**4. Insecticide resistance mechanisms in insect pests of tea**

Insects come across with numerous toxins as they go through their life cycle. Some of these toxins are naturally produced by plants (plant allelochemical) and others by humans (synthetic insecticides). To protect themselves against the natural toxins, insects have evolved various detoxification mechanisms [41] These mechanisms also cross-protect insect pests when they are exposed to synthetic insecticides [25]. Herbivorous insect groups (agricultural pests) are significantly more diverse than their non-herbivorous sister groups [46]. The role of plant in promoting diversification in insects has occurred through co-evolutionary 'defence strategies' among them [47]. This diversification could also have been a result of insects 'tracking' plant phylogenies, with minor chemical changes in plants allowing the evolving populations of insects to change and speciate accordingly, which probably has occurred long after chemical changes in plants [48]. Evolution to herbivory preceded via mixed feeding on reproductive parts or spores, dead tissues of plants and animals and fungi. This progression implies that omnivory preceded generalized herbivory and the evolution of specialization on specific plant taxa was a later accomplishment [49].

Among sucking insect herbivores, the actual food used, i.e. digested whole tissue particularly parenchyma (as in *Helopeltis* sp.), cell content (thrips) and phloem flow (*Empoasca* sp.), influences both the feeding mechanism and feeding behaviour [48]. While the chewing insects (looper caterpillar complex) cause extensive damage, the sucking insects cause modest to barely perceptible damage. However, sucking insects, particularly phloem and digested tissue feeder,impose an additional challenge to the plants as they deplete photosynthates, act as vector of viruses and introduce chemical and protein effectors that alter plant defence mechanisms (signalling) and development [50]. When these attributes are combined with a broad host range, breeding strategies that promote invasiveness, highly evolved feeding strategies, the ability to adapt to a wide range plant habitats and the emergence of insecticide resistance, it is not surprising that sucking insects cause heavy losses in agriculture and horticulture [51].

Insecticide resistance is a genetic change in response to selection pressure of toxicants that impair pest control in the field [52]. Insecticide resistance does not occur unless a structural genetic change occurs that is heritable. Therefore, insecticide resistance is an evolutionary phenomenon that results under the selection pressure of a new toxicant in the environment [8].Thus insecticide resistance is different from insecticide tolerance. Insecticide tolerance is the natural ability of a population to withstand the toxic effect of a particular insecticide. It can develop within one generation as a result of physiological adaptation, i.e. induction of xenobiotic detoxifying enzymes. Hence, variation within a population may include individu‐ als with genetic traits that make them better adapted to survive in exposure to an insecticide. If these individuals survive the insecticide exposure, then the tolerance traits can be passed on to the next generation, thereby enriching the gene pool with those genes. The mechanisms of development of insecticide tolerance can be divided into four levels:

*Altered behaviour – avoidance of contact with the insecticide*

tophos, acephate, dimethoate, phosalone, carbaryl and triazophos [32]. Recently, several insecticides have been tested on *S. dorsalis* in chili ecosystem in USA and found limited success with chlorfenpyr, spinosad and imidacloprid [33, 34]. The performance of novaluron, aba‐ mectin, spiromesifen, cyfluthrin, methiocarb and azadirachtin failed to provide effective

Similarly, in another emerging sucking insect pest of tea, tea greenfly, *E. flavescens*, repeated management failure and biochemical insecticide resistance in tea ecosystem from Northeast India have been reported [11, 24]. In China, chemical insecticides including fenvalerate, cyfluthin, cypermethrin and imidacloprid are sprayed to control the leafhoppers as frequently as seven times annually or even more frequently [36–37]. A high level of resistance against many insecticides has been reported in related species, *E. vitis* [38]. The resistance to thiame‐ thoxam was highest and to cyperemethrin was lowest in *E. vitis.* Recently, in Fujian province of China, a regional diversity of resistance to eight insecticides in *E. vitis* has been reported in tea ecosystem with higher resistance level to bifenthrin, acetamiprid, imidacloprid, cartap and

A high level of insecticide resistance in folivores, such as black hairy caterpillar, bunch caterpillar, looper pest complex (*Hyposidra talaca, H. infixaria, Buzura suppressaria, Eturesia magnifica*) and in termite of tea ecosystem, has been reported with reduced susceptibility

Detoxification of insecticides is an important toxicokinetic mechanism for insect pests to tolerate regularly applied insecticides [8, 40–42]. Susceptibility levels against insecticides change mainly due to metabolic detoxification of the insecticides through the induction of some detoxifying enzymes under the stress of different management practices [43–45].

Generally, three principal enzymes, general esterases (GEs), glutathione S-transferases (GSTs) and cytochrome P450-mediated monooxygenases (CYP450s), are involved in the process of metabolic detoxification of insecticides [41]. Estimation of the activities of these metabolic defence-related detoxifying enzymes gives information on the level of tolerance/resistance of the insect pest population to insecticides and is a useful tool in monitoring the tolerance/ resistance to insecticides at population level of the pest. The early detection of metabolic threats related to tolerance/resistance to insecticides in pest specimens is of crucial importance for devising pest control techniques that would minimize the development of tolerant/resistant

Insects come across with numerous toxins as they go through their life cycle. Some of these toxins are naturally produced by plants (plant allelochemical) and others by humans (synthetic insecticides). To protect themselves against the natural toxins, insects have evolved various detoxification mechanisms [41] These mechanisms also cross-protect insect pests when they are exposed to synthetic insecticides [25]. Herbivorous insect groups (agricultural pests) are significantly more diverse than their non-herbivorous sister groups [46]. The role of plant in

forms and prevent any undesirable wastage of insecticide, money and manpower.

**4. Insecticide resistance mechanisms in insect pests of tea**

control of this pest [35].

352 Insecticides Resistance

chlorfenapyr [39].

against different insecticides.

*Development of barrier tissues – reduced penetration of the insecticide though the integument*

*Enhanced detoxification – higher metabolism of the insecticide*

*Alteration of receptor – at the target site for the insecticide.*

The first level, at which insecticide tolerance can develop, is when the insect encounters an insecticide. An altered behaviour helps the insect to avoid coming into contact with the insecticide. Once the insect comes in contact with the insecticide, a reduced and delayed penetration through the cuticle will reduce the effect of the insecticide at the target site; this is yet another level of resistance. Within the insect's body, the insecticide may be enzymatically metabolized and thereby inactivated. At the third level of resistance mechanism, three systems of xenobiotic detoxification enzymes operate: esterases, glutathione S-transferases and cytochrome P450-dependent monooxygenases. The increased activity of one of these enzyme systems in metabolizing insecticides will result in insecticide tolerance. Alterations at the target site for the insecticide are the last level of insecticide resistance mechanisms. Different classes of insecticides bind to specific target sites and reduced binding at the target site, or increased number of target site molecules may confer insecticide resistance.

#### **4.1. Behavioural resistance**

Behavioural resistance mechanisms are the least studied resistance mechanisms in insects, but this is not to say that behavioural resistance is the least significant. Behavioural resistance can be defined as '*evolved behaviours that will reduce an insect's exposure to toxic compounds or that allows an insect to survive in what would otherwise be a toxic and fatal environment*' [53]. Behavioural resistance has been observed in more than 30 species of insects [53]. Avoidance is the first step in the evolution of behavioural resistance [54]. In *H. theivora*, this kind of resistance has been seen [55]. *H. theivora* shows a different egg-laying strategy to avoid insecticide exposure. Even *E. flavescens* avoids exposure to direct sunlight and therefore prefers to stay on the underside of the tea leaves. This behaviour cross-protects it from the direct insecticide exposure in conventional tea plantations during spraying [8]. The same has been found in *S. dorsalis* which resides inside the leaf bud during development and underside of the leaf during adult stage, thereby avoiding direct exposure to insecticides [8].

#### **4.2. Reduced penetration**

Reduced penetration of insecticides through barrier tissues of insects is another way in which an insect can modify the effective dose of insecticide at the target site. The mechanism may not prevent the insecticide from eventually entering the insect, but it can reduce the rate at which the insecticide reaches the target site. Reduced penetration has been shown to function as a resistance mechanism to many different insecticides, and, by the nature of this mechanism, cross-resistance is often found [56]. The rate of penetration of insecticides through the insect cuticle or other barriers (peritrophic membrane) depends on the physicochemical properties of the insecticide and the barrier. A reduced penetration contributes to DDT resistance in the tobacco budworm, *Heliothis virescens* F. (Lepidoptera: Noctuidae). DDT-resistant larvae had an altered composition of the cuticle. The protein and lipid contents are greater in the cuticle of resistant larvae and, furthermore, the cuticle of the resistant larvae probably had a higher degree of sclerotization [57]. In *M. domestica*, two resistant strains, with reduced penetration as one of the resistance mechanisms, also showed increased cuticular lipid content; more total lipids, mono-glycerides, fatty acids, sterols and phospholipids were present in the resistant strains compared to a susceptible strain [58]. Reduced penetration has been documented as a resistance mechanism only at the level of the insect cuticle, but any biological membrane may serve as a barrier and thereby give resistance [59]. As a single resistance mechanism it usually only confers low levels (less than threefold) of resistance [59]. Reduced penetration has been shown to function as a resistance mechanism to many different insecticides, including insecticides of the three major classes, OPs, carbamates and pyrethroids. Reduced penetration of OPs through the cuticular barrier has been reported, for example, diazinon in *M. domesti‐ ca* [60], azinphos-methyl in the pear psylla, *Psylla pyricola* Foester (Hemiptera: Psyllidae) [61] and profenofos in *H. virescens* [62]. However, by slowing the penetration rate of insecticides, this mechanism reduces the risk that the insects' detoxification systems become overloaded, and the dose of insecticide reaches to a lethal level at the target site. In female *H. theivora*, a higher level of body lipid has been found which effectively reduces the penetration of insecticide to the target site [63]. No studies on resistance due to reduced penetration in *E. flavescens* and *S. dorsalis* or any other tea pests have been reported to date. The studies had shown that when different resistance mechanisms are combined in the same individuals, a synergistic effect, resulting in a high level of resistance, may arise [64, 65]. Therefore, even a small degree of reduced penetration can contribute significantly to the overall insecticide resistance of the insect pests.

#### **4.3. Metabolic detoxification**

metabolized and thereby inactivated. At the third level of resistance mechanism, three systems of xenobiotic detoxification enzymes operate: esterases, glutathione S-transferases and cytochrome P450-dependent monooxygenases. The increased activity of one of these enzyme systems in metabolizing insecticides will result in insecticide tolerance. Alterations at the target site for the insecticide are the last level of insecticide resistance mechanisms. Different classes of insecticides bind to specific target sites and reduced binding at the target site, or increased

Behavioural resistance mechanisms are the least studied resistance mechanisms in insects, but this is not to say that behavioural resistance is the least significant. Behavioural resistance can be defined as '*evolved behaviours that will reduce an insect's exposure to toxic compounds or that allows an insect to survive in what would otherwise be a toxic and fatal environment*' [53]. Behavioural resistance has been observed in more than 30 species of insects [53]. Avoidance is the first step in the evolution of behavioural resistance [54]. In *H. theivora*, this kind of resistance has been seen [55]. *H. theivora* shows a different egg-laying strategy to avoid insecticide exposure. Even *E. flavescens* avoids exposure to direct sunlight and therefore prefers to stay on the underside of the tea leaves. This behaviour cross-protects it from the direct insecticide exposure in conventional tea plantations during spraying [8]. The same has been found in *S. dorsalis* which resides inside the leaf bud during development and underside of the leaf during adult stage,

Reduced penetration of insecticides through barrier tissues of insects is another way in which an insect can modify the effective dose of insecticide at the target site. The mechanism may not prevent the insecticide from eventually entering the insect, but it can reduce the rate at which the insecticide reaches the target site. Reduced penetration has been shown to function as a resistance mechanism to many different insecticides, and, by the nature of this mechanism, cross-resistance is often found [56]. The rate of penetration of insecticides through the insect cuticle or other barriers (peritrophic membrane) depends on the physicochemical properties of the insecticide and the barrier. A reduced penetration contributes to DDT resistance in the tobacco budworm, *Heliothis virescens* F. (Lepidoptera: Noctuidae). DDT-resistant larvae had an altered composition of the cuticle. The protein and lipid contents are greater in the cuticle of resistant larvae and, furthermore, the cuticle of the resistant larvae probably had a higher degree of sclerotization [57]. In *M. domestica*, two resistant strains, with reduced penetration as one of the resistance mechanisms, also showed increased cuticular lipid content; more total lipids, mono-glycerides, fatty acids, sterols and phospholipids were present in the resistant strains compared to a susceptible strain [58]. Reduced penetration has been documented as a resistance mechanism only at the level of the insect cuticle, but any biological membrane may serve as a barrier and thereby give resistance [59]. As a single resistance mechanism it usually only confers low levels (less than threefold) of resistance [59]. Reduced penetration has been shown to function as a resistance mechanism to many different insecticides, including

number of target site molecules may confer insecticide resistance.

thereby avoiding direct exposure to insecticides [8].

**4.1. Behavioural resistance**

354 Insecticides Resistance

**4.2. Reduced penetration**

Metabolic detoxification of insecticides is an important toxicokinetic mechanism for insects to tolerate the toxic effects of insecticides. Generally, lipophilic (hydrophobic) insecticides are rapidly detoxified. Organophosphates, organochlorines, carbamates and pyrethroids are lipophilic compounds, and detoxification enzymes transform these insecticides to more hydrophilic and less biologically active compounds so that can be eliminated more easily by excretion. Increased detoxification of insecticides has often been reported in many resistant populations [40]. Three enzyme systems are generally recognized as the major detoxification systems involved in insecticide resistance in insects. These are carboxylesterases, cytochrome P450-dependent monooxygenases and glutathione S-transferases [40, 41].

#### **4.4. Alteration at the target site for insecticide (target site insensitivity)**

The biochemical sites for insecticide action differ for different insecticides and are a potential field of research for developing insecticides, which can act specifically or more efficiently on insect biochemical sites compared to mammals. The target site receptor for action of organo‐ phosphates, carbamates, organochlorines and pyrethroids is in the nervous system. The enzyme acetylcholinesterases (AChEs) (EC 3.1.1.7) are the target sites for organophosphates and carbamates, and voltage-gated sodium channel of the nerve membrane is the target of pyrethroids and DDT. Neurotoxic insecticides such as cyclodienes (e.g. dieldrin and endosul‐ fan), y-HCH (lindane) and fipronil target gamma-aminobutyric acid (GABA)-receptor [68, 69] and nicotinyl insecticides (imidacloprid and nicotine) target the nicotinic acetylcholine receptor (nACHR) [70]. Alteration at the target site, to less a sensitive target for neurotoxic insecticides, is an important toxicodynamic resistance mechanism in insects [71].

In insects, the potent inhibitors of AChE are organophosphates and carbamates. These compounds inhibit the activity of AChE by forming a stable covalent intermediate, preventing the enzyme to hydrolyse acetylcholine. An accumulation of acetylcholine keeps the ion channel of the receptor permanently open, which eventually kills insect. OPs and carbamates are quasiirreversible inhibitors of AChE. The organophosphates and carbamates phosphorylate and carbamylate the active site serine of AChE, respectively [72]. Generally, the reactivation time of phosphorylated or carbamylated AChE is long. However, the half-lives of reactivation vary considerably, from minutes to several days, depending on the compound interacting with AChE [73]. Carbamylated AChE generally reactivates faster than phosphorylated AChE. Reduced sensitivity of AChE to inhibition by OPs and carbamates is an important resistance mechanism in insects and is often referred to as altered or insensitive AChE [74]. The presence of insensitive AChE conferring resistance was first noticed in OP-resistant mites, *Tetranychus urticae* Koch (Acari: Tetranycidae) [74] and also found in several insect populations resistant to these compounds [75–77]. Insecticide susceptible and resistant insect pest populations differ in the level of AChE activity [78–80]. A higher level of AChE activity has been reported in *H. theivora* sampled from conventional tea plantations than from organic garden indicating the presence of resistance to insecticides in conventional tea ecosystems [81, 82]. There is no such report on *S. dorsalis* and *E. flavescens* in conventional tea ecosystems to date.
