Molecular Insights into Resistance Mechanisms of Lepidopteran Insect

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Molecular Insights into Resistance Mechanisms of Lepidopteran Insect Pests against Toxicants Vishal V. Dawkar, Yojana R Chikate, Purushottam R Lomate, Bhushan B Dholakia, Vidya S. Gupta, and Ashok P Giri J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr400642p • Publication Date (Web): 04 Oct 2013 Downloaded from http://pubs.acs.org on October 8, 2013

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Journal of Proteome Research

Molecular Insights into Resistance Mechanisms of Lepidopteran Insect Pests against Toxicants

Vishal V. Dawkar#, Yojana R. Chikate#, Purushottam R. Lomate#, Bhushan B. Dholakia#, Vidya S. Gupta and Ashok P. Giri* Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008 (MS), India

*Address for correspondence: Ashok P. Giri, Plant molecular Biology Unit, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune- 411 008 (MS), India E-mail address: [email protected] Tel.: +91 (0)20 25902710 Fax: +91 (0)20 25902648 #

Equal contribution

1 ACS Paragon Plus Environment


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ABSTRACT: Insect pests remain as a major reason for crop loss worldwide despite extensive use of chemical insecticides. Greater than 50% of all insecticides are organophosphates, followed by synthetic pyrethroids, organochlorines, carbamates and biopesticides, and their continued use may have many environmental, agricultural, medical and socioeconomic issues. Importantly, only a countable number of insects have acquired the status of crop pests, mostly due to monoculture of crop plants and polyphagous nature of the insects. In this review, we focus on adaptations of Lepidopteran insects to phytochemicals and synthetic pesticides in native and modern agricultural systems. Due to heavy use of chemical insecticides a strong selection pressure is imposed on insect populations, resulting in the emergence of resistance against candidate compound(s). Current knowledge suggests that insects generally implement a threetier system to overcome the effect of toxic compounds at physiological, biochemical and genetic level. Further, we have discussed whether the adaptation to phytochemicals provides an advantage to the insect while encountering synthetic insecticide molecules. Specific metabolic pathways employed by insects to convert deterrents into less toxic forms or their removal from the system are highlighted. Using proteomics approach insect proteins interacting with insecticides can be identified and their modification in resistant insects can be characterized. Also, systems biology studies can offer useful cues to decipher the molecular networks participating in the metabolism of detrimental compounds. KEYWORDS: Lepidoptera, insect-pests, proteomics, field crops, insecticide, resistance mechanisms


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1. LEPIDOPTERAN INSECTS: A MAJOR THREAT TO AGRICULTURAL CROPS Lepidoptera is the largest order of insects that includes butterflies, skippers and moths; some of them are major pests of crops. They cause significant damage to important crops, hence, efficient strategy is required for their control.1Although Lepidopteran larvae damage valuable plants, adults of many species play important role in pollination and some species, such as the silkworm (Bombyx mori), can even be beneficial to humans. Lepidopterans are highly adaptive and these adaptations can be against various kinds external perturbations such as climate, environment and food. High adaptive value and varied physiological situations in the environment have benefited Lepidopterans in evolving multiple survival mechanisms. 2. NATURAL PLANT DEFENSE MECHANISMS Plants have evolved various dynamic defense strategies to improve their survival and reproduction. Plants can produce various secondary metabolites (phytochemicals), which affect the performance and endurance of herbivores.2,3 Plant defense mechanisms vary widely, ranging from mechanical to specific chemical effects that include digestibility reducers and toxins.4,5 One advantage of inducible defense mechanisms over that of constitutive ones is that they are initiated only when needed, and are therefore, potentially less costly.6,7 Plant secondary metabolites include phenolics, terpenoids, alkaloids and their derivatives. Co-evolution with herbivores have allowed

plants to recognize key metabolic processes involved in insects

physiology such as nervous (neurotransmitter synthesis, receptor activation, enzymes involved in signal transduction), digestive and endocrine systems and produce, specialized plant defense molecules targeted against these processes8-10 Essential oils and phenolics act by disrupting the endocrine system of insects and inhibiting important enzymes, respectively.11-13 For example, the



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well known cotton phenolic pigment gossypol is a repellent for several insects due to its toxic effects 14,15 Terpenoids are chemically the the most diverse class of bioactive natural products (over 50,000 structures), produces by plants, which have key role against insects as antifeedants, repellents and toxins.16 For instance, phytoecdysones steroids from the common fern (Polypodium vulgare) disrupt insect molting by mimicking molting hormones.17 In case of Lepidopteran larvae, plant drimane sesquiterpenes block stimulatory effects of glucose and inositol on chemosensory receptor cells located on insect mouthparts.18Alkaloids (over 12000 structures) can modulate insect enzymes, or alter carbohydrate and fat storage by inhibiting the formation of phosphodiester bonds.19,20 By disrupting nerve transmission in insects, alkaloids can also affect cell membrane and cytoskeletal structures, causing cells to weaken, collapse, or even leak.21,22 Interestingly, certain alkaloids such as pyrrolizidine occur naturally in many plants as non-toxic N-oxides, which are converted into the toxic, uncharged, hydrophobic tertiary alkaloids in alkaline digestive tracts of insects.23 Upon ingestion by insects, plant cyanogenic glycosides are metabolized by β-glucosidases, resulting in the formation of the sugar and cyanohydrin moieties, and the latter spontaneously decomposing to toxic hydrogen cyanide.24 Hydrogen cyanide inhibits the insect mitochondrial respiratory pathway enzyme cytochrome coxidase .25-27 Similar to cyanogenic glycosides, glucosinolates (GSL) are protected in vacuoles from thioglucosidases called myrosinases28 that hydrolyze GSL into toxins and feeding repellents such as isothiocyanates, nitriles and thiocyanates. Plants also produce several defense proteins that limit the rate of enzymatic conversion of ingested food in insects, by either altering physical availability or chemical identity.29 The major classes of such defense proteins are proteinase inhibitors (PIs), α-amylase inhibitors, lectins, polyphenol oxidases (PPO), threonine


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deaminase and arginase. PIs inhibit digestive proteases in insects leading to reduced growth performance, weight loss or even death of insects.30-32 Lectins are sugar-binding proteins found particularly in storage organs and protective structures of plants and inhibit nutrient absorption in insects.33 Plant PPOs are known to be induced upon wounding and this is suggestive of their role in defense. For example, Spodoptera litura showed decreased growth with increased mortality upon feeding on transgenic tomato plants

overexpressing a PPO.34 Further, PPO activity

produces highly reactive O-quinones that might reduce the nutritive value of plant proteins by covalently modifying free amino groups.35,36 Threonine deaminase and arginase deplete the level of amino acids Thr and Arg, respectively, leading to adverse effect on insects.37,38 3. SYNTHETIC INSECTICIDES AND THEIR TARGETED ACTIONS Various control strategies including mechanical (ploughing), biological (plant formulations spraying), chemical (insecticides) and cultural control (inter cropping) of crop pest management have been practiced throughout the world. In 1939, Swiss chemist Paul Hermann Müller developed the first synthetic pesticide dichlorodiphenyltrichloroethane (DDT). Further, technological advancement led to the development of more efficient pest control strategies. Currently, several classes of synthetic insecticides are available in the market viz. organochlorides, pyrethroids, neonicotinoids, ryanoids, organophosphates, carbamates etc. (Table 1). In general, these pesticides cause membrane disruption, inhibition of nutrient and ion transport, signal transduction processes, inhibition of metabolism, or the disruption in hormonal control of physiological processes in insect pests.39 4. RESISTANCE MECHANISMS IN INSECTS: KEY TO SURVIVAL Insect employ a variety of resistance mechanisms exist in insects, including detoxification, target site modifications and nerve insensitivity. Detoxification occurs when



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toxins are modified by reduction, oxidation and conjugation reactions, resulting in the excretion of modified toxin molecules. Metabolic defense system in insects generally involves three main groups of enzymes acting in three phases against a number of insecticides and phytochemicals. Phase I involves reduction of toxicity of substrates by cytochrome P450 monooxygenases (CYPs). In phase II, hydrophobic toxic compounds are converted to hydrophilic products by action

of

the

glutathione

S-transferases

(GSTs),

uridinediphosphate

(UDP)-

glucuronosyltransferases (UGTs), and carboxylesterases (COEs) facilitating their excretion. Finally, phase III includes ATP binding cassette (ABC) and major membrane transporters that actively pump conjugated xenobiotics out of the cell. Usually, insects deal with toxic chemicals via avoidance,40 sequestration,41 excretion, target site mutation, alteration of sensitivity, overexpression and producing multiple isoforms of detoxifying enzymes41-44 (Figure 1). The predominant biochemical mechanism for metabolic detoxification of toxic chemicals involves CYPs or COEs43 mediated reactions, resulting in the reduction or oxidation of toxin. GSTs then convert the detoxified molecule into a more water soluble form by glutathione conjugation which facilitate their rapid removal from the cell.45 This can be achieved by either overexpression44 (Figure 1A) or expressing duplicated isoforms of these enzymes39 (Figure 1B). Alternatively, the modification of target site (mutation of amino acid residue) might result in insensitivity or adaptation of insects to toxic chemicals46,47 (Figure 1C, D). Physiological adaptations involve (i) sequestration which refers to selective transport and storage of toxic chemicals preventing their interference in normal physiological processes of insects (stored toxin can be subsequently used for defense against predators by insect)41,48-53 (Figure 1E). Plant defensive chemicals are often targeted to specialized secretory compartments, most of which are stored systemically in the body or localized in the peripheral tissues leading to


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selective compartmentalization. Insects avoid potentially detrimental allelochemicals from sensitive sites by exoskeletal segregation.41 (ii) early inactivation of the toxic chemical (e.g. by insect oral secretion) or avoidance of toxic chemicals (e.g. by vein cutting), most of which are usually detected by visual, olfactory or contact mechanisms40,54 and (iii) excretion (the toxin is readily expelled by insect)55 (Figure 1E). Sequence based transcript expression profiling studies have revealed that in Helicoverpa armigera fed with various host plants, differential expression was identified for genes involved in primary and secondary metabolism, environmental response, cellular processes, xenobiotic metabolism and extracellular matrix-receptor pathways.56 Similarly, in Plutella xylostella, whole genome sequencing revealed duplications in ABC transporter gene families along with CYPs, GSTs and COEs involved in xenobiotic metabolic pathways.57 Interestingly, insects adapt to plant derived toxic compounds, such as cardenolides, by making a single residue change in Na, K-ATPases and high-level molecular convergence of these genes have helped insects to attain reduce sensitivity towards cardenolides.58 Another possible factor responsible for metabolizing such defensive compounds could be the alkaline pH of the herbivore gut that might inhibit plant β-glucosidases.24 Resistance pathways against particular plant secondary metabolites [pyrrolizidinealkaloids (PA), GSL, furanocoumarin etc.] governed by specific insect enzymes (CYPs, COEs, GSTs, nitrilase etc.) are illustrated in Figure 2. Benzoxazinoids or hydroxamic acids are known to be highly toxic plant defensive compounds against the Lepidopteran insect species.59 However, Mythimna separata caterpillars can survive high levels of such benzoxazinoid compounds by possibly into glucosylating them. UGTs are catalysts for the transfer of glycosyl group from UDP-glucose to a variety of acceptor molecules. For example, in Manduca sexta and B. mori, metabolism of phytochemicals such as



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ﬂavonoids and coumarins, and their removal has been shown to occur through UGTs.60 Papilio polyxenes has adapted to feeding on toxin-containing host plants through diversification of the CYPs involved in detoxification and through its furanocoumarin responsive regulatory cascades (Figure 2A).60 Recent studies using plant-mediated RNAi have shown that silencing of H. armigera CYP6AE14 transcripts results in impaired tolerance to gossypol and reduced larval growth.61,62 Lepidopterans sequester plant secondary metabolites such as terpenes, phenols and many nitrogen-containing compounds and uses them as toxic or unpalatable molecules for predators.41 M. sexta accumulates nicotine synthesized by tobacco plants in its own body and uses it as a deterrent to parasitoids. Some Lepidopteran insects such as Utetheisea ornatrix can detoxify alkaloids, store them in their bodies and use them in defense against their own predators such as the lacewing Ceraeochrysa cubana.23 Similarly, metabolic pathway via N-oxygenase or flavin-dependent monooxygenases has been shown to detoxify PAs in Estigmene acrea and Tyria jacobaeae (Figure 2B).63,64 Larvae of Zygaena filipendulae can sequester cyanogenic glucosides and/or synthesize them de novo, and use them as defense molecules.26,65 Some insects redirect the hydrolysis of GSL, that is usually catalyzed by myrosinase, towards the formation of less toxic nitriles, which eventually inhibit the production of toxic isothiocyanates production. This process is found in Pieris rapae, which utilize the nitrile specifier protein to excrete nitriles in their frass.66 Conversely, P. xylostella uses a GSL sulfatase enzyme instead of a nitrile specifier protein to convert isothiocyanates and nitriles into desulfoglucosinolates, which are less toxic and easily excretable (Figure 2C).67,68 Insect can also overcome the adverse effects of plant proteinaceous molecules in myriad ways. They can minimize the effects of PIs by (i) over expressing insensitive proteinases, (ii) regulating the level of existing proteinases or (iii) degrading the


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PIs.69-71 Switching enzymes with altered substrate specificity and upregulation of chymotrypsinlike activity to counter trypsin inhibitor has been reported in H. armigera.32,72 Chemical insecticides kill the pests, while the pests become resistant and eventually require increased doses of chemicals for their control. An overview of classes of chemical insecticides, their representative compounds and modes of action, along with potential insect resistance mechanisms are presented in Figure 3 and 4. Different classes of insecticides, acting via different molecular pathways, can target similar or common insect physiological and biochemical mechanisms. For example, pyrethroids and organochlorides target sodium channels of nervous cells whereas organophosphates and carbamates target acetyl cholinesterases (AChEs) in insect nervous system (Figure 3). Organophosphorus insecticides irreversibly inhibit carboxyl/cholin esterase family enzymes.73,74 Resistance to organophosphates is associated with decreased COEs activity in insect species (Figure 4A).74 Two classes of compounds, organophosphates and carbamates are commonly used to inhibit AChE. However, target-site mutations in AChE are known to compensate for these insecticides and sodium channel mutations confer pyrethroid resistance (Figure 3).75,76 Intensive insecticide use has resulted in the emergence of several resistant insect species possessing altered AChEs.77 Conventional insecticides are designed to target majorly the ligand-gated ion channels, voltage-gated ion channels and AChEs. DDT and pyrethroids are old and widely used pesticides known to inhibit voltage-gated sodium channels. Inherited resistance arises from acquired target-site mutations in the insects. Genetic variation can also result in overproduction of detoxification enzymes, either by gene amplification or gene duplication events.78 Several mutations in voltage-gated sodium channel locus have been reported for pyrethroid resistance in insects. For example, a single amino acid change (from Leu to Phe) is



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known to provide knockdown resistance (kdr) to pyrethroids, as first reported in house flies.79 In case of Cydia pomonella, a single amino acid substitution in an AChE target-site was shown to counteract insecticides.80 Moreover, DNA variation among 38 sequences of para sodium channel gene revealed a unique kdr mutation (L1014F) involved in pyrethroid resistance.47 In Heliothis virescens resistance is associated with mutations encoding Leu to His or Val to Met changes in voltage-gated sodium channel.46,81 Konus et al. (2013)82 have reported that H. armigera can metabolize pyrethroids by modulating energy metabolism by CYPs, ATP synthase and arginine kinase (Figure 4B). DDT resistance has been associated with CYP6G, where the resistant alleles show insertion of partial Accord transposable element at the 5’end (Figure 4C).83 Amplification or production of alternative truncated forms of esterases is also known to be involved in organophosphate resistance. A rich understanding of the relevant biology of insecticide resistance at molecular level will pave a way to design a better and more effective insect control strategy. 5.

IDENTICAL

RESISTANCE

MECHANISMS

ATTRIBUTED

TO

COMMON

TARGETS During the natural phenomenon of plant-insect co-evolution, both the organisms have either developed novel machinery or mechanisms, or accomplished favorable adaptations using existing machinery. Knowledge of plant defensive molecules has contributed to the development of chemical insecticides, since their targets and mode of action in insects are quite similar to that of phytotoxins. For instance, several chemical insecticides target insects’ cellular processes such as signal transduction, membrane trafficking of nutrients and ions, hormonal responses, enzyme activity and other physiological processes, in similar fashion to that of defensive plant secondary metabolites. Examples are, (i) pyrethroids (commercial insecticide) and natural compounds like


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pyrethrins produced by the flowers of pyrethrums (Chrysanthemum cinerariaefolium);84 (ii) certain plant chemicals such as essential oils, terpenoids and organophosphates/carbamates; (iii) neonicotinoids, which are synthetic analogues of the natural insecticide nicotine and which behaves as nicotinic acetylcholine receptor agonists;85-87 and (iv) ryanoids, which are synthetic analogues with the same mode of action as that of ryanodine, a naturally occurring insecticide extracted from Ryania speciosa. Ryanoids bind to Ca2+ channels in cardiac and skeletal muscle, blocking nerve transmission.88-90 Recently, comparative genomics studies have revealed that when compare to B. mori, P. xylostella harbor large set of insecticide resistance genes that may have been acquired by continuous exposure to insecticides.57 Natural mechanisms of resistance against phytochemicals in insects have taken millions of years to evolve. However, the extra selection pressure exerted by the current indiscriminate use of chemical insecticides has greatly accelerated this process, eventually leading to the emergence of stronger resistance mechanisms in insects. Many Lepidopteran species, which have recently became polyphagous, could be considered as an example for this phenomenon. 6. Bacillus thuringiensis (Bt) CRY TOXINS: AN EFFECTIVE BIOINSECTICIDE Transgenic crops expressing Bt Cry proteins are presently the most significant and commonly used strategy against insect pests. The mode of action of Bt toxins in midgut of insects involves several steps, including solubilization of Bt crystals, proteolytic processing of Bt pro-toxin by proteinases, binding of activated toxin to the receptors, and insertion of the toxin molecule into the epithelial cell membrane to create pores.91 Cry toxin first binds to a cadherinlike protein, resulting in a conformational change.92 After proteolytic activation and oligomerization, toxin displays increased binding affinity for aminopeptidase N, which facilitates the insertion of toxins into membrane, along with concomitant pore formation on cell membrane



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which results in cell death by osmotic shock. In an alternative model, Cry toxin monomers bind to a cadherin-like receptor, through which a protein kinase A dependent oncotic signaling pathway is activated leading to cell death.93-95 The most commonly documented Bt resistance mechanism in insects is the alteration of midgut receptor binding for Bt toxins.96,97 However, several studies have reported association of Bt resistance with reduced activity of digestive enzymes involved in the solubilization and activation of Bt pro-proteins. Resistance to Cry1Ac and Cry1Ab in insects falls into this category. Another mechanism reported is overexpression of certain proteases involve in degradation of the Cry toxins.98 7. PROTEOMIC STUDIES ON RESISTANCE MECHANISMS IN INSECTS Numerous insect proteins, in particular enzymes, are known to be key players in resistance mechanisms against phytochemicals and synthetic insecticides. An untargeted ‘Omics’ approach can provide molecular insights for better understanding the evolution of resistance mechanisms in insect pests and enable us to freeze the targets. However, there are only few systematic studies carried out on comparative proteome analysis of insects challenged by phytochemicals or insecticides, which have thus far only add incremental knowledge to existing information, rather than providing new information. Proteomic studies published on plant-insect interactions with an agricultural perspective are listed in Table 2. Detailed phylogenetic, zymographic and quantitative studies have identified specific esterases associated with insecticide resistance in H. armigera.99 Han et al., (2012)100 have reported a major role for members of clade 1 esterases in monocrotophos resistance in H. armigera. Similarly, B. mori larval exposure to pyridalyl [2, 6-dichloro-4 (3-, 3-dichloroallyloxy) phenyl 3-(5trifluoromethyl)-2-pyridyloxy propyl ether] resulted in the identification of thiolperoxiredoxin and proteasomes as the major induced candidates.101


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Proteome mapping of H. armigera midgut lumen revealed multiple isoforms of digestive enzymes (proteases, amylases and lipases) and proteins with functions such as (i) peritrophic matrix binding, (ii) lipocalin-like, (iii) human allergenic enzymes and (iv) insect immune system.102 Interaction studies of recombinant PIs from Capsicum annuum with H. armigera midgut proteases using intensity fading-MALDI-TOF-MS showed gradual processing of multidomain PIs into single domain inhibitors by the action of gut proteases.103 Systematic proteomic analyses of gut, haemolymph and frass of H. armigera upon feeding on non-host plant Cassia tora have provided potential molecular basis for adaptation to plant allelochemicals.104 Defense related proteins were upregulated in haemolymph and substantial amount of proteins involved in energy metabolism were found in frass. These findings were further supported by a recent report from Stevens et al., (2013)105 which provided detailed analysis of insect frass proteins upon exposure of larvae to plant PIs. Using shotgun HPLC-ESI-MS proteomic approach, 2043 peptides were identified from the midgut of S. litura larvae wherein most of the catalytic proteins were hydrolases, oxidoreductases and transferases.106 Proteomic analyses

of Cry toxin fed

Plodia interpunctella larva revealed approximately 300 proteins involved in Bt resistance.107,108 Genomic tilling arrays and differential proteomics of Tribolium castaneum challenged by diflubenzuron revealed that UDP-N-acetylglucosamine, pyrophosphorylase and glutathione synthetase were significantly upregulated.109 In the sweet potato whitefly Bemisia tabaci, transcript and protein profiling studies have revealed a molecular basis for thiamethoxam resistance.110 Birner-Gruenberger et al. (2012)111 have employed a functional proteomics approach to identify and characterize Drosophila melanogaster esterases found to be involved in insecticide tolerance, lipid metabolism and lifespan control. Proteomic mapping studies on D. melanogaster



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exposed to insecticides demonstrated that insects can adapt to feeding in the presence of toxin through regulation of enzymes, which are associated with ion transport, lipid and sugar metabolism pathways.112 Changes in protein metabolism in response to azadirachtin were studied in the pupae of S. litura113 and ten proteins, including ecdysone receptor, were found to be significantly affected. This indicates a potential interaction between azadirachtin and the ecdysteroid hormonal system, which can cause major disruption in the growth and development of an insect.113 An investigation of o-sec-butyl phenyl methyl carbamate (BPMC) toxicity in the brown planthopper showed differential expression of eighteen proteins with appearance of four new proteins.114 Altogether, the above mentioned studies have targeted the differential expression of numerous proteins rather than any one specific functional component or mechanism. These reports demonstrate the utility of proteomics approach to identify proteins interacting with insecticides and characterize modifications in the target-site of proteins responsible for the emergence of insecticide-resistance in insects. Future efforts using proteomics and system biology approaches can further improve our knowledge of insect adaptation and their resistance mechanisms. 8. FUTURE PROSPECTIVE In the perquisite of society, researchers need to identify and develop new insecticides to control various kinds of insects, which will not be perilous for environment. Bt transgenic crops have been greatly successful in controlling insect infestation and helping to reduce the use of chemical pesticides. However, evolution of insect resistance threatens the continued success of Bt transgenic crops.115 Under these circumstances, generation of insecticides that are less harmful to beneficial insects and environment is necessary. Plant originated proteins such as PIs, lectins or bacterial vegetative insecticidal proteins could serve as the potential insect controlling


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agents in near future. Mithofer and Boland (2012)116 have suggested some alternative approaches which might help in developing new strategies to protect crop plants from insect pests. It is necessary to know the exact basis of insect resistance mechanism that will ultimately help to design better insect control strategies. For example, following the discovery that Bt resistance in insects is associated with the change in Cry toxin binding receptor, future work can focus on developing toxins that can elicit their activity without the need for receptor binding.117 Furthermore, identification of the key molecules involved in the resistance mechanism would probably require high-throughput analytical techniques and methodologies. Modern integrated ‘Omics’ approaches (such as genomics, proteomics and metabolomics) are certainly providing the ways to go forward in this area. Proteomics could be a good approach to understand the biochemical mechanisms of insecticide resistance in insects. Before insecticide resistance in insects gets out of control, there is a need to establish robust resistance management plans in place, based on existing knowledge and ongoing studies using cutting age technology, in order to save the cops. ACKNOWLEDGEMENTS VVD and YRC are thankful to the Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi for Research Associateship and Senior Research Fellowship respectively. PRL acknowledges support from Department of Biotechnology (DBT), Government of India, New Delhi for Research Associateship. Authors are thankful to Dr. D. Shanmugam for critical suggestions in manuscript. We apologize to all researchers whose publications have not been included in this review due to space limitation. Project funding under CSIR network programs in XII plan (BSC0107 and BSC0120) is greatly acknowledged.



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110. Yang, N.; Xie, W.; Yang, X.; Wang, S.; Wu, Q.; Li, R.; Pan, H.; Liu, B.; Shi, X.; Fang, Y.; Xu, B.; Zhou, X.; Zhang, Y. Transcriptomic and proteomic responses of sweetpotato whitefly, Bemisia tabaci, to thiamethoxam. PLoS ONE 2013, 8, e61820. 111. Birner-Gruenberger, R.; Bickmeyer, I.; Lange, J.; Hehlert, P.; Hermetter, A.; Kollroser, M.; Rechberger, G. N.; Kuhnlein, R. P. Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster alpha-Esterase-7. Insect Biochem. Mol. Biol. 2012, 42, 220-229. 112. Pedra, J. H. F.; Festucci-Buselli, R. A.; Sun, W. L.; Muir, W. M.; Scharf, M. E.; Pittendrigh,

B.

R.

Profiling

of

abundant

proteins

associated

with

dichlorodiphenyltrichloroethane resistance in Drosophila melanogaster. Proteomics 2005, 5, 258-269. 113. Huang, Z. W.; Shi, P.; Dai, J. Q.; Du, J. W. Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin. Pest. Biochem. Physiol. 2004, 80, 85-93. 114. Sharma, R.; Komatsu, S.; Noda, H. Proteomic analysis of brown planthopper: application to the study of carbamate toxicity. Insect Biochem. Mol. Biol. 2004, 34, 425-432. 115. Tabashnik, B. E.; Gassmann, A. J.; Crowder, D. W.; Carriere, Y. Insect resistance to Bt crops: Evidence versus theory. Nat. Biotechnol. 2008, 26, 199-202. 116. Mithofer, A.; Boland, W. Plant defense against herbivores: Chemical aspects. Annu. Rev. Plant Biol. 2012, 63, 431-450. 117. Pardo-Lopez, L., Soberon, M, Bravo, A. Bacillus thuringiensis insecticidal threedomain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiol. Rev. 2013, 37, 3-22.


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118. Newcomb, R. D.; Campbell, P. M.; Ollis, D. L.; Cheah, E.; Russell, R. J.; Oakeshott, J. G. A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 7464-7468. 119. Krishnamoorthy M.; Jurat-Fuentesa, J. L., McNall R. J.; Andacht, T.; Adanga, M. J. Identification of novel Cry1Ac binding proteins in midgut membranes from Heliothis virescens using proteomic analyses. Insect Biochem. Mol. Biol. 2007, 37, 189-201. 120. Teese, M. G.; Campbell, P. M.; Scott, C.; Gordon, K. H. J.; Southon, A.; Hoyan, D.; Robin, C.; Russell, R. J.; Oakeshott, J. G. Gene identification and proteomic analysis of the esterases of the cotton bollworm, Helicoverpa armigera. Insect Biochem. Mol. Biol. 2010, 40, 1-16. 121. Courtiade, J. M., A; Svatos, A.; Heckel, D. G.; Pauchet, Y. Comparative proteomic analysis of Helicoverpa armigera cells undergoing apoptosis. J. Proteome Res. 2011, 10, 2633-2642. 122. Carinhas, N. Robitaille, A. M.; Moes, S.; Carrondo, M. J. T.; Jenoe, P. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection. PLoS ONE 2011, 6, e26444. 123. Celorio-Mancera, Mde. L.; Courtiade, J.; Muck, A.; Heckel, D. G.; Musser, R. O. Sialome of a generalist lepidopteran herbivore: identification of transcripts and proteins from Helicoverpa armigera labial salivary glands. PLoS ONE 2011, 6, e26676. 124. Jurat-Fuentes, J. L.; Karumbaiah, L.; Jakka, S. R. K.; Ning, C.; Liu, C. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to cry toxins from Bacillus thuringiensis. PLoS ONE 2011, 6, e17606.



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125. Soulages, J. L; Firdaus, S. J.; Hartson, S.; Chen, X.; Howard, A. D.; Arrese, E. L. Developmental changes in the protein composition of Manduca sexta lipid droplets. Insect Biochem. Mol. Biol. 2012, 42, 305-320. 126. Zhang, Q.; Lu, Y.; Xu, W. Integrated proteomic and metabolomic analysis of larval brain associated with diapause induction and preparation in the cotton bollworm, Helicoverpa armigera. J. Proteome Res. 2012, 11, 1042-1053.


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FIGURE LEGENDS Figure 1. General defense mechanisms in insects. Insect pests employ the above mentioned mechanisms to overcome plant allelochemicals or insecticides. Increased resistance to insecticides can be due to overproduction of detoxifying enzymes such as GST, CYP and COE (A), gene duplication (multiple isoforms of enzymes) (B), Mutations ( or

insertion,

deletion

substitution) in the target that result into insensitivity to insecticide (C) or target site

modification

causing

reduced

insecticide

binding

(D),

sequestration

(selective

compartmentalization and storage of toxic chemicals), avoidance by visual, olfactory signals or by contact and excretion (ready removal of toxicants) (E). Figure 2. Detoxification pathways for certain classes of plant allelochemicals. Furanocoumarin (A), pyrrolizidine alkaloid (B) and glucosinolate (C) are reproduced from reported studies. FMO: flavin-containingmonooxygenase, NSP: nitrile specifier protein. Figure 3. Major classes of chemical insecticides, their mode of action and resistance mechanism in insects against these insecticides. Internal blue colored circle shows the class of insecticide and external pink circle illustrates the target of a corresponding insecticide in insect. The ring and ball structure of representative insecticide (e.g. acephate for organophosphates, cypermethrin for pyrethroids, DDT for organochlorides, imidacloprid for neonicotinoids, rynaxypyr for ryanoids and aldicarb for carbamates) from each class has been placed in between two circles. External green triangles indicate the mechanism of detoxifications used by the insects to cope up with the adverse effects of insecticides. Figure 4. Defence pathways of insect pests against particular synthetic insecticide. Phase I and II reactions for organophosphate (A), pyrethroid (B) and organochlorines (C) are reproduced



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from

reported

studies.

DDT:

Dichlorodiphenyltrichloroethane,

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DDD:

dichlorodiphenyldichloroethane and DDE: Dichlorodiphenyldichloroethylene.


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Table 1. Mode of action of major insecticides, their target insects and resistance mechanisms in insect pests. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Class

Insecticide

Toxicity

Organochlorides39,99

DDT Endosulfan Aldrin Methoxychlor Dieldrin Chlorpyrifos Acephate Monocrotophos Parathion Piperonyl butoxide Aldicarb Methomyl Carbaryl Carbofuran Formetanate Cypermethrin Deltamethrin Fenvalerate Tetramethrin Etofenprox Acetamiprid Clothianidin Imidacloprid Nitenpyram Nithiazine Rynaxypyr

Broad spectrum

Organophosphates118

Carbamates118

Pyrethroids84

Neonicotinoids85-87

Ryanoids88-90

Mode of action

Insect resistance status Modulate sodium channel of Resistance plasma membrane of the nervous cells

Resistance mechanism Modification of para sodium channel, enzymatic detoxification, strong nerve insensitivity, penetration resistance Target site insensitivity mutations, knockdown resistance, enzymatic detoxification

Broad spectrum

Acetylcholine esterase Inhibitors

Resistance

Broad spectrum

Acetylcholine esterase Inhibitors

Resistance

Target site insensitivity mutations, knockdown resistance

Broad spectrum

Sodium channel modulators

Resistance

Modifications of the sodium channel protein, penetration resistance, strong nerve insensitivity

Broad spectrum

Nicotinic Acetylcholine receptor agonists / antagonists

Resistance

Overexpression of microsomal oxidases, target site resistance, oxidative detoxification

Lepidoptera

Activators of Ryanodine receptor of Ca2+ channels in cardiac and skeletal muscles

No resistance

Resistance not reported yet



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Table 2. Proteomics studies on Lepidopteran insects related to agriculture. Different platforms and tools were used to identify general, specific or 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

interacting proteomes.

Insect pest M. sexta108

Source Larval midgut

•

P. interpunctella107 S. litura113

Larval midgut

•

Pupae

•

Nilaparvata lugens114 H. virescens119 H. armigera102

Whole larvae

•

S. litura106 H. armigera120 H. armigera103 H. armigera104 H. armigera121 S. frugiperda122 H. armigera123 H. virescens, H. armigera, S. frugiperda124 M. sexta125

Highlights Combination of mass spectroscopy and western blotting leads to identification of two new B. thuringiensis Cry1Ac binding proteins Protein modifications and changes in the synthesis of specific proteins helps to insects in physiological adaptations against Bt Cry proteins Identified potential interaction between azadirachtin and the ecdysteroid hormonal system responsible for arresting growth and development Use of proteomic data for the identification of target genes and predicting their functions

Larval midgut Larval midgut

• de novo sequencing approach to identify additional Cry1Ac binding proteins • Proteome mapping of the larval midgut lumen • Characterization of the nutritional and defensive functions of important intraorganismal extracellular proteins Larval midgut • Relationships between physiological events and functions of the proteins Larval gut • Role of carboxyl/cholinesterase in organophosphates and synthetic pyrethroids resistance Larval gut • Response to PIs by expressing protease variants Gut, hemolymph • Dissected adaptation and/or detoxification mechanisms by regulating specific metabolic and frass of pathways larvae Cell line of • Discovered apoptotic pathways in Lepidoptera are highly conserved with mammals or pupal ovaries Eukaryote lineage Larvae • Highlights possible regulatory bottlenecks associated with the described cell density effect on viral replication Salivary glands • Identification of secreted proteins from salivary gland • Comprehensive understanding of enzymes responsible for digestion and plant offense Larval midgut • Identification of midgut membrane-bound alkaline phosphatase as a target for development of biomarkers for Bt Cry toxin resistance Lipid droplets of • Lipid droplets proteome revealed involvement of complex organelles in the function of 36 ACS Paragon Plus Environment

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H. armigera H. armigera100 T. castaneum109

larval fat body Larval brain Larval midgut Larval midgut

B. tabaci110

Whole fly

H. armigera82

Larval midgut

126

• • •

• • •

cellular metabolism Larval brain proteins/molecules programs pupal diapause Major role of esterases in the monocrotophos resistance along with CYPs or GSTs Found enzymes involved in chitin metabolism remain unaffected upon exposure to diflubenzuronwhile UDP-N-acetylglucosaminepyrophosphorylase and glutathione synthetase were upregulated Expression of a suite of Phase I and Phase II detoxification enzymes Identification of proteins involved in metabolism of thiamethoxam Increased activity of energy metabolism related proteins for compensating the costs of energy-consuming detoxification processes



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Graphical Abstract

C B

D Death

Change in binding site

Multiple gene copies

Survival

Target site modifications

A

E Sequestration

High enzyme production

Avoidance ACS Paragon Plus Environment

Inside insect Outside insect

Excretion

Figure 2

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ACS Paragon Plus Environment


Figure 3

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Figure 4

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Graphical Abstract

C B

D Death

Change in binding site

Multiple gene copies

Survival

Target site modifications

A

E Sequestration

High enzyme production

Avoidance ACS Paragon Plus Environment

Inside insect Outside insect

Excretion

Molecular Insights into Resistance Mechanisms of Lepidopteran Insect

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