboxylesterases (ALiE) Regarding Resistance to Insecticides: Case Study of Colorado Potato Beetle...
carbamates, and DDT and with EPN it synergizes dicrotophos, dimethoate, and phorate in
Anthonomus grandis .
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.
Dierences between the enzymes in susceptible and resistant insects are quantitative, and in
Tetranychus urticae, they are also qualitative .
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 specicity
is less pronounced in the enzymatic products from Musca domestica and Blaella germanica.
Relative hydrolysis speed is much higher in mammals than in insects, which is most likely the
basis of the selective toxicity of pyrethroids .
Figure 2. Hydrolysis of dierent OPs pesticides by OPH + CbE enzymes .
Insect Physiology and Ecology164
Lile 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
ecient in carbaryl hydrolysis than enzymes in other mammals. In insects, perhaps, it is about
a nonspecic esterase, aromatic esterase, primarily responsible for carbamate hydrolysis .
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 eect of eld. Hence, malathion-α-monoacid is inactive. The metabolism of malathion in
insects and mammals is quantitatively similar, but qualitatively quite dierent. 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 detoxication of mevinphos, the carboxyl esterase plays no or a very
lile 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,
houseies, 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 .
Besides, oxidative metabolites with a still intact carbamate ester bond are also subjected to
hydrolysis by the same or perhaps dierent enzymes. In many cases, it is impossible to
determine whether oxidation happens before or after hydrolysis . N-substituted carba-
mates, the products of oxidative N-dealkylation are hydrolytically more unstable than N-
methyl carbamates (about 100 times as much).
Role of Carboxylesterases (ALiE) Regarding Resistance to Insecticides: Case Study of Colorado Potato Beetle...
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 diculties 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 .
Pyrethrins and pyrethroids are quite dierently hydrolyzed in living organisms. The
dierences 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 spliing of the ester bond, but oxidation is also
important. Hydrolysis is, however, irrelevant and lile 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
aects the metabolism of these compounds. When the substituent is transposed to the carboxyl
group at C-1, spliing 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
. Like in mammals and in insects, the main primary metabolic process of pyrethroids
(permethrin, deltamethrin, cypermethrin, etc.) in plants (beans, coon, etc.) is the spliing of
the ester bond. Fenvalerate acts similarly in dierent plants (tomatoes, tobacco, leuce,
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
spliing 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). Spliing of the permethrin ester bond is more dicult
in sh. Insects (cockroaches, houseies, 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 spliing 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 houseies. In
Spodoptera lioralis 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 laer 20 times (T. ni), i.e., three times for both
compounds (S. lioralis).
Figure 4. Esterase-mediated hydrolysis of the pyrethroid bifenthrin .
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 eect [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 . In vertebrates, these enzymes are located only in the liver, and they are
primarily related to the microsome fraction. Dierent divalent cations and nucleoids do not
aect the enzymatic activity. The oxidative derivative of dimethoates is not hydrolyzed by
carboxyamides but inhibits this enzyme. Dimethoate of amidases from dierent sources diers
in susceptibility to EPN-induced inhibition in vivo. Hence, housey 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. .
Breaking the amide bond among organophosphates is primarily determined for dimethoate,
dicrotophos, and vamidothion . When compared to other pathways, spliing of the amide
bond of dimethoates by hydrolysis amounts to 27.3% in houseies, 2.5% in rat liver, and 0.0%
of rice leaves in vitro. Spliing of the S─C bond in dimethoate is hydrolytic. Vamidothion is
similarly metabolized. It has been determined that spliing 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.
lioralis, Spodoptera exigua, etc.), it has been recorded that these compounds (diubenzuron,
chloruazuron, diubenzuron, etc.) degrade rapidly. For example, diubenzuron is rapidly
eliminated from insects (t½ about 7 h), and chloruazuron slowly (t½ > 100 h). Adding
hydrolase inhibitors (DEF and related compounds) in food prolongs the retention time (t½
from 7 to 18 h for diubenzuron) and increases the toxicity of diubenzuron for T. castaneum
and S. lioralis. 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 . Therefore, DEF
and prophenophos express a synergetic activity with diubenzuron in S. exigua that has
developed resistance to them, ranging from 3.7 (to DEF) to 5.2 times to prophenophos [34,
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 dierent 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 . They show a pronounced
specicity for substrates, and there is a great variation among the dierent species. The
mechanism of hydration is not clear enough. Perhaps it includes a nucleophilic aack of the
OH group on oxirane carbon . The EH activity from liver microsomes does not depend on
NADPH or CO2 and this enzyme is not aected by BDO-type synergists . 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
. Specic 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 .
EHs catalyze the spliing of the epoxide ring of dierent insecticides and other compounds.
Thereby, they form certain trans-diols that are less toxic, so these enzymes are included in
epoxide detoxication processes. These processes are most studied in cyclodienes. Spliing 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 spliing of the epoxide ring . Epoxides of various alkenes and arenas are enzymatically
hydrolyzed, thus forming trans-di-hydro diols .
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 . Average
enzyme was prepared out of 40 CBP fourth-instar larvae, using 40 ml of phosphate buer
Insect Physiology and Ecology168
(0.02 M, pH7). Incubation of the average enzyme of Colorado potato beetle with dierent
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
Activity of ALiE at varying concentrations of the substrate 1-NA showed that the increasing
the concentration of the substrate aects the increase in 1-N amount, and this dependence is
linear. Statistical analysis has obtained high-correlation coecient (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 specicity 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 dierent 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.
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 dierent concentration of 1-N are
shown in Table 1 and Chart 1. In order to avoid the eect 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 coecient is 0.98, which indicates a high dependence of investi-
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 buer (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 aects the increase in creation of 1-N, and this
dependence is linear. Statistical analysis shows high correlation coecient and a very small
statistical error (as shown in Table 2), which indicates the high dependency of investigated
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 specicity of the
investigated enzyme regarding substrate.
Correlation coecient 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 detoxication. DEF is a specic inhibitor of ALiE esterases, while the ES is specic
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 signicant dierences 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 signicantly decreased activity of the
enzyme. Dierences in the enzyme activity without inhibitor and pre-incubation with DEF are
very signicant. There are very signicant dierences 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 signicant dierences
between the basic enzyme activity and its activity after pre-incubation with ES. Duncan’s test
proved that all the variants belong to dierent groups.
Dierent 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 specic to this group of enzymes, these results indicate their distinguished
activity in potato beetle. Incubating the average enzyme with dierent inhibitors has been
proven in the case of Colorado potato beetle that degradation of 1-NA into 1-N comes under
the inuence 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 eect
of oxidase or glutathione transferase (GST), but due to the eect of esterase (Table 3).
Treatment Absorbance per replication Average Median Variance Standard deviation Duncan test
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 eect on the activity of CPB larvae ALiE.
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 ecient 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, nonspecic, 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 conrmed 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 . 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 specic to this group of enzymes, these
results indicate their distinguished activity in Colorado potato beetle.
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.
Sladjan Stankovic1* and Miroslav Kostic2
*Address all correspondence to: firstname.lastname@example.org
1 Institute for Science Application in Agriculture, Belgrade, Serbia
2 Institute for Medicinal Plant Research “Dr Josif Pancic”, Belgrade, Serbia
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