==== Front Front Physiol Front Physiol Front. Physiol. Frontiers in Physiology 1664-042X Frontiers Media S.A. 10.3389/fphys.2020.594845 Physiology Original Research Genome Mining and Expression Analysis of Carboxylesterase and Glutathione S-Transferase Genes Involved in Insecticide Resistance in Eggplant Shoot and Fruit Borer, Leucinodes orbonalis (Lepidoptera: Crambidae) Kariyanna B. 12 Prabhuraj A. 1 Asokan R. 3 Agrawal A. 2 Gandhi Gracy R. 2 Jyoti P. 2 Venkatesan T. 2 Bheemanna M. 1 Kalmath B. 1 Diwan J. R. 4 Pampanna Y. 5 Mohan M. 2* 1Department of Agricultural Entomology, University of Agricultural Sciences, Raichur, India 2ICAR-National Bureau of Agricultural Insect Resources, Bengaluru, India 3ICAR-Indian Institute of Horticultural Research, Bengaluru, India 4Department of Genetics and Breeding, University of Agricultural Sciences, Raichur, India 5Department of Horticulture, University of Agricultural Sciences, Raichur, India Edited by: Kai Lu, Fujian Agriculture and Forestry University, China Reviewed by: Wei Dou, Southwest University, China; Yuji Yasukochi, National Agriculture and Food Research Organization (NARO), Japan *Correspondence: M. Mohan, Mohan.M@icar.gov.inThis article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology 19 11 2020 2020 11 59484514 8 2020 22 10 2020 Copyright © 2020 Kariyanna, Prabhuraj, Asokan, Agrawal, Gandhi Gracy, Jyoti, Venkatesan, Bheemanna, Kalmath, Diwan, Pampanna and Mohan.2020Kariyanna, Prabhuraj, Asokan, Agrawal, Gandhi Gracy, Jyoti, Venkatesan, Bheemanna, Kalmath, Diwan, Pampanna and MohanThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major cause of low productivity in eggplant and insecticides being the mainstay of management of L. orbonalis. However, field control failures are widespread due to the evolution of insecticide resistance. Taking advantage of the whole genome sequence information, the present study investigated the level of insecticide resistance and the expression pattern of individual carboxylesterase (CE) and glutathione S-transferases (GSTs) genes in various field collected populations of L. orbonalis. Dose-mortality bioassays revealed a very high level of resistance development against fenvalerate (48.2–160-fold), phosalone (94-534.6-fold), emamectin benzoate (7.2–55-fold), thiodicarb (9.64–22.7-fold), flubendiamide (187.4–303.0-fold), and chlorantraniliprole (1.6–8.6-fold) in field populations as compared to laboratory-reared susceptible iso-female colony (Lo-S). Over-production of detoxification enzymes viz., CE and GST were evident upon enzyme assays. Mining of the draft genome of L. orbonalis yielded large number of genes potentially belonging to the CE and GST gene families with known history of insecticide resistance in other insects. Subsequent RT-qPCR studies on relative contribution of individual genes revealed over-expression of numerous GSTs and few CEs in field populations, indicating their possible involvement of metabolic enzymes in insecticide resistance. The genomic information will facilitate the development of novel resistance management strategies against this pest. Leucinodes orbonalisinsecticide resistancegenomearboxylesterasegene expressionglutathione S-transferasecarboxylesteraseIndian Council of Agricultural Research10.13039/501100001503Department of Science and Technology, Ministry of Science and Technology, India10.13039/501100001409 ==== Body Highlights - High levels of multiple insecticide resistance in field collected Leucinodes orbonalis populations. - Enhanced midgut activities of carboxylesterase and glutathione S-transferase enzymes. - Qualitative changes in esterase banding pattern in resistant populations. - Mining and retrieval of 94 carboxylesterase and 33 glutathione S-transferase full length gene sequences from draft assembled genome and transcriptome of L. orbonalis. - Over-expression of large number of glutathione S-transferase and few carboxylesterase genes in resistant field collected populations. Introduction Eggplant or aubergine (Solanum melongena), also called as brinjal, is one of the most popular vegetable crops in south-east Asia especially India, China, and Bangladesh. The most important limiting factor in brinjal cultivation is the damage caused by shoot and fruit borer, Leucinodes orbonalis Guenee (Lepidoptera: Crambidae). It was first described from India and now it is distributed all over Asia, Africa, and in few parts of Europe (Mally et al., 2015). In India and Bangladesh, it causes severe yield losses up to 93% despite best management practices (Kodandaram et al., 2017; Prodhan et al., 2018). Surveys in India and Bangladesh indicated that farmers spray chemical insecticides up to 84 times during a 6–7 month cropping season. A very high level of pesticide load at a concentration 40–450 times higher than the maximum residue limit (MRL) in marketed brinjal fruits poses major health concerns (Srinivasan, 2008; Latif et al., 2010; Kariyanna et al., 2020b). It is also considered as a pest of quarantine significance to the number of countries outside its native range. L. orbonalis has high reproductive potential with overlapping generations. Studies reporting high level of insecticide resistance development in L. orbonalis might be attributed to the long-term indiscriminate use of various insecticides on eggplant (Kodandaram et al., 2017; Shirale et al., 2017). Resistant insects overcome the toxic effect of insecticide molecules by adopting one or more mechanisms ranging from cuticular thickening, nerve impenetration, altered production of metabolic enzymes, target site insensitivity, enhanced excretion through ABC transporters as well as by gut symbionts (Mohan et al., 2015). The enhanced detoxification of insecticides by metabolic enzymes viz., carboxylesterases (CE) and glutathione S-transferases (GSTs) are commonly reported in resistant populations of many insect species and mites (Ranson et al., 2002; Enayati et al., 2005; Oakeshott et al., 2005; Perry et al., 2011; Das and Dutta, 2014; Akıner and Ekşi, 2015; Benoit et al., 2015). The resistant individuals metabolize the insecticides faster due to higher catalytic rate or enhanced production of the enzyme as a consequence of increased transcription or gene duplication (Panini et al., 2016; Kariyanna et al., 2020a). The GSTs are phase II metabolic enzymes and are distributed in most of the animals (Hayes and Pulford, 1995). Among several subclasses of GST, the enzymes which are frequently associated with the insecticide resistance are Delta and Epsilon classes (Friedman, 2011; Lumjuan et al., 2011). Similarly, enhanced metabolism of organophosphates, carbamates, and pyrethroids are frequently associated with CEs through gene amplification, upregulation, mutations by coding sequence, or a combination of all these mechanisms (Zhang et al., 2012; Cui et al., 2015). Considering the economic and social impact of L. orbonalis, genetically modified eggplant expressing insecticidal cry1Ac gene from Bacillus thuringiensis has been developed (Shelton et al., 2018), but, a moratorium on its commercialization was imposed by Indian Government due to public concern. Though Bt eggplant is commercialized in Bangladesh way back in 2014, yet the area under cultivation is <2500 Ha (Shelton et al., 2018) due to various reasons. Baring Bt brinjal, the insecticides remain the sole method of controlling L. orbonalis in all the eggplant growing countries. With the availability of genome sequence, the present study investigated the level of insecticide and the expression pattern of CE and GST genes from field collected insecticide resistant L. orbonalis populations, to pinpoint the key metabolic genes involved in insecticide degradation. Materials and Methods Insect Collection and Maintenance The field populations of L. orbonalis larvae were collected during 2017–2018 from intensive eggplant growing regions of India viz., Raichur (16.2120° N, 77.3439° E), Dharmapuri (12.0933° N, 78.2020° E), Bhubaneshwar (20.2961° N, 85.8245° E), Pune (18.5204° N, 73.8567° E), and Varanasi (25.3176° N, 82.9739° E). All the field populations were reared under laboratory conditions at 27 ± 2°C, 60–70% relative humidity (RH), and a photoperiod of 14:10 h (L:D) on a natural diet and the F1 individuals were used for bioassay and biochemical studies. The insecticide susceptible iso-female line (National Accession number: NBAIR-IS-CRA-01A), designated as Lo-S (65th generation) was originally derived from L. orbonalis collected near Bengaluru (12.9716° N, 77.5946° E) and maintained at insect genomic resources laboratory at ICAR-NBAIR. Insecticide Resistance Bioassays Insecticides fenvalarate (20% EC), emamectin benzoate (5% EC), phosalone (35%), thiodicarb (75%), flubendiamide (20%), and chlorantraniliprole (18.5%) were selected for conducting dose-mortality bioassays based on their usage history on brinjal by farmers. Filter paper residue assay (Cheng et al., 2010) with essential modifications was used for insecticide resistance bioassays on early second instar larvae of L. orbonalis. Five to seven appropriate concentrations of each of the selected insecticides were prepared based on the dose bracketing technique. The filter paper discs (4.5 cm diameter) were dipped in the appropriate dilutions and dried vertically under shade for 1 h. Then, the discs were placed individually in plastic containers (5 cm diameter) and 10 larvae were released in each container. Moistened filter paper discs without insecticide were used in control. After 24 h, the larvae were transferred to untreated natural diet. All the assays were replicated and repeated at least thrice on alternate days. The mortality of the larvae was assessed after 48 h. The pooled larval mortality data were subjected to probit analysis using the software POLO (LeOra, 1987) and the lethal concentration to kill 50% of the test larvae (LC50) was calculated for each population. Resistance ratios (RRs) were calculated with the following formula: LC50 of field population/LC50 of insecticide susceptible Lo-S population. Preparation of Midgut Homogenate The starved late second instar larvae were used for the preparation of midgut homogenate. Midguts were dissected and homogenized with homogenization buffer (0.1 M sodium phosphate buffer pH 7.8 containing 1 mM each of DTT, EDTA, PTU, and PMSF). The content was centrifuged at 12,000 rpm for 20 min and the clear supernatant was used as a enzyme source for estimating the titers of carboxylesterase and GST. The total protein content of the preparations was assessed by Coomassie brilliant blue G-250 dye-binding method using bovine serum albumin (BSA) as the standard (Bradford, 1976). Assays on Carboxylesterase and Glutathione S-Transferase The activity of carboxylesterase was determined using the method described by van Asperen (1962) with essential modifications and α-naphthyl acetate as a substrate. The assay mixture in a 96 well microplate (iMark, Biorad microplate reader) consisted of an appropriate amount of midgut homogenate, 0.1 M sodium phosphate buffer pH 7.8 and 800 μl of 3 mM α-naphthyl acetate containing 0.3 mM eserine. The mixture was incubated at 30°C for 30 min. Finally, 200 μl of 0.1% tetrazotized o-dianisidine (Fast blue B) in 3.5% sodium dodecyl sulfate was added and incubated for 20 min at room temperature in dark. The α-naphthol formation was measured at 590 nm and the enzyme activity was computed from α-naphthol standard curve. Glutathione S-transferase activity was determined using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate (Kao et al., 1989). The assay mixture composed of 50 mM each of CDNB and L-glutathione reduced and 10 μl gut homogenate. The change in absorbance was measured at 340 nm for 5 min and the enzyme activity in terms of nmoles of CDNB conjugated per minute per mg protein using the molar extinction coefficient of 5.3 mM–1 (optimized for the path length: 0.55 cm). Esterase Isozyme Studies The native polyacrylamide gel electrophoresis (PAGE) with 10% resolving gel was performed as per Davis (1964) to visualize the esterase isozymes from the 2nd instar midgut larval homogenates of the L. orbonalis populations tested. Midgut homogenates with 20 μg protein were loaded onto the native PAGE and run at a constant voltage of 70 for 1.5 h. Gel was briefly stained with freshly prepared 0.05% (w/v) α-naphthyl acetate and 0.1% (w/v) fast blue B in 50 mM phosphate buffer pH 7.8. Genome Mining and Phylogenetic Analysis The draft assembled genome (NCBI Bioproject ID: PRJNA377400) of L. orbonalis was used as query for extracting the gene sequences of glutathione S-transferases (GSTs) and carboxylesterases (CEs). The hmm model for carboxylesterase and GST (CE-PF00135, PF02230, and GST-C PF00043, GST-N PF02798, GST-PF13417, PF14497, PF17171, PF17172) were obtained from PFAM database1. A total of 94 and 33 putative CE and GST sequences were mined using hmm search with HMMER3 software package2. Among them, the genes with known history of involvement in insecticide resistance in other insects were selected and further confirmed with NCBI BLASTX. The GST and CE protein sequences of Drosophila melanogaster was downloaded from its genome database (Marygold et al., 2013), GenBank3 and Uniprot4. We combined these putative GST protein sequences with D. melanogaster’s annotated protein sequences for each of the six subclasses of GST (Epsilon, Sigma, Omega, Delta, Zeta, and Theta). The sequences were multiple aligned using CLUSTALW and a maximum likelihood tree with 1000 bootstrap value was constructed using MEGA75 and TreeDyn online server6 was used to visualize the tree. Similarly, 94 CE protein sequences under seven subclasses (α-esterases, gliotactin, glutactin, neurolignin, juvenile hormone esterases, acetylcholinesterases, and cricklet) were combined, multiple aligned using MAFFT online server using conserved domain feature and phylogenetic tree of CE genes from L. orbonalis using D. melanogaster as a reference were constructed with the help of CLC workbench (QIAGEN Bioinformatics7) using maximum likelihood approach. Quantitative Real-Time PCR The late 2nd instar larvae were used for RNA extraction (ISOLATE II RNA mini kit) by following the manufacturer’s guidelines (Bioline). Purity and concentration of total RNA were measured in a spectrophotometer (NanoDrop Lite, Thermo Fisher Scientific) and denatured agarose gel (Masek et al., 2005). RNA samples with an A260/A280 ratio ranging from 1.8 to 2.0 and A260/A230 ratio >2.0 were used for cDNA preparation. First strand of complementary DNA was synthesized from 4 μg of total RNA using Revert AID first strand cDNA synthesis kit (Thermo Fisher ScientificTM, Lithuania) following the suppliers guidelines and stored at −80°C till further use. qPCR primers for 25 GST and 16 CE gene sequences were designed using Primer 3.0 software. The detailed information of the primers used in the current study is listed in Tables 1, 2. To remove the primer dimer and confirm the efficiency, OligoEvaluatorTM sequence analysis tool (accessed on July 20188) was used. Primer specificity analysis was observed by single peak in the melting curve and single band from the agarose gel of the all candidate genes of CE and GST ranged from 90.36 to 104.58% (Tables 1, 2). Quantitative real-time PCR (RT-qPCR) amplifications were performed in 20 μL reaction consisted of 10 μL 2×SYBR® Premix EX TaqTM II (Tli RNaseH Plus, TAKARA®, Japan), 1 μl cDNA and gene specific primer pair. The parameters used for RT-qPCR are: One cycle of 95°C for 3 min; 35 cycles of 95°C for 30 s, 53°C for 45 s, and 72°C for 1 min; a final cycle of 72°C for 10 min using Roche 480II machine. All the samples including control (no template) and internal control were performed in triplicates. The PCR products were electrophoresed on 2.0% agarose gel in a 1.0×TAE buffer. Relative expression levels for the CE and GST genes were calculated by 2–ΔΔCT method. The 28SR3 (28S ribosomal protein S3 mitochondrial) (Kariyanna et al., 2019) gene was used as an internal control to normalize the expression of target genes. TABLE 1 Primer details for carboxylesterase (CE) genes. Sl. No Sequence Primer sequence 5′→3′ Length GC (%) Tm (°C) Sequence length Product size E (%) 1. Contig8459-0.14 F GTGTCAAACGTGTTCAGAATC 21 43 54 2796 100 91.43 R TACCCAGGAAAGTAGTGTAGT 21 43 54 2. Contig12636-0.8 F CTTGCACTCTGTTCACTAACT 21 43 54 2712 95 88.17 R TCTGTCGTCGTTGTTGTATTC 21 43 54 3. Contig11486-0.8 F TCACATGAGACTGGGTAGAA 20 48 55 3936 95 103.22 R CTCTGTAGTGTCTGTCGTAGA 21 45 54 4. Contig4177-0.23 F AACCTGTTCTCACAAGCTATC 21 48 55 1146 95 98.05 R AGTCTAGTACCTCTCAGGATTG 22 45 55 5. Contig5480-0.2 F TCCCTCACACTAACGAAAGA 20 45 55 6498 95 98.12 R TCCAGGTATTCAGGAGTGATAG 22 45 55 6. Contig11205-0.1 F AACGTGGAACCTTACTTCAC 20 45 54 597 100 97.60 R CACCTTCGTCTTCGTTGTAA 20 45 54 7. Contig4653-0.41 F TGCAAGTGACTGTGAACGAAG 21 48 58 2417 179 101.88 R AGCGTTACCAGGGATGTCTTT 21 48 58 8. Contig132-0.69 F ATCCCTCTGGAACCTCAAAAA 21 43 54 3375 179 88.81 R GGGAACATTCTGAAAGGGAAG 21 48 54 9. Contig3761-0.1 F TGTGGGCTAACTTCGCTAAAA 21 43 55 681 233 90.70 R TTCGATTTTTCTCACCACACC 21 43 56 10. Contig3761-0.2 F CCTGGCTCTGAAACTGACTTG 21 52 55 1251 150 99.31 R TTGATAGGAGGAGCAGCGTAA 21 43 55 11. Contig9545-0.25 F CAAAGAACTTCAACTTCACT 20 35 49 555 193 101.67 R TTTTTCGTCAGGTTTGTGAGG 21 43 55 12. Contig9545-0.26 F TCCTTCATTCCTGCAAAGAGA 21 43 55 804 167 100.15 R AGTAGTCACCCACCACGTCAG 21 57 60 13. Contig11588-0.37 F CTGGTAACGCTGGTATCAAA 20 45 54 1269 111 97.11 R CAGCTGATTGACCGAAGATAG 21 48 55 14. Contig5480-0.52 F ACCAATCAGACGACTCACACC 21 52 59 1863 157 90.48 R GCGATGTTCACCAGTTTTGAT 21 43 55 15. Contig5480-0.3 F GGTCCTGTGAAAGGTTACAA 20 45 55 663 94 92.15 R TGTATTTGTCAGCACCAGTAG 21 43 55 16. Contig11205-0.4 F GAAGGTGAAATCGTGAACAAC 21 43 54 225 165 97.11 R TTGGTATGATGATGAACCGTGT 22 41 56 TABLE 2 Primer details for glutathione S-transferase (GST) genes. Sl. No Sequence Primer sequence 5′→3′ Length GC% Tm (°C) Sequence length Product size E (%) 1. Contig7206-0.56 F GCCTCAAGACTGCATGAAAAG 21 48 56 600 223 98.59 R GTCAGCCAGAGTGATTTCGTC 21 52 58 2. Contig2323-0.22 F TTCACCTCCTTGCAGATCAGT 21 48 58 375 160 87.80 R GAAGTCACCGTCTTTCAGCAG 21 52 58 3. Contig8855-0.81 F ACGACATCCTGAACACTCTGC 21 52 59 657 165 98.93 R TTCTCCATTGTCTCACCCAAG 21 48 57 4. Contig2841-0.35 F CGCTATGAGATTCTGCCCTTAC 22 50 57 762 120 104.58 R GAGAAGTCGAACAGCCATTCA 21 48 57 5. Contig2596-0 F TGCTACCTGGTGGACAAATTC 21 48 57 639 236 92.92 R CCCATTTAGTGTCGTTCAGGA 21 48 56 6. Contig14202-0.24 F TATCCTGGCTCTGAACGCTAA 21 48 58 717 171 105.55 R TCGTCCAGGTATTCCACAGTC 21 52 59 7. Contig1970-0.34 F GACGCTCTGAGACTGTTCGAC 21 57 60 564 190 96.96 R CTTCGTAACCAGGAGCAGTTG 21 52 58 8. Contig1510-0.48 F ATCGGTTGGCTGAACACTATG 21 48 57 726 227 95.00 R AAAGTAGCCAGCATTTGAGCA 21 43 57 9. Contig2478-0.25 F TCTGGGACTCACACGCTATCT 21 52 56 654 208 100.59 R ATTTGAGCCAGGTTTTCAGGT 21 43 60 10. Contig3755-0.39 F CGCTATCATGCAATACGTGTG 21 48 56 654 183 102.61 R AGACCCAGAGGAGTTCTTTCG 21 52 59 11. Contig3537-0.3 F ACAAATTCAACCTGCTGCCTA 21 43 56 423 227 96.58 R CAGTTTACCAGCGAAGTGACC 21 52 58 12. Contig10769-0.3 F TCACTGGCTATCGCTAGATACA 22 45 57 621 116 98.89 R CGTTTGACCAGAAGTCGAAGAT 22 45 57 13. Contig1010-1.18 F GACGCTCTGAGACTGTTCGAC 21 57 60 648 190 98.00 R CTTCGTAACCAGGAGCAGTTG 21 52 58 14. Contig9166-0.22 F ACAAATCAGTGGCTGCTAAA 20 40 54 345 97 95.31 R GGCAGGTAGTACAGTTTGATAG 22 45 54 15. Contig6423-0.33 F TGCTACCTGGTGGACAAATTC 21 48 57 645 236 89.22 R CCCATTTAGTGTCGTTCAGGA 21 48 56 16. Contig199-0.91 F TGTGAAAGCTCTGGGTGAATC 21 48 57 567 233 99.14 R GAAGTCCACGTTCATGTCGAT 21 48 57 17. Contig8048-0.1 F TCAATCGAAGAAGACCTGGAA 21 43 55 486 150 98.83 R CAGCAGTTGAGGGTACACGTT 21 52 60 18. Contig11878-0.11 F TGCTACCTGGTGGACAAATTC 21 48 57 540 236 98.55 R CCCATTTAGTGTCGTTCAGGA 21 48 56 19. Contig7006-0 F CGCTAGATACCTGGCTAACAAA 22 45 56 606 98 92.55 R CCCAGAAGTCGTAGATGTTCAG 22 50 57 20. Contig3023-0.16 F GCTGCTTACGAAGGTAGAATG 21 48 55 618 173 99.14 R GCAGGGAAGTGTTCTTTAGGG 21 52 58 21. Contig2596-0 F TGCTACCTGGTGGACAAATTC 21 48 57 639 235 101.31 R CCCATTTAGTGTCGTTCAGGA 21 48 56 22. Contig2478-0.15 F CATCACACGCTATCACTACTT 21 43 54 456 102 97.25 R GCAGTCTTTGGTCGATTCT 19 47 54 23. Contig3291-0.7 F TATCGTGTCAGGTATCCAAC 20 45 53 402 179 92.16 R GTCAGCCAGAGTCAGTTGGTC 21 57 60 24. Contig4841-1.1 F TGGACGACACTAGACCTAAA 20 45 54 327 81 93.78 R GTTGGATACCTGACACGATAG 21 48 54 25. Contig2323-0.23 F CTCACACGCTATCACTACTTAC 22 45 54 645 101 95.56 R GCAGTCTTTGGTCGATTCT 19 47 54 Results Insecticide Resistance Monitoring The LC50 values against fenvalerate, phosalone, thiodicarb, emamectin benzoate, flubendiamide and chlorantraniliprole indicated that there is a large shift in susceptibility of field-populations of L. orbonalis as compared to the laboratory reared susceptible iso-female colony (Lo-S). The field collected populations exhibited 3.6–160-fold resistance against fenvalerate, 7.3–534.6-fold resistance against phosalone, 7.0–55.0-fold resistance against emamectin benzoate, 2.0–22.7-fold resistance against thiodicarb, 29.5–303.0-fold resistance against flubendiamide, and 1.6–8.6-fold resistance against chlorantraniliprole. However, the susceptibility level of all the field populations and the Lo-S did not vary significantly against chlorantraniliprole based on the overlapping fiducial limit values. High level of resistance development against phosalone and flubendiamide was observed in L. orbonalis collected from Varanasi whereas other field populations, the RRs were non-significant as compared to Lo-S. L. orbonalis populations from Bhubaneswar showed a significantly very high levels of resistance against fenvalerate, phosalone and flubendiamide as compared to Lo-S. The population of L. orbonalis collected from high hills near Pune recorded less RR and the susceptibility status was almost on par with Lo-S population (Table 3). TABLE 3 Resistance pattern of field populations of L. orbonalis against various insecticides. Insecticide Population LC50 (ppm) Slope ± SE Fiducial limits χ2 heterogeneity (DF) Resistance ratio Lower Upper Fenvalerate Bhubaneswar 259.2 2.6 ± 0.52 125.61 623.8 7.01 (3) 160 Raichur 93.2 3.1 ± 0.2 2 56.1 125.8 3.92 (3) 57.5 Dharmapuri 78.4 2.5 ± 0.23 53.2 145 2.81 (3) 48.3 Pune 5.9 2.1 ± 0.11 2.51 16.32 2.91 (3) 3.6 Varanasi 153.2 1.6 ± 0.23 75.2 432.4 3.37 (4) 94.6 Bengaluru (Lo-S) 1.62 2.2 ± 0.42 0.85 3.57 2.62 (4) – Phosalone Bhubaneswar 641.5 2.61 ± 0.22 283.51 2564.6 6.90 (3) 534.6 Raichur 190.2 1.9 ± 0.2 1 110.1 280.5 3.90(3) 158.5 Dharmapuri 113 3.3 ± 0.56 56.4 203.2 0.89 (3) 94.2 Pune 8.81 1.7 ± 0.08 4.34 24.81 3.48 (3) 7.3 Varanasi 435.8 1.9 ± 0.21 210.5 1202.8 1.37 (3) 363.2 Bengaluru (Lo-S) 1.2 1.2 ± 0.21 0.521 2.42 1.91 (3) – Emamectin benzoate Bhubaneswar 0.072 1.4 ± 0.09 0.291 0.204 3.4 (3) 7.2 Raichur 0.1 1.1 ± 0.10 0.07 0.2 1.19 (3) 10 Dharmapuri 0.07 1.5 ± 0.31 0.03 0.1 2.66 (3) 7 Pune 0.012 1.8 ± 0.09 0.005 0.045 1.07 (3) 1.2 Varanasi 0.55 1.2 ± 0.28 0.231 1.821 6.89 (4) 55 Bengaluru (Lo-S) 0.01 1.4 ± 0.07 0.004 0.051 3.17 (3) – Thiodicarb Bhubaneswar 211.6 1.71 ± 0.83 116.23 621.78 1.91 (3) 18.2 Raichur 263.2 2.1 ± 0.21 121.3 467.9 2.3 (3) 22.7 Dharmapuri 161.9 2.05 ± 0.30 83.82 341.7 7.2 (3) 13.9 Pune 23.6 2.0 ± 0.12 32.01 184.8 1.3 (3) 2.03 Varanasi 112 2.2 ± 0.18 56.82 456.8 1.7 (4) 9.6 Bengaluru (Lo-S) 11.61 1.73 ± 0.32 5.462 23.56 4.7 (3) – Flubendiamide Bhubaneswar 69.7 1.20 ± 0.08 36.34 173.35 3.7 (3) 303.0 Raichur 43.1 1.72 ± 0.10 22.39 93.72 2.9 (3) 187.4 Dharmapuri 61.5 1.3 ± 0.13 35.65 129.39 7.2 (3) 267.4 Pune 6.79 1.10 ± 01.0 3.02 17.93 14.6 (4) 29.5 Varanasi 61.3 1.79 ± 0.08 20.32 173.8 17.5 (4) 266.5 Bengaluru (Lo-S) 0.23 – – – – – Chlorantraniliprole Bhubaneswar 2.29 0.66 ± 0.16 0.28 6.4 4.08 (3) 6.9 Raichur 0.80 0.59 ± 0.16 0.02 2.9 2.80 (3) 2.4 Dharmapuri 2.84 0.63 ± 0.15 0.41 7.94 4.92 (3) 8.6 Pune 1.76 0.6 ± 0.16 0.12 5.59 3.08 (3) 5.3 Varanasi 0.60 0.47 ± 0.15 0.002 3.2 5.68 (3) 1.6 Bengaluru (Lo-S) 0.33 0.81 ± 0.26 0.003 1.24 1.55 (3) – Activities of Detoxification Enzymes Quantitative differences in the titer of GST and CE were determined using α-naphthyl acetate and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates, respectively. Significant level of elevated GST activities (4.1–8.9-fold) was observed in field collected L. orbonalis as compared to susceptible Lo-S population. However, the overproduction of CE has not pronounced much (1.5–3.2-fold), but there was a significant level of overproduction in two field populations based on Mann–Whiteney U-test. The results indicated that GST activity was altered more profoundly than the CE (Table 4). TABLE 4 Metabolic enzymes activities in the midgut of L. orbonalis populations. L. orbonalis population Glutathione -S- transferase Carboxylesterase Specific activity (nMoles/min/mg protein) Fold variation as compared to Lo-S Specific activity (μMoles/mg/min) Fold variation as compared to Lo-S Bhubaneswar 70.5 ± 3.6* 5.6 6.02 ± 0.21* 2.8 Raichur 73.0 ± 3.6* 5.8 6.83 ± 0.21* 3.2 Dharmapuri 112.3 ± 11.2* 8.9 5.78 ± 0.33* 2.7 Varanasi 51.8 ± 2.8* 4.1 3.52 ± 3.4 1.7 Pune 103.4 ± 9.5* 8.2 3.28 ± 0.33 1.5 Bengaluru (Lo-S) 12.62 ± 1.3 – 2.12 ± 0.13 – *Indicates significant differences by Mann–Whiteney U-test (p < 0.05) as compared to Lo-S population.The qualitative differences in carboxylesterase isozymes were visualized under native PAGE after incubation of the gels in the α-naphthyl acetate as a substrate. Among the four major esterase activity bands (E1 to E4), the high molecular weight E1 and E2 bands were absent in Lo-S and Pune populations and faint in case of Varanasi population. However, the E1 band was very prominent and intense in other field populations indicating its over-production nature (Figure 1). FIGURE 1 The Native PAGE of the esterase isozyme pattern of L. orbonalis (1. Lo-S, 2. Pune, 3. Varanasi, 4. Raichur, 5. Bhubaneswar, 6. Dharmapuri). Differential Expression of CE Genes Ninety-four CE genes were identified from the genome and transcriptome sequences of L. orbonalis. They were classified under seven subfamilies viz., α-esterases, gliotactin, glutactin, neurolignin, juvenile hormone esterases, acetylcholinesterases, and cricklet like orthologs (Figure 2). The length of the gene sequences ranged from 225 to 6498 bp. Expression profiling of 16 genes (homologous to resistant genes in other insects with ≥90% query coverage) was examined from the mRNA samples derived from late second instar larvae of five field-collected L. orbonalis populations by RT-qPCR. The CE genes contig11486-0.8 and contig4653-0.41 were over-expressed more than 10-fold as compared to susceptible Lo-S population (Figures 3, 4). FIGURE 2 Phylogenetic relationships of 94 CE proteins of L. orbonalis with selected D. melanogaster protein representative of seven characterized carboxylesterase classes. Putative CE proteins in L. orbonalis were identified using hmmsearch against PFAM CE families (CE PF00135). The Maximum likelihood tree was constructed using CLC workbench. FIGURE 3 Transcription profiling of carboxylesterase genes in field collected populations of L. orbonalis depicted as fold change over the susceptible Lo-S colony. (A) Bhubaneswar, (B) Dharmapuri, (C) Pune, (D) Raichur, (E) Varanasi. FIGURE 4 Expression profiles (fold changes over Lo-S population) of carboxylesterase genes across field collected L. orbonalis populations (P, Pune; D, Dharmapuri; R, Raichur; B, Bhubaneswar; V, Varanasi). The fold changes are indicated in different shades indicate significant difference as per Mann–Whiteney U-test p-value < 0.05. Differential Expression of Glutathione S-Transferase Genes Mining of genome and transcriptome data of L. orbonalis yielded 33 unigenes codings for GSTs. Lengths of the identified GST unigene sequences ranged from 370 to 5,048 bp. The phylogenetic analysis revealed that the predicted GST genes are represented under all six classes of GST viz., Epsilon (10 genes), Sigma (6 genes), Omega (5 genes), Delta (8 genes), Zeta (3 genes), and Theta (1 gene) (Figure 5). RT-qPCR analysis of 25 GST genes (homologous to other insects resistant genes) revealed that many of them were over-expressed in larvae collected from the field. The GST genes named contig2323-0.22, contig14202-0.24, contig3755-0.39, contig3537-0.3, contig199-0.91, contig3023-0.16, contig3291-0.7, contig4841-1.1, and contig2323-0.23 were very highly expressed (>50-fold) in one or more resistant field-collected L. orbonalis populations (Figures 6, 7) over the susceptible Lo-S population. FIGURE 5 Phylogenetic tree of 34 predicted L. orbonalis glutathione S-transferase proteins with D. melanogaster genes representative of the six characterized GST classes. Putative GST proteins in L. orbonalis were identified using hmmsearch against PFAM GST families (GST-C PF00043, GST-N PF02798), the Maximum Likelihood tree with 1000 bootstraps was constructed using MEGA7 and TreeDyn online server was used to visualize the tree. Number on each node is bootstrap values in percent. Scale = 1 amino acid substitution per site. FIGURE 6 Transcription profiling of glutathione -S- transferase genes in field collected populations of L. orbonalis depicted as fold change over the susceptible Lo-S colony. (A) Bhubaneswar, (B) Dharmapuri, (C) Pune, (D) Raichur, (E) Varanasi. FIGURE 7 Expression profiles (fold changes over Lo-S population) of glutathione -S- transferase genes across field collected L. orbonalis populations (P, Pune; D, Dharmapuri; R, Raichur; B, Bhubaneswar; V, Varanasi). The fold changes are indicated in different shades indicate significant difference as per Mann–Whiteney U-test p-value < 0.05. Discussion Damage caused by L. orbonalis is the major limiting factor in realizing maximum productivity of eggplant in many Asian countries including India, China, and Bangladesh. The high reproductive potential and shorter life cycle of the pest coupled with the evolution of multiple insecticide resistance pose a major challenge in profitable cultivation of eggplant (Prodhan et al., 2018). The insecticide resistance levels detected in the field collected populations ranged from low (>10-fold) to moderate (10–100-fold) and high (>100-fold) against fenvalerate (48.2–160-fold), phosalone (94–534.58-fold), emamectin benzoate (7–55-fold), thiodicarb (9.64–22.67-fold), and flubendiamide (29.51–363.91-fold). The resistance levels reflect the occurrence of differential selection pressure associated with long term history of insecticide use against L. orbonalis. However, the response to chlorantraniliprole was similar among the resistant field collected populations with RRs below 10-fold. The efficacy of chlorantraniliprole has been reported earlier (Kodandaram et al., 2013; Munje et al., 2015). The chlorantraniliprole and flubendiamide are anthranilic and phthalic diamides, respectively, are the newer insecticides with little or no cross-resistance among them and also with other classes of insecticides. They are the activators of the ryanodine receptor. Insect employs various mechanisms to nullify the toxic effect of xenobiotic compounds. Insect metabolic enzymes play a major role in detoxification of plant allelochemicals and pesticidal compounds in addition to other physiological roles. Insect pests have evolved large reservoirs of various detoxification enzymes. Glutathione S-transferases mediated detoxification can be direct or by the metabolism of secondary products generated from other detoxification enzymes. The insecticides are metabolized rapidly into readily excretable water-soluble metabolites by reductive dehydrochlorination or by conjugation with reduced glutathione (Pavlidi et al., 2018). The carboxylesterase is a phase-1 detoxification enzyme that mainly hydrolyses organophosphates, carbamates, and synthetic pyrethroids. The quantitative mechanisms underlying metabolic resistance is characterized by over-production due to gene amplification or transcriptional upregulation (Mohan et al., 2015; Feyereisen, 2015; Pavlidi et al., 2018). A qualitative mechanism occurs as a result of changes in the enzymatic characteristics. The present investigation on the enhanced metabolism of insecticides in resistant populations of L. orbonalis resistance is very prominent as inferred by elevated GST (1.2–2.6-fold) and carboxylesterases (6.3–13.1-fold) titers. The involvement of CEs and GSTs in insecticide resistance is commonly reported in many other insect species (Mohan et al., 2015; Pavlidi et al., 2018). With the advent of genome and transcriptome information, multiple GSTs and carboxylesterases encoding genes have been characterized from insects and mites such as Plutella xylostella, Culex spp., Tribolium castaneum, Bombyx mori, Leptinotarsa decemlineata, Tetranychus cinnabarinus, Musca domestica and in many other insect species (Yang and Liu, 2011; Wang et al., 2014; Cui et al., 2015; Meisel and Scott, 2018; Pavlidi et al., 2018). L. orbonalis genome has large expansion of CE and GST genes which is comparable to many other insects. However, the GSTs have prominent role in insecticide resistance as compared to CEs. mRNA level of at least nine GSTs were over-expressed >50-fold where as none of the CEs showed very high levels of expression in the field collected resistant populations. The over-expression of few CE genes in field populations, especially from Varanasi, Raichur, and Bhubaneswar, also aligned with additional high molecular weight esterase band (E1) probably contributed to overall resistance. A similar high molecular weight esterase band associated with B-esterase type was reported from diamondback moth (Mohan and Gujar, 2003). In addition to qualitative changes in esterase banding pattern, there is a significant increase in the production of the CEs in at least three field populations. The over-production and appearance of new esterase isozymes might be the consequence of increased transcription or gene duplication events (Malathi et al., 2015; Panini et al., 2016). Most of the GST gene expansions are from epsilon and delta subclasses together accounting for 18 genes (54.5%) out of 33 genes identified from the draft genome of L. orbonalis. These two GST subclasses are insect-specific, widely expanded in many other insect species whereas other classes have wider taxonomic distribution (Friedman, 2011). There were at least nine GSTs over-expressed >50-fold in one or more of the field-collected resistant populations of L. orbonalis. In Anopheles gambiae, Apis mellifera, B. mori, D. melanogaster, Nasonia vitripennis, and T. castaneum respectively 28, 8, 23, 37, 19, 35 GSTs and 51, 24, 76, 35, 41, 49 CEs were identified (Yu et al., 2008, 2009; Oakeshott et al., 2010). Similarly, the gene expansion of cytochromoe p450 monooxygenases (CYP) and their role in insecticide resistance in L. orbonalis were recently documented (Kariyanna, 2019; Kariyanna et al., 2020a). Together with the results of present and earlier studies it is very much evident that multiple metabolic enzymes contribute toward overall insecticide resistance in L. orbonalis. However, in many insect species including L. orbonalis, it is difficult to distinguish whether the expansion of these metabolic genes and their involvement in insecticide detoxification are driven from ancestry or adaptation to stress or both. Conclusion In conclusion, the present study provides the first genomic level information on GST and CE gene families of L. orbonalis and their role in insecticide resistance. The information further supports insect resistance management through use of reduced risk pesticides including biologicals. Data Availability Statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. Author Contributions BKr: conducted the experiment, analysis, manuscript writing, materials and methods, and hypothesis. AP: analysis, material methods, and corrections. RA: analysis and technical suggestion, manuscript correction, and resource supply. AA and PJ: bioinformatics and analysis. RG and BKl: resources supply and, technical guidance and suggestions. TV: resources supply, technical guidance and suggestions, and reviews and literatures. MB and JD: analysis, technical guidance and suggestions, and reviews and literatures. MM: designing experiment, resources supply, manuscript corrections, analysis, material and methods, technical guiding, and suggestions. YP: technical guidance and suggestions, and reviews and literatures. All authors contributed to the article and approved the submitted version. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors profusely thank Indian Council of Agricultural Research for generous funding under ICAR-CRP Genomics project. The first author acknowledges the award of DST-Inspire fellowship (2016/IF160900) and UAS Raichur for necessary support. Authors are thankful to Director, ICAR-NBFGR for timely release of fund. The facilities extended by Director, ICAR-NBAIR, and Director, ICAR-IIHR, Bengaluru are gratefully acknowledged. 1 http://pfam.xfam.org 2 http://hmmer.janelia.org 3 http://www.ncbi.nlm.nih.gov/ 4 http://www.uniprot.org/ 5 http://www.megasoftware.net 6 www.treedyn.org 7 https://www.qiagenbioinformatics.comproductsclc-main-workbench 8 http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/calculator.html Supplementary Material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys.2020.594845/full#supplementary-material Supplementary Table 1 Highlights of the research. Click here for additional data file. Supplementary Table 2 Sequence information GST and CE from L. orbonalis. Click here for additional data file. Supplementary Table 3 Resistance genes of GST and CE from L. orbonalis. Click here for additional data file. Supplementary Table 4 Detailed sequence information of CE from L. orbonalis. Click here for additional data file. Supplementary Table 5 Detailed sequence information of GST from L. orbonalis. Click here for additional data file. Supplementary Table 6 NCBI ID and description for the Reference protein taken in GST and CE phylogeny. Click here for additional data file. Supplementary Table 7 Comparative characterization L. orbonalis CE genes with other group based on the nr hit. Click here for additional data file. Supplementary Table 8 Descriptive information of the L. orbonalis CE sequences. Click here for additional data file. Supplementary Presentation 1 Graphical representation of the manuscript. Click here for additional data file. ==== Refs References Akıner M. M. Ekşi E. (2015 ). Evaluation of insecticide resistance and biochemical mechanisms of Culex pipiens L. in four localities of east and middle mediterranean basin in Turkey. Int. J. Mosq. Res. 2 39 –44 . Benoit J. Adelman B. Z. N. Reinhardt K. Dolan A. Poelchau M. Jennings E. C. (2015 ). Unique features of the bed bug, a global human ectoparasite, identified through genome sequencing. Nat. Commun. 7 :10165 . 10.1038/ncomms10165 26836814 Bradford M. M. (1976 ). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 248 –254 . 10.1016/0003-2697(76)90527-3 942051 Cheng X. Chang C. Daia S.-M. (2010 ). Responses of striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae), from Taiwan to a range of insecticides. Pest Manag. Sci. 66 762 –766 . 10.1002/ps.1939 20232286 Cui F. Li M. X. Chang H. J. Mao Y. Zhang H. Y. Lu L. X. (2015 ). Carboxylesterase-mediated insecticide resistance: quantitative increase induces broader metabolic resistance than qualitative change. Pestic. Biochem. Physiol. 121 88 –96 . 10.1016/j.pestbp.2014.12.016 26047115 Das M. Dutta P. (2014 ). Status of insecticide resistance and detoxifying enzyme activity of Aedes albopictus population in Sonitpur district of Assam. India. Int. J. Mosq. Res. 1 35 –41 . Davis B. J. (1964 ). Disc electrophoresis II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121 404 –427 . 10.1111/j.1749-6632.1964.tb14213.x 14240539 Enayati A. A. Ranson H. Hemingway J. (2005 ). Insect glutathione transferases and insecticide resistance. Insect Molec. Biol. 14 3 –8 . 10.1111/j.1365-2583.2004.00529.x 15663770 Feyereisen R. (2015 ). Insect P450 inhibitors and insecticides: challenges and opportunities. Pest Manag. Sci. 71 793 –800 . 10.1002/ps.3895 25404103 Friedman R. (2011 ). Genomic organization of the glutathione S-transferase family in insects. Molec. Phylogenet. Evol. 61 924 –932 . 10.1016/j.ympev.2011.08.027 21930223 Hayes J. D. Pulford D. J. (1995 ). The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Molec. Biol. 30 445 –600 . 10.3109/10409239509083491 8770536 Kao C. H. Hung C. F. Sun C. N. (1989 ). Parathion and methyl parathion resistance in diamondback moth (Lepidoptera: Plutellidae) larvae. J. Econ. Entomol. 82 1299 –1304 . 10.1093/jee/82.5.1299 Kariyanna B. (2019 ). Identification and Functional Analysis of Genes Involved in Insecticide Resistance in Leucinodes orbonalis Guenee (Lepidoptera: Crambidae). PhD thesis, University of Agricultural Sciences, Raichur , Karnataka , 1 –199 . Kariyanna B. Prabhuraj A. Asokan R. Babu P. Jalali S. K. Venkatesan T. (2019 ). Identification of suitable reference genes for normalization of RT-qPCR data in eggplant fruit and shoot borer (Leucinodes orbonalis Guenée). Biologia 75 289 –297 . 10.2478/s11756-019-00346-4 Kariyanna B. Prabhuraj A. Asokan R. Ramkumar G. Venkatesan T. Gracy R. G. (2020a ). Genomewide analysis and expression patterns of cytochrome P450 genes involved in insecticide resistance in Leucinodes orbonalis (Lepidoptera: Crambidae). Biotechnol. Appl. Biochem. 1 –12 . 10.1002/bab.1997 Kariyanna B. Prabhuraj A. Bheemanna M. Kalmat B. Pamapanna Y. Mohan M. (2020b ). Insecticide usage pattern and evolution of resistance in brinjal shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) in India. Plant Arch. 20 (Suppl. 2 ), 1255 –1261 . Kodandaram M. H. Rai A. B. Sharma S. K. Singh B. (2017 ). Shift in the level of susceptibility and relative resistance of brinjal shoot and fruit borer Leucinodes orbonalis (Guen) to diamide insecticides. Phytoparasitica 45 151 –154 . 10.1007/s12600-017-0584-z Kodandaram M. H. Sujoy Saha A. B. R. Prakash S. N. (2013 ). Compendium on Pesticide use in Vegetables. Extension Bulletin No. 50. Varanasi : Indian Institute of Vegetable Research , 133. Latif M. A. Rahman M. M. Alam M. Z. (2010 ). Efficacy of nine insecticides against shoot and fruit borer, Leucinodes orbonalis Guenee (Lepidoptera: Pyralidae) in eggplant. J. Pest Sci. 83 391 –397 . 10.1007/s10340-010-0309-2 LeOra S. (1987 ). ). Polo-Plus, POLO for Windows. Petaluma, CA : LeOra Software . Lumjuan N. Rajatileka S. Changsom D. Wicheer J. Leelapat P. Prapanthadara L. A. (2011 ). The role of the Aedes aegypti Epsilon glutathione transferases in conferring resistance to DDT and pyrethroid insecticides. Insect Biochem. Molec. Biol. 41 203 –209 . 10.1016/j.ibmb.2010.12.005 21195177 Malathi V. M. Jalali S. K. Gowda D. K. Mohan M. Venkatesan T. (2015 ). Establishing the role of detoxifying enzymes in field-evolved resistance to various insecticides in the brown planthopper (Nilaparvata lugens) in South India. Insect Sci. 24 35 –46 . 10.1111/1744-7917.12254 26200805 Mally R. Korycinska A. Agassiz D. J. L. Hall J. Hodgetts J. Nuss M. (2015 ). Discovery of an unknown diversity of Leucinodes species damaging Solanaceae fruits in sub-Saharan Africa and moving in trade (Insecta. Lepidoptera, Pyraloidea). Zookeys 472 117 –162 . 10.3897/zookeys.472.8781 25632252 Marygold S. J. Leyland P. C. Seal R. L. Goodman J. L. Thurmond J. Strelets V. B. (2013 ). FlyBase: improvements to the bibliography. Nucleic Acids Res. 41 D751 –D757 .23125371 Masek T. Vopalensky V. Suchomelovam P. Pospisek M. (2005 ). Denaturing RNA electrophoresis in TAE agarose gels. Anal. Biochem. 336 46 –50 . 10.1016/j.ab.2004.09.010 15582557 Meisel R. P. Scott J. G. (2018 ). Using genomic data to study insecticide resistance in the house fly. Musca domestica. Pestic. Biochem. Physiol. 151 76 –81 . 10.1016/j.pestbp.2018.01.001 30704717 Mohan M. Gujar G. T. (2003 ). Local variation in susceptibility of the diamondback moth, Plutella xylostella (Linnaeus) to insecticides and role of detoxification enzymes. Crop Prot. 22 495 –504 . 10.1016/s0261-2194(02)00201-6 Mohan M. Venkatesan T. Sivakumar G. Yandigeri M. S. Verghese A. (2015 ). Fighting Pesticide Resistance in Arthropods. New Delhi : Westville Publishers . Munje S. S. Salunke P. B. Botre B. S. (2015 ). Toxicity of newer insecticides against Leucinodes orbonalis (Guen.). Asian J. Biol. Sci. 10 106 –109 . 10.15740/has/ajbs/10.1/106-109 Oakeshott J. G. Claudianos C. Campbell P. M. Newcomb R. D. Russell R. J. (2005 ). “Biochemical genetics and genomics of insect esterases ,” in Comprehensive Molecular Insect Science-Pharmacology , Vol. 5 eds Gilbert L. I. Latrou K. Gill S. S. (Oxford : Elsevier ), 309 –381 . 10.1016/b0-44-451924-6/00073-9 Oakeshott J. G. Johnson R. M. Berenbaum M. R. Ranson H. Cristino A. S. Claudianos C. (2010 ). Metabolic enzymes associated with xenobiotic and chemosensory responses in Nasonia vitripennis. Insect Biochem. Molec. Biol. 19 147 –163 . 10.1111/j.1365-2583.2009.00961.x 20167025 Panini M. Manicardi G. C. Moores G. Mazzoni E. (2016 ). An overview of the main pathways of metabolic resistance in insects. Invertebrate Surviv. J. 13 326 –335 . Pavlidi N. Vontas J. Leeuwen T. V. (2018 ). The role of glutathione S-transferases (GSTs) in insecticide resistance in crop pests and disease vectors. Curr. Opi. Insect Sci. 27 97 –102 . 10.1016/j.cois.2018.04.007 30025642 Perry T. Batterham P. Daborn P. J. (2011 ). The biology of insecticidal activity and resistance. Insect Biochem. Molec. Biol. 41 411 –422 . 10.1016/j.ibmb.2011.03.003 21426939 Prodhan M. Z. Hasan H. M. T. Chowdhury M. M. I. Alam M. S. Rahman M. L. Azad A. K. (2018 ). Bt eggplant (Solanum melongena L.) in Bangladesh: fruit production and control of eggplant fruit and shoot borer (Leucinodes orbonalis Guenee), effects on non-target arthropods and economic returns. PLoS One 13 :e0205713 . 10.1371/journal.pone.0205713 30462653 Ranson H. Claudianos C. Ortelli F. Abgrall C. Hemingway J. Sharakhova M. V. (2002 ). Evolution of supergene families associated with insecticide resistance. Science 298 179 –181 . 10.1126/science.1076781 12364796 Shelton A. M. Hossain M. J. Paranjape V. Azad A. K. Rahman M. L. Khan A. S. M. M. R. (2018 ). Bt eggplant project in bangladesh: history, present status, and future direction. Front. Bioeng. Biotechnol. 6 :106 . 10.3389/fbioe.2018.00106 30123795 Shirale D. Patil M. Parimi S. (2017 ). Insecticide resistance in field populations of Leucinodes orbonalis (Lepidoptera: Crambidae) in India. Can. Entomol. 149 399 –407 . 10.4039/tce.2017.3 Srinivasan R. (2008 ). Integrated pest management for eggplant fruit and shoot borer (Leucinodes orbonalis) in south and southeast Asia: past, present and future. J. Biopesticides. 1 105 –112 . van Asperen K. (1962 ). A study of house fly esterase by means of a sensitive colorimetric method. J. Insect Physiol. 8 401 –416 . 10.1016/0022-1910(62)90074-4 Wang Y. Zhao S. Shi L. Xu Z. He L. (2014 ). Resistance selection and Biochemical mechanism of resistance against cyflumetofen in Tetranychus cinnabarinus (Boisduval). Pestic. Biochem. Physiol. 111 24 –30 . 10.1016/j.pestbp.2014.04.004 24861930 Yang T. Liu N. (2011 ). Genome analysis of cytochrome P450s and their expression profiles in insecticide resistant mosquitoes. Culex quinquefasciatus. PLoS One 6 :e29418 . 10.1371/journal.pone.0029418 22242119 Yu Q. Lu C. Li B. Fang S. Zuo W. Dai F. (2008 ). Identification, genomic organization and expression pattern of glutathione S-transferase in the silkworm, Bombyx mori. Insect Biochem. Molec. Biol. 38 1158 –1164 . 10.1016/j.ibmb.2008.08.002 19280710 Yu Q. Y. Lu C. Li W. L. Xiang Z. H. Zhang Z. (2009 ). Annotation and expression of carboxylesterases in the silkworm, Bombyx mori. BMC Genomics 10 :553 . 10.1186/1471-2164-10-553 19930670 Zhang Y. Wang L. Guo H. Li G. Zhang Z. Xie L. (2012 ). A transcriptome-based screen of carboxylesterase-like genes that are involved in chlorpyrifos resistance in Laodelphax striatellus (Fallen). Pestic. Biochem. Physiol. 104 224 –228 . 10.1016/j.pestbp.2012.08.006