**4. Insect resistance to insecticides**

According to IRAC (Insecticide Research Action Committee), resistance may be defined as "a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recom‐ mendation for that pest species."

Cross-resistance may be described as an ability to simultaneously develop tolerance to substances they have never been exposed to (resistance to one insecticide confers resistance to another, newly introduced insecticide). Due to large populations and numerous descendants (they breed quickly), there is always a risk that insecticide selection pressure will ultimately result in insecticide resistance. Insecticide resistance may evolve rapidly, especially when field application of insecticides is misused or overused.

The resistance occurs with behavior change, when the insects avoid contacts with the toxicant [39], or physiological change [40], where they survive toxicant exposure. Most important mechanisms of resistance are reduced cuticle penetration, increased excretion, increased metabolic detoxification and altered target-site sensitivity. Knowing molecular basis for emergence and development of resistance is very important for developing appropriate measures and strategies to slow down the resistance [41].

There is never only one cause of resistance. Hence, regardless of the type, the cause of resistance to different pesticides can vary from substance to substance.

According to Hassall [42], factors that lead to resistance are:

1) morphological, 2) physiological and biochemical, and 3) behavioral.

#### **4.1. Morphological changes**

to a wide range of insecticides, including arsenic compounds, organochlorine compounds, carbamates, organophosphates, and pyrethroids [1, 19] and more recently to neonicotinoids [13, 23, 26, 27, 28, 29]. Experimental proofs on development of resistance to *Bacillus thuringien‐*

In Balkan region, a significant level of CPB resistance was detected in 1967 to insecticides from the class of chlorinated hydrocarbons [32], which was proved for most localities of ex-Yugoslavia [33], where the resistance to organophosphorus insecticides and carbamates was detected in some CPB populations. Studying CPB resistance was continued in the years to follow [34, 35]. Remarkably high levels of resistance of the fourth instar larvae to quinalphos and carbaryl were recorded [36, 37]. Research on insecticide resistance level of CPB to most

The rate of resistance development increases progressively with the introduction of new, synthetic insecticides. When it comes to pyrethroids, this resistance occurred 2–4 years after pyrethroids were put into practice and widely used. Physiological and genetic mechanisms of CPB resistance have been little studied. It is expected that in the future CPB will develop

According to IRAC (Insecticide Research Action Committee), resistance may be defined as "a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recom‐

Cross-resistance may be described as an ability to simultaneously develop tolerance to substances they have never been exposed to (resistance to one insecticide confers resistance to another, newly introduced insecticide). Due to large populations and numerous descendants (they breed quickly), there is always a risk that insecticide selection pressure will ultimately result in insecticide resistance. Insecticide resistance may evolve rapidly, especially when field

The resistance occurs with behavior change, when the insects avoid contacts with the toxicant [39], or physiological change [40], where they survive toxicant exposure. Most important mechanisms of resistance are reduced cuticle penetration, increased excretion, increased metabolic detoxification and altered target-site sensitivity. Knowing molecular basis for emergence and development of resistance is very important for developing appropriate

There is never only one cause of resistance. Hence, regardless of the type, the cause of resistance

*sis* products and to transgenic plants, were also obtained [19, 30, 31]

commonly used insecticides is ongoing [6, 17, 18, 38].

resistance to all newly introduced insecticides [1].

application of insecticides is misused or overused.

measures and strategies to slow down the resistance [41].

to different pesticides can vary from substance to substance. According to Hassall [42], factors that lead to resistance are:

1) morphological, 2) physiological and biochemical, and 3) behavioral.

**4. Insect resistance to insecticides**

mendation for that pest species."

22 Insecticides Resistance

Such changes lead to a reduction of the amount of a pesticide that comes into an insect's body in a unit of time, compared to the amount that comes into the body of a susceptible insect. The change in lipid content of the insect's cuticle can result in reduced absorption, as a factor for overall resistance development. This so-called penetration resistance is quite significant since it often goes along with other resistance mechanisms, enhancing their effect. In its simplest form, this can be explained by the fact that different speed of intake is a factor that, combined with excretion, provides a high level of resistance, even during usual enzyme activity [43].

#### **4.2. Metabolic changes**

Populations of resistant insects may detoxify or destroy 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 efficient forms of these enzymes. In addition to being more efficient, these enzyme systems also may have a broad spectrum of activity (i.e., they can degrade many different insecticides).

Changes in insect metabolism could be manifested as:


#### *4.2.1. Gene amplification*

Gene amplification is the multiple copying of structural genes that manage the synthesis of enzymes, thus ensuring hundreds of copies of structural genes. Increased detoxification can be a result of better supply of enzymes, and this quantitative increase is caused by gene amplification [45]. Gene amplification has been determined as a key factor for increased esterase production or change in sensitivity of AChE and Na+ channels (the target-site of 90% of insecticides), but not GST [46].

In the first 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 detoxification [6].

#### *4.2.1.1. Oxidative processes*

Oxidative processes have a dominant role in the metabolism of insecticides present in living organisms. These reactions are catalyzed by enzymes of multifunctional oxidases (MFO) or mono-oxygenises, located in the endoplasmic reticulum, i.e., in microsoms. Cyt P450 is the active center of MFO. MFO mechanism has evolved due to the need of living organisms to protect themselves from many natural toxicants they are constantly exposed to [47, 48]. It is clear that several forms of Cyt P450 exist both in resistant and susceptible strains, while there is a quality difference in different strains [49].

MFO activity of CPB (epoxidation, N- and O-demethylation) is 2–3 times higher in resistant species than in susceptible species. Resistant species have two different types of mfo. Type 1 mfo provides resistance to permethrin, and weak cross-resistance to azinphosmethyl and carbofuran. Type 2 provides resistance azinphosmethyl and carbofuran, but not to perme‐ thrin [50]. This mechanism is comprised in the resistance of CPB larvae and adults to imidaclopr‐ ide [29].

#### *4.2.1.2. Hydrolytic processes*

Insecticide detoxification primarily unfolds through molecule hydrolysis on different sites, thereby breaking ester, carboxyl-ester, amide, and other chemical bonds. Pyrethrins, pyreth‐ roids, organophosphates, carbamates, and other insecticides are degraded by hydrolysis. This is the basis for the selective effect of insecticides and for insects' resistance mechanisms. The most important hydrolytic enzymes are phosphoric triesters and carboxylesterases (ALiE esterases, nonspecific or B-esterases) [50].

Esterase-related insect resistance is based on the following:


The impact of nonspecific esterase on the level of resistance to carbamates has not been confirmed [52], which was also [36] indicated in the case of CPB. The role of esterase in CPB resistance was confirmed [29, 36, 53].

#### *4.2.1.3. Conjugation processes*

Forming of conjugates almost always implies detoxification, but sometimes there are some cases of toxicant reactivation. The most important conjugation reactions are: glutathione conjugation, glucoside or glucuronide conjugation and amino acids conjugation. A change in GST activities depends on modifying a series of enzymes, rarely on only one enzyme, as in esterase. In this class of enzymes, there is no proof that enzyme amplification and resistance are related [54]. When determining the amount of GST-metabolite of azinphosmethyl in resistant CPB strains, no direct impacts of GST to resistance were found. The authors think that the share of GST in total resistance is manifested through the transformation of toxic oxidative metabolites of azinphosmethyl, whose levels are higher in resistant strains [46].

#### *4.2.2. Change in target-site sensitivity*

*4.2.1.1. Oxidative processes*

24 Insecticides Resistance

ide [29].

enzymes.

*4.2.1.2. Hydrolytic processes*

is a quality difference in different strains [49].

esterases, nonspecific or B-esterases) [50].

resistance was confirmed [29, 36, 53].

*4.2.1.3. Conjugation processes*

Esterase-related insect resistance is based on the following:

Oxidative processes have a dominant role in the metabolism of insecticides present in living organisms. These reactions are catalyzed by enzymes of multifunctional oxidases (MFO) or mono-oxygenises, located in the endoplasmic reticulum, i.e., in microsoms. Cyt P450 is the active center of MFO. MFO mechanism has evolved due to the need of living organisms to protect themselves from many natural toxicants they are constantly exposed to [47, 48]. It is clear that several forms of Cyt P450 exist both in resistant and susceptible strains, while there

MFO activity of CPB (epoxidation, N- and O-demethylation) is 2–3 times higher in resistant species than in susceptible species. Resistant species have two different types of mfo. Type 1 mfo provides resistance to permethrin, and weak cross-resistance to azinphosmethyl and carbofuran. Type 2 provides resistance azinphosmethyl and carbofuran, but not to perme‐ thrin [50]. This mechanism is comprised in the resistance of CPB larvae and adults to imidaclopr‐

Insecticide detoxification primarily unfolds through molecule hydrolysis on different sites, thereby breaking ester, carboxyl-ester, amide, and other chemical bonds. Pyrethrins, pyreth‐ roids, organophosphates, carbamates, and other insecticides are degraded by hydrolysis. This is the basis for the selective effect of insecticides and for insects' resistance mechanisms. The most important hydrolytic enzymes are phosphoric triesters and carboxylesterases (ALiE

**•** 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

**•** Change in their activity – by altering structural genes that directly determine the nature of

The impact of nonspecific esterase on the level of resistance to carbamates has not been confirmed [52], which was also [36] indicated in the case of CPB. The role of esterase in CPB

Forming of conjugates almost always implies detoxification, but sometimes there are some cases of toxicant reactivation. The most important conjugation reactions are: glutathione conjugation, glucoside or glucuronide conjugation and amino acids conjugation. A change in GST activities depends on modifying a series of enzymes, rarely on only one enzyme, as in esterase. In this class of enzymes, there is no proof that enzyme amplification and resistance are related [54]. When determining the amount of GST-metabolite of azinphosmethyl in

organism [51] or amplification of genes responsible for DNA methylation.

There are four basic groups of macromolecules, depending on the neurotoxic insecticide targetsite:


#### *4.2.2.1. Acetylcholinesterase (AChE)*

AChE is a target-site for organophosphate and carbamate insecticides. The structure of AChE has undergone some changes that resulted in different levels of transformation of differently structured AChEs. Modified forms of AChE differ among species. As a result, many different forms of cross-resistance are possible [42]). It is important to determine kinetic constants, especially Michaelis constant (Km). It is a constant that measures the enzyme's affinity toward the substrate (ACh, butyrylcholine, and ATCh). During the 1980s and 1990s, some authors [42, 55, 56, 57] indicated that altered AChE causes the resistance to carbamates and organophos‐ phate compounds.

Studies on resistance to organophosphates and carbamates have shown that AChE activity of CPB is quite pronounced and easily measured. The AChE activity of the fourth instar larvae was determined by measuring the absorption, at 585μm wavelength. Total AChE activity was correlated with the determined resistance to carbamate insecticides [36].

#### *4.2.2.2. Na+ channels – Sodium channels*

In some cases where resistance cannot be explained by other causes, one can assume there has been a modification in the target-site structure. The exception is the resistance of flying insects that can lead to diminishing of the knockdown effect. In houseflies, this property is carried by the kdr gene and it can be associated with the alternation of receptors in the nerve cell membrane. Pyrethroids can predominantly affect synaptic sites, which are less sensitive in resistant housefly strains. There is some evidence that the term "change in target-site sensi‐ tivity" was coined to explain the kdr resistance factor [58, 59, 60]. The target-site inactivity of motor nerves' ends to permethrin and deltamethrin is also proved. The insensitivity of bindingsite of resistant strains can be a result of multiple insecticide receptors. It is also possible that weakened binding can be a result of structural changes in proteins or changes in the structure of lipids adjacent to ion pumps in kdr-resistant strains, which can cause minor problems in the mechanism of the ion pump or diminish the process of repetitive polarization, typical for unchanged receptors. It is not clear whether the changes detected in lipid structures of neural membranes of kdr and super-kdr strains of houseflies are a factor that reduces the sensitivity of Na+ channels or these are just compensation changes, necessary for normal functioning of modified Na+ channels [56, 61], as a cause of pyrethroid resistance, which point out changes in the target-site (modification of Na+ channels), detoxification increased by oxidation, hydrolysis, and specific proteins.

#### *4.2.2.3. GABA receptors*

By using subcellular products of the neural tissue of insects, several studies have shown that cyclodienes and lindane have a neurotoxic impact by blocking the GABA receptor complex. Studies on brain tissues of cockroaches showed that resistant strains had 90% lower sensitivity of GABA receptors to cyclodienes. It was found that mutations of Rdl-genes that encode the GABAa receptor subunit caused the resistance of Hypothenemus hampei Ferrari to endosul‐ phan [62].

#### *4.2.2.4. ACh receptors*

Acetylcholine (ACh) is a neurotransmitter that regulates a large number of vital functions. Its activity is enabled by two types of postsynaptic ACh receptors (muscarinic and nicotinic – nAChR). Nicotinic receptors mainly act as ACh activity modulators [63, 64]. The activity of insecticides is manifested through nAChR activation or blocking. The basic structure of nAChR consists of five protein subunits, mostly two identical alpha-subunits and three beta-subunits that give it a pentagonal shape. So far, scientists have detected ten different nAChR genes in insects. The number of nAChR genes implies there are much more nAChR protein subunits, whose main role is recognition when binding the receptor on one side and ACh or insecticide on the other side [65].

Every modification in the protein structure, even the smallest one, can reduce the affinity of nAChR. This mechanism can be a reason for reduced CPB sensitivity to imidacloprid when oxidative and hydrolytic enzymes are blocked [29].

#### **4.3. Behavioral changes**

This type of resistance implies the evolution of behavior, manifested in reduced exposure to toxic compounds or in the insect's ability to survive in toxic or some other kind of fatal environment. Flying insects can acquire the instinct not to dwell long on contaminated surfaces [42]. This resistance mechanism has been recorded for many insecticide classes [66]. Insects simply stop feeding or leave the treated surface.

In CPB, where management is increasingly based on growing transgenic potatoes that contain Bacillus thurigiensis δ- endotoxin, the correlation between these two resistance mechanisms is very significant. More pronounced physiological resistance was recorded [67] in the larvae that avoided transgenic potato crops [31] grown in the same field with nontransgenic potato crops.

#### **4.4. Cross-resistance and multiresistance**

mechanism of the ion pump or diminish the process of repetitive polarization, typical for unchanged receptors. It is not clear whether the changes detected in lipid structures of neural membranes of kdr and super-kdr strains of houseflies are a factor that reduces the sensitivity of Na+ channels or these are just compensation changes, necessary for normal functioning of modified Na+ channels [56, 61], as a cause of pyrethroid resistance, which point out changes in the target-site (modification of Na+ channels), detoxification increased by oxidation,

By using subcellular products of the neural tissue of insects, several studies have shown that cyclodienes and lindane have a neurotoxic impact by blocking the GABA receptor complex. Studies on brain tissues of cockroaches showed that resistant strains had 90% lower sensitivity of GABA receptors to cyclodienes. It was found that mutations of Rdl-genes that encode the GABAa receptor subunit caused the resistance of Hypothenemus hampei Ferrari to endosul‐

Acetylcholine (ACh) is a neurotransmitter that regulates a large number of vital functions. Its activity is enabled by two types of postsynaptic ACh receptors (muscarinic and nicotinic – nAChR). Nicotinic receptors mainly act as ACh activity modulators [63, 64]. The activity of insecticides is manifested through nAChR activation or blocking. The basic structure of nAChR consists of five protein subunits, mostly two identical alpha-subunits and three beta-subunits that give it a pentagonal shape. So far, scientists have detected ten different nAChR genes in insects. The number of nAChR genes implies there are much more nAChR protein subunits, whose main role is recognition when binding the receptor on one side and ACh or insecticide

Every modification in the protein structure, even the smallest one, can reduce the affinity of nAChR. This mechanism can be a reason for reduced CPB sensitivity to imidacloprid when

This type of resistance implies the evolution of behavior, manifested in reduced exposure to toxic compounds or in the insect's ability to survive in toxic or some other kind of fatal environment. Flying insects can acquire the instinct not to dwell long on contaminated surfaces [42]. This resistance mechanism has been recorded for many insecticide classes [66]. Insects

In CPB, where management is increasingly based on growing transgenic potatoes that contain Bacillus thurigiensis δ- endotoxin, the correlation between these two resistance mechanisms is very significant. More pronounced physiological resistance was recorded [67] in the larvae that avoided transgenic potato crops [31] grown in the same field with nontransgenic potato

hydrolysis, and specific proteins.

*4.2.2.3. GABA receptors*

26 Insecticides Resistance

*4.2.2.4. ACh receptors*

on the other side [65].

**4.3. Behavioral changes**

crops.

oxidative and hydrolytic enzymes are blocked [29].

simply stop feeding or leave the treated surface.

phan [62].

Insect populations exposed to certain insecticides have an ability to simultaneously develop tolerance to other substances they have never been exposed to. Insecticide resistance is model of rapid evolution within populations and typical example of directional selection. Eradication of susceptible genotypes from field populations increases both the frequency of resistant genes, which became dominant especially in the absence of susceptible insect refugees, and the application dose of insecticide, needed to keep pest below economically damaging levels. Same physiological or biochemical mechanisms of resistance to one group of insecticides, in some cases, leads to resistance to insecticides from other group/class; such phenomenon is com‐ monly known as cross-resistance [4].

Cross-resistance enables resistant insects to survive the exposure to insecticides with similar chemical composition to the one they are resistant to. In general, cross-resistance results in detoxifying or changing sensitivity to common biochemical and physiological damage. It also happens when one enzymatic system detoxifies more than one class of insecticides [68]. Hence, cross-resistance does not necessarily spread to all members of the same group of insects or it is limited to only closely related pesticides. Similarities in their mode of action or, sometimes the similarity of their enzymatic systems, are more important for their degradation.

On the other hand, multiresistance is resistance to insecticides from different classes. It depends on different mechanisms, so insects can develop resistance to a large number of insecticides from different classes, regardless of their chemical structure. Each new insecticide can cause one or more resistance mechanism to develop, and each developed mechanism results in resistance to similar insecticides. Multiresistance can occur when the organism develops more than one mechanisms of resistance, such as change in AChE sensitivity combined with multienzymatic detoxification, as in the case of organophosphates [68]. Rapid development of multiresistance is the most important reason why organisms quickly become resistant to new compounds introduced to replace inefficient insecticides, which is a result of persistent R-genes and their interactions manifested in several mechanisms of resistance [69].

Recorded cases of negative cross-resistance are very important. Increased resistance to one compound can lead to increased susceptibility to another [70]. Negative cross-resistance has still not been commercially exploited in field conditions, but knowledge on this mechanism can potentially be very important in practice.

Cross-resistance limits the choice of available insecticide, whereas multiresistance represents a rapid overview of insecticide selection that prevents us from reusing insecticides on resistant species for a longer period [69].
