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Major putative pesticide receptors, detoxification enzymes, and transcriptional profile of the midgut of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae)
Abstract
Insecticide resistance mechanisms, including those for Cry proteins (Bt), in Heliothis virescens are not well understood. Sequencing of midgut transcriptomes may facilitate the discovery of the genes responsible for resistance development. In this study, a total of 5856 Sanger sequences were obtained and assembled to 1687 contigs (464) and singletons (1233) with average length of 507 bp. Blast similarity search showed that 1372 cDNAs from this study matched different genes or cDNAs in the GenBank and other sequence databases. Blast2go annotation identified 611 highly similar proteins with metabolic and cellular processes as major biological functions and catalytic activity and binding as major molecular functions. At least 143 contigs and singletons were associated with pesticide activation, detoxification, and resistance development. These cDNAs, with average length of 601 bp, matched nine groups of pesticide resistance related genes. At least 80 cDNAs coded for Bt resistance related enzymes and potential receptors, including 58 proteinases, 4 cadherins, 13 aminopeptidase, and 5 alkaline phosphatases. Other putative detoxification enzymes included 20 cytochrome P450 oxidases, 11 glutathione S-transferases, 9 esterases, 8 sodium channels, and 15 cytochrome oxidases. Of the 143 contigs and singletons, 111 cDNA sequences seemed to be new resistance candidate gene transcripts in GenBank because they either priorly matched resistance candidate cDNAs of other species, or had low sequence identity with those previously sequenced from H. virescens. This study provides a foundation for future research to develop a gut-specific DNA microarray for analysis of the global changes of gene expression in response to biological and chemical pesticides. Future development resistance management strategies could benefit from this study and help continue research to identify key genes targetable by classic and novel approaches.
... The alimentary canal, in insects, mainly divides into foregut, midgut and hindgut, and is the primary organ of digestive absorption, detoxification metabolism, immune defense and oxidative stress response (Hakim et al., 2010;Azevedo et al., 2013;Borges et al., 2014). Generally, the midgut occupies the majority of the digestive tract and is also considered to be the center of physiological functions in most insects (Dow, 1987;Hakim et al., 2010;Zhu et al., 2011). For instance, most of the enzymes of digestion and detoxification are largely produced and secreted in the midgut of insects, such as Anoplophora glabripennis and Plutella xylostella (Scully et al., 2013;Xia et al., 2016). ...
... As is well known, xenobiotic detoxification is a crucial adaptive mechanism that permits insects to overcome complex environmental stresses including a variety of toxic substances, such as insecticides, mycotoxins, phytotoxins and other xenobiotics (Ranson et al., 2002;Zhu et al., 2011;Xu et al., 2013;Zhang et al., 2016). Normally, oxidation-reduction, conjugation and hydrolysis are three central and correlative pathways in xenobiotic detoxification (Xu et al., 2013). ...
... Another important aspect for grazers is the protection of the intestinal mucosa from hazardous substances and possible mechanical impacts, especially of the midgut epithelium. In insects and other arthropods, the food bolus in the midgut is surrounded by a non-cellular envelope, the peritrophic membrane (PM), which often contains chitin fibrils and proteins and represents the first barrier against pathogens or hazardous particles (Peters 1967(Peters , 1969(Peters , 1992Zhu et al. 2011;Salvador et al. 2014). Furthermore, recent studies showed that the midgut epithelium produces a variety of detoxification enzymes (Zhu et al. 2011;Salvador et al. 2014). ...
... In insects and other arthropods, the food bolus in the midgut is surrounded by a non-cellular envelope, the peritrophic membrane (PM), which often contains chitin fibrils and proteins and represents the first barrier against pathogens or hazardous particles (Peters 1967(Peters , 1969(Peters , 1992Zhu et al. 2011;Salvador et al. 2014). Furthermore, recent studies showed that the midgut epithelium produces a variety of detoxification enzymes (Zhu et al. 2011;Salvador et al. 2014). PM lies between the food particles in the lumen and the epithelium of the midgut. ...
Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis ( L. stagnalis ) and the mayfly Rhithrogena semicolorata ( R. semicolorata ). In L. stagnalis , bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata , irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs ( p < 0.01).
... Changes in the transcription of genes involved in resistance appear common among insects where resistance has evolved (Liu & Scott, 1998;Zhu et al., 2013). Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a major pest of citrus in the United States, China, Pakistan, Brazil and Pacific island countries (Grafton-Cardwell et al., 2013;Kanga et al., 2016;Qasim et al., 2018a;Qasim et al., 2018b;Chen et al., 2019). ...
... Little is known about thiamethoxam-induced gene expression related to resistance, and specifically, the response of D. citri to thiamethoxam. Transcriptomics is a useful approach for the identification of new genes and the exploration of gene functions related to insecticide resistance (Zhu et al., 2011). RNA-sequencing (RNA-seq) is now a common method used to analyze gene expression (Hrdlickova et al., 2017), and this method has been used to identify insecticide metabolism-related genes in several species including Aedes aegypti L. (David et al., 2010), Plutella xylostella L. Pan et al., 2018) and Sitobion avenae Fabricius (Wei et al., 2019). ...
Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits
the causal pathogen of huanglongbing and is a global pest of citrus. D. citri populations
exhibit resistance to multiple insecticide modes of action in areas where these
chemicals have been overused. We performed genome-wide transcriptional analysis for
two field populations of D. citri (Wauchula and Lake Alfred, Florida, USA) that exhibit
1300-fold resistance to the neonicotinoid insecticide, thiamethoxam, and compared it to
that of susceptible psyllids collected from the same area and without imposed selection.
The Lake Alfred population responded to insecticide resistance by up-regulation of 240
genes and down-regulation of 148 others. The Wauchula population exhibited similar patterns
to the Lake Alfred population with up-regulation of 253 genes and down-regulation
of 115 others. Gene Ontology annotation associated with cellular processes, cell, and catalytic
activity were assigned to differentially expressed genes (DEGs). The DEGs from
Lake Alfred and Wauchula populations were mapped to Kyoto Encyclopedia of Gene and
Genomes pathways and implicated enrichment of metabolic pathways, oxidative phosphorylation,
extracellular matrix-receptor interaction, terpenoid backbone biosynthesis,
and insect hormone biosynthesis in the resistant populations. Up-regulation of 60s ribosomal
proteins, UDP-gluscoyltransferases, cytochrome c oxidases, and CYP and ABC
transporters among thiamethoxam-resistant D. citri implicates a broad array of novel and
conventionally understood resistance mechanisms.
... In insects, the midgut is an organ that not only is relevant in the digestion and absorption of nutrients but also serves as a physico-chemical barrier against pathogens and chemical stressors such as insecticides (Billingsley and Lehane 1996;Zhu et al. 2011;Catae et al. 2014;Scudeler et al. 2016;Silva et al. 2016). The lumen in insect midgut is lined by a noncellular envelop, the peritrophic matrix that is the first barrier against pathogens, and a wide variety of detoxificative enzymes is produced by the midgut epithelium (Zhu et al. 2011;Salvador et al. 2014) that is consisted of one layer of digestive, regenerative, and endocrine cell types (Billingsley and Lehane 1996;Chapman 2013). ...
... In insects, the midgut is an organ that not only is relevant in the digestion and absorption of nutrients but also serves as a physico-chemical barrier against pathogens and chemical stressors such as insecticides (Billingsley and Lehane 1996;Zhu et al. 2011;Catae et al. 2014;Scudeler et al. 2016;Silva et al. 2016). The lumen in insect midgut is lined by a noncellular envelop, the peritrophic matrix that is the first barrier against pathogens, and a wide variety of detoxificative enzymes is produced by the midgut epithelium (Zhu et al. 2011;Salvador et al. 2014) that is consisted of one layer of digestive, regenerative, and endocrine cell types (Billingsley and Lehane 1996;Chapman 2013). Due to these crucial roles, the midgut has become an important and toxicologically relevant organ for monitoring environmental pollution in trophic route of aquatic insects (Saouter et al. 1991;Chand 2014;Gutiérrez et al. 2016). ...
In insects, the midgut performs multiple physiologic functions (e.g., digestion and nutrients absorption) and serves as a physical/chemical barrier against pathogens and chemical stressors such as deltamethrin, a pyrethroid insecticide, commonly used in insect control that are agricultural pests and human disease vectors. Here, we described the midgut cell ultrastructure of Callibaetis radiatus nymphs, which are bioindicators of water quality and the ultrastructural alterations in midgut under sublethal exposure to deltamethrin at three different periods (1, 12, 24 h). The digestive cells of deltamethrin-unexposed nymphs had long microvilli, many mitochondria in the apical cytoplasm, a rough endoplasmic reticulum, a basal labyrinth with openings for hemocele, and the midgut peritrophic matrix which is classified as type I. Nymphs exposed to deltamethrin exhibited digestive cells rich in autophagic vacuoles, basal labyrinth loss, and microvilli disorganization since the first hour of contact with deltamethrin. However, these midgut tissues underwent to autophagic cellular recovery along the 24 h of exposure to deltamethrin. Thus, the sublethal exposure to deltamethrin is sufficient to disturb the ultrastructure of C. radiatus midgut, which might reduce the abilities of these insects to survive in aquatic environments contaminated by pyrethroids.
... The alimentary canal, in insects, mainly divides into foregut, midgut and hindgut, and is the primary organ of digestive absorption, detoxification metabolism, immune defense and oxidative stress response (Hakim et al., 2010;Azevedo et al., 2013;Borges et al., 2014). Generally, the midgut occupies the majority of the digestive tract and is also considered to be the center of physiological functions in most insects (Dow, 1987;Hakim et al., 2010;Zhu et al., 2011). For instance, most of the enzymes of digestion and detoxification are largely produced and secreted in the midgut of insects, such as Anoplophora glabripennis and Plutella xylostella (Scully et al., 2013;Xia et al., 2016). ...
... As is well known, xenobiotic detoxification is a crucial adaptive mechanism that permits insects to overcome complex environmental stresses including a variety of toxic substances, such as insecticides, mycotoxins, phytotoxins and other xenobiotics (Ranson et al., 2002;Zhu et al., 2011;Xu et al., 2013;Zhang et al., 2016). Normally, oxidation-reduction, conjugation and hydrolysis are three central and correlative pathways in xenobiotic detoxification (Xu et al., 2013). ...
The physiological functions of insect foregut, especially in xenobiotic detoxification, are scarcely reported because of the unimportance in appearance and insufficient molecular information. The cockroach Periplaneta americana, an entomological model organism, provides perfect material to study physiological functions of foregut tissue due to its architectural feature. Through Illumina sequencing of foregut tissue from P. americana individuals (control) or insects treated with cycloxaprid, as novel neonicotinoid insecticide, 54 193 166 clean reads were obtained and further assembled into 53 853 unigenes with an average length of 366 bp. Furthermore, the number of unigenes involved in xenobiotic detoxification was analyzed, mainly including seventy cytochrome P450s, twelve glutathione S-transferases (GSTs), seven carboxylesterases (CarEs) and seven ATP-binding cassette (ABC) transporters. Compared to control, the expression of twenty-two xenobiotic detoxification unigenes was up-regulated after cycloxaprid application, mainly containing eighteen P450s, one GST, two CarEs and one ABC transporter, indicating that the oxidation-reduction was the major reactive process to cycloxaprid application. Through qRT-PCR analysis, the expression of selected unigenes (six P450s, one GST and one CarE) was up-regulated at least 2.0-fold following cycloxaprid treatment, and was generally in agreement with transcriptome data. Comparing to the previous midgut transcriptome of P. americana, it looks like that the expressive abundance of the xenobiotic detoxification unigenes might be important factors to the detoxifying functional differences between foregut and midgut. In conclusion, insect foregut would also play important roles in the physiological processes related to xenobiotic detoxification. This article is protected by copyright. All rights reserved.
... These included catalytic proteins with hydrolase (esterase/carboxylesterase), oxidoreductase (cytochrome b), and transferase (flavin adenine dinucleotide [FAD] synthetase) activities ( Fig. 2 and Table S2). Insect metabolic and catalytic genes were demonstrated to function in insecticide resistance when they were identified in the midgut transcriptome profile of tobacco budworm, Heliothis virescens, exposed to biological and chemical pesticides (Zhu et al., 2011). In another report, their upregulation led to a substantial increase of metabolic detoxification in acephate-resistant tarnished plant bug, Lygus lineolaris . ...
... Carboxylesterase proteins display active catalytic triad, which contains the active center essential for esterase activity. Besides their functions in insecticide resistance (Guerrero et al., 1999;Guerrero, 2000;Li et al., 2007;Zhu et al., 2011Zhu et al., , 2012, their involvement in insect immune responses to bacterial (Shiotsuki & Kato, 1999) and fungal infection or mechanical wounding (Serebrov et al., 2001) have been reported. It was proposed that these immune inducible carboxylesterases function in degrading toxic substances generated during microbial infection. ...
Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40 % of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of eighteen selected EST clones were confirmed by RT-PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed. This article is protected by copyright. All rights reserved
... Meanwhile, genes encoding candidate Bt-binding proteins, including cadherin protein (Tabashnik et al., 2006;Xu et al., 2005), aminopeptidases N gene (Tiewsiri and Wang, 2011), alkaline phosphatase (ALP) (Jurat-Fuentes and Adang, 2007), membrane glycolipids (Griffitts et al., 2005), and ABCC2 of ABC transporters (Baxter et al., 2011;Gahan et al., 2010), have been studied extensively in recent years. In addition, glycoproteins (Hossain et al., 2004), chymotrypsin, proteinase/protease and trypsin have also been implicated in the Bt resistance (Zhu et al., 2011). Bt resistance in P. xylostella, however, has not been linked to cadherin mutations or to any other Cry1Ac receptors other than ABCC2 (Baxter et al., 2008;Cerda et al., 2006). ...
... Similar dominance of catalytic genes was also observed in the midgut transcriptome of sugarcane borer (Diatraea saccharalis) (Guo et al., 2012) and tobacco budworm (H. virescens) (Zhu et al., 2011). Moreover, majority of these DEUs were up-regulated, which suggested that lipid metabolism and bioconversion were enhanced in resistant strains. ...
... Soberón et al. (2007;[13]) demonstrated that susceptibility to Bt toxin Cry1Ab was reduced by cadherin gene silencing with RNA interference in Manduca sexta, and suggested cadherin increases Bt toxicity by facilitating toxin oligomerization. A global transcriptome-based analysis of Bt-resistance development in Heliothis virescens by Zhu et al. (2011;[14]) revealed genes potentially related to Bt activation and resistance (proteinases, cadherins, aminopeptidases, and alkaline phosphatases) as well as detoxification (cytochrome P450 oxidases, glutathione S-transferases, esterases, sodium channels, and cytochrome oxidases). ...
... The quantitative results highlight a variety of larval-stage L. dispar genes that respond within 24 hours to infection by Bacillus thuringiensis. Bt-responsive midgut genes have been discussed in other studies of lepidopteran pests, such as the beet armyworm [25] and tobacco budworm [14], and our similar findings support the shared core set of responsive midgut genes operating across this order (e.g., lectin, cytochrome p450, amylase, chymotrypsin). Interestingly, the general pattern of downregulation in metabolism-associated genes, and a mixed response among immune-related genes, corresponds very well to observations recently made of a Coleopteran species, Tenebrio molitor, following ingestion of Cry3Aa protoxin [38]. ...
Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which 838, 1,248 and 3,305 were respectively partitioned into high-, mid- and low-quality tiers on the basis of homology information. Digital gene expression profiles suggested genes differentially expressed at 24 hours post infection, and qRT-PCR analyses were performed for verification. The differentially expressed genes primarily associated with digestive function, including α-amylase, lipase and carboxypeptidase; immune response, including C-type lectin 4; developmental genes such as arylphorin; as well as a variety of binding proteins: cellular retinoic acid binding protein (lipid-binding), insulin-related peptide binding protein (protein-binding) and ovary C/EBPg transcription factor (nucleic acid-binding). This is the first study conducted to specifically investigate gypsy moth response to a bacterial infection challenge using large-scale sequencing technologies, and the results highlight important genes that could be involved in biopesticide resistance development or could serve as targets for biologically-based control mechanisms of this insect pest.
... Similarly, Lactobacillales possess the ability to synthesize enzymes including pectindegrading, glycoside hydrolase and polysaccharide lyases, thereby positively influencing pesticide resistance (Warnecke et al., 2007;Xiang et al., 2006). Pseudomonas was also found to be involved in the host immunity and may similarly play similar role in C. medinalis immune response (Potrikus & Breznak, 1977;Warnecke et al., 2007;Zhu et al., 2011). Additionally, Wolbachia has been recognized as ...
To understand the effect of diet on gut bacteria of Cnaphalocrocis medinalis (rice leaf folder, RLF), we compared the composition and diversity of gut bacterial community in C. medinalis larvae collected from three different rice variety fields of Minghui 82 (RLF1), Youngyong 15 (RLF2) and Minghui 2155 (RLF3), using culture‐independent PCR (V3 variable region)‐denaturing gradient gel electrophoresis (PCR‐DGGE) methods and hypervariable region (V4) of 16S rRNA sequenced by Illumina HiSeq platform. The DGGE result showed that the bacteria genera Asaia , Bacillus , Stenotrophomonas , Achromobacter and Serratia coexisted in the guts of RLF1, RLF2 and RLF3, while Cedecea , Burkholderia and Acinetobacter coexisted in the guts of RLF2 and RLF3. However, only the genus Enterococcus existed in the guts of RLF1, and Pantoea , Wolbachia and Tumebacillus in RLF3 larvae. A total of 25 bacterial phyla, 48 classes, 127 orders, 223 families, and 406 genera were identified when using 16s RNA sequencing, with 35 genera coexisted in RLF1, RLF2 and RLF3 larval guts, while 83, 36, 141 unique genera existed in RLF1, RLF2 and RLF3, respectively. The indices of ACE and Shannon were not significantly different among RLF1, RLF2 and RLF3. The dominant bacterial taxa were Proteobacteria, Bacteroidetes and Firmicutes at the phylum level, and Acinetobacter and Wolbachia at the genus level. The relative genus abundance for the genera with relative abundance (≥0.01) was significantly different among RLF1, RLF2 and RLF3. The findings indicated that different rice varieties had significant effects on the relative abundance of gut bacteria in RLF, which could provide new insights into the relationship between insect gut bacteria and their associated host plants.
... There are two key reasons for the limited toxicity of insecticides against pests and other harmful insects. The first is their metabolic degradation in the midgut and the reduced penetration of insecticides through midgut walls (Cheng Zhu et al., 2011). Several enzymes are involved in the detoxification process to reach metabolic degradation of the insecticides (Ibrahim & Ali, 2018;Ibrahim et al., 2023;Wang et al., 2020). ...
Background
Mosquitoes cause a variety of health problems in humans and pets. So, the control of mosquito larvae is one of the best ways to avoid health problems arising from diseases transmitted by these insects. There are various control mechanisms including mechanical, biological and chemical control. The latter, despite the presence of some obstacles associated with its use, is preferred because of its ability to supply rapid management results.
Result
A novel laboratory-synthesized chemical compound containing pyrazole and pyridine moieties (pyrazole–pyridine derivatives, PPD) was used to control and address the biological effects on Culex pipiens mosquito second larval instar. A sublethal concentration (LC30) of PPD inhibited larval growth by about 50%. Furthermore, the developmental time of larvae into pupae and the emergence of adults from the pupal stages were increased by about 20% and 17%, respectively. The ultrastructural studies on the midgut cells revealed that treated larvae suffered dramatic degeneration in the gastric caeca and the posterior midgut cells, while the anterior midgut epithelium appeared with an abundance of lysosomal activities. Additionally, treated larvae showed fluctuated activities in the levels of the detoxifying enzymes and increased levels in total antioxidants.
Conclusions
These results clearly show that pyrazole and pyridine moieties containing compounds can be used against larval stages of C. pipiens.
... As reported, the insect midgut is the indispensable barrier for xenobiotics to enter and plays a vital role in the digestion and absorption of nutrients in insects (Canton & Bonning 2020). Several studies have reported that the midgut is enriched with most of the detoxification enzymes (Zhu et al. 2011). Meanwhile, the midgut is considered as the critical tissue engaged in defending against exogenous harmful substances including chemical insecticides ). ...
... As reported, the insect midgut is the indispensable barrier for xenobiotics to enter and plays a vital role in the digestion and absorption of nutrients in insects (Canton & Bonning 2020). Several studies have reported that the midgut is enriched with most of the detoxification enzymes (Zhu et al. 2011). Meanwhile, the midgut is considered as the critical tissue engaged in defending against exogenous harmful substances including chemical insecticides ). ...
... Wang et al. revealed that the glycolysis and the TCA cycle showed significant responses to fenpropathrin in B. mori [19]. Additionally, the midgut also plays a major role in insecticide resistance [20]. Most detoxification-related enzymes are produced and secreted in the midgut of insects, including in Anoplophora glabripennis and Plutella xylostella [21,22]. ...
Coumarin and its derivatives are plant-derived compounds that exhibit potent insecticidal properties. In this study, we found that natural coumarin significantly inhibited the growth and development of Spodoptera litura larvae through toxicological assay. By transcriptomic sequencing, 80 and 45 differentially expressed genes (DEGs) related to detoxification were identified from 0 to 24 h and 24 to 48 h in S. litura after coumarin treatment, respectively. Enzyme activity analysis showed that CYP450 and acetylcholinesterase (AChE) activities significantly decreased at 48 h after coumarin treatment, while glutathione S-transferases (GST) activity increased at 24 h. Silencing of SlCYP324A16 gene by RNA interference significantly increased S. litura larval mortality and decreased individual weight after treatment with coumarin. Additionally, the expression levels of DEGs involved in glycolysis and tricarboxylic acid (TCA) cycle were inhibited at 24 h after coumarin treatment, while their expression levels were upregulated at 48 h. Furthermore, metabonomics analysis identified 391 differential metabolites involved in purine metabolism, amino acid metabolism, and TCA cycle from 0 to 24 h after treated with coumarin and 352 differential metabolites associated with ATP-binding cassette (ABC) transporters and amino acid metabolism. These results provide an in-depth understanding of the toxicological mechanism of coumarin on S. litura.
... mRNA was purified from total RNA using the NucleoTrap mRNA purification kit (BD Bioscience Clontech, Palo Alto, CA, USA). The Creator Smart cDNA Library Construction Kit (BD Bioscience Clontech) was used for cDNA library construction by following the manufacturer's instructions and modified protocols described by Zhu et al. [21]. Approximately 30,000 clones were obtained and sequenced with an M13 forward primer on an ABI 3730XL sequencer. ...
Frequent sprays on cotton prompted resistance development in the tarnished plant bug (TPB). Knowledge of global gene regulation is highly desirable to better understand resistance mechanisms and develop molecular tools for monitoring and managing resistance. Novel microarray expressions of 6688 genes showed 3080 significantly up- or down-regulated genes in permethrin-treated TPBs. Among the 1543 up-regulated genes, 255 code for 39 different enzymes, and 15 of these participate in important pathways and metabolic detoxification. Oxidase is the most abundant and over-expressed enzyme. Others included dehydrogenases, synthases, reductases, and transferases. Pathway analysis revealed several oxidative phosphorylations associated with 37 oxidases and 23 reductases. One glutathione-S-transferase (GST LL_2285) participated in three pathways, including drug and xenobiotics metabolisms and pesticide detoxification. Therefore, a novel resistance mechanism of over-expressions of oxidases, along with a GST gene, was revealed in permethrin-treated TPB. Reductases, dehydrogenases, and others may also indirectly contribute to permethrin detoxification, while two common detoxification enzymes, P450 and esterase, played less role in the degradation of permethrin since none was associated with the detoxification pathway. Another potential novel finding from this study and our previous studies confirmed multiple/cross resistances in the same TPB population with a particular set of genes for different insecticide classes.
... In spite of efforts to develop chemical and biological pesticides, insects are also looking for ways to neutralize the effects of these pesticides. ESTs and GSTs are the two important groups of detoxifying enzymes that involve in the breakdown and neutralization of toxic compounds entering insect body (Zhu et al., 2011). Findings of our study support results of earlier investigations regarding the effect of fungal conidia on detoxifying enzymes of insects (Xia et al., 2000(Xia et al., , 2001Serebov et al., 2001Serebov et al., , 2006Dubovskiy et al., 2008;Zibaee et al., 2009;Fan et al., 2013;Petlamul et al., 2019). ...
The current study aimed to determine antioxidant and detoxifying responses of Chilo suppressalis Walker (Lepidoptera: Crambidae) to Beauveria bassiana (Strains BBRR1, BBAL1, BBLN1, BBLN2), Metarhizium anisopliae and Hirsutella subulata. The interactions of insect humoral immune responses with the entered conidia of entomopathogenic fungi in addition to nodule formation and melanization caused the production of several reactive oxygenate species (ROS), such as hydrogen peroxidase (H2O2), hydroperoxides (ROOH), superoxide radicals (O 2−), and hydroxyl radical (OH −). The highest activity of catalase was recorded by BBRR1 and BBAL1, treatment after 48 to 96 h while the larvae treated by BBRR1 showed the highest peroxidase activity. Both ascorbate peroxidase and glucose-6-phosphate dehydrogenase showed the highest activity in the larvae treated by BBRR1 after 48-96 h. The highest concentration of Malondialdehyde (MDA) reported in the larvae treated by BBRR1, BBAL1 and BBLN1, after 48 hours. The highest activity of general esterases was recorded in the larvae treated by BBRR1 after 48-96 hours. Similar results were recorded in the activity of glutathione-S-transferase but the enzyme had also the highest activity in the larvae treated by BBAL1 and BBLN2 after 48 hours. The larvae treated by BBRR1 and BBLN1 showed the highest activity of acid phosphatase (ACP) after 72 and 96 hours while the highest activity of alkaline phosphatase (ALP) was obtained in the larvae only treated by BBRR1 after 48-96 hours. The results clearly revealed that BBRR1 significantly and severely induced antioxidant and detoxifying systems of C. suppressalis larvae implying on virulence and immune induction of BBRR1 against the larvae.
... 18,19 In addition, insects also produce a variety of detoxifying enzymes in response to adversity stress induced by invading pathogens to digest exogenous poisons and enhance their adaptability to adversity stress. 20 For instance, the activities of carboxylesterase (CarE), glutathione S-transferase (GST), and acetylcholinesterase (AChE) in Tenebrio molitor were induced by H. beicherriana 21 ; the activities of AChE and GST in Holotrichia oblita larvae were increased after feeding on Cry8Gal. 22 Recently, RNA sequencing (RNA-Seq) has been widely used in the study of insect stress response, and this technique has emerged as a reliable method for identifying insect antioxidant and detoxifying enzyme genes. ...
Background
Combining the entomopathogenic nematode (EPN), Heterorhabditis beicherriana LF strain, and Bacillus thuringiensis (Bt) HBF‐18 strain is a practical strategy to manage the larvae of Holotrichia parallela Motschulsky (white grubs). However, the mechanisms underlying the larval defense response to this combined biocontrol strategy are unknown.
Results
The activities of some antioxidant enzymes (SOD, POD, CAT) and some detoxifying enzymes (AChE, P‐450, CarE, GST) in grubs showed an activation–inhibition trend throughout the EPN‐Bt exposure time course. Eight potentially key antioxidant and detoxifying enzyme genes in response to EPN‐Bt infection were identified from the midgut of grubs through RNA sequencing. After silencing CAT, CarE18, and GSTs1, the enzyme activities were significantly decreased by 30.29%, 68.80%, and 34.63%, respectively. Meanwhile, the mortality of grubs was increased by 18.40%, 46.30%, and 42.59% after exposure to EPN‐Bt for 1 day. Interestingly, the PI3K/Akt signaling pathway was significantly enriched in KEGG enrichment analysis, and the expression levels of phosphatidylinositol 3‐kinase (PI3K), protein kinase B (Akt), cap ‘n’ collar isoform‐C (CncC), kelch‐like ECH‐associated protein 1 (Keap1), and CarE18 were all up‐regulated when exposed to EPN‐Bt for 1 day. Furthermore, RNAi‐mediated PI3K silencing showed a similar down‐regulated trend between PI3K/Akt/CncC and CarE18. Moreover, silencing PI3K rendered grubs more susceptible to EPN‐Bt and accelerated symbiotic bacteria multiplication in grubs.
Conclusion
These results suggest that the PI3K/Akt/CncC pathway mediates the expression of CarE18 and participates in the defense response of H. parallela larvae against EPN‐Bt infection. Our data provide valuable insights into the design of appropriate management strategies for this well‐known agricultural pest. © 2022 Society of Chemical Industry.
... The midgut of insects is an organ for digestion and absorption of nutrients, but also functions as a physical-chemical barrier against pathogens and chemicals (Zhu et al., 2011;Salvador et al., 2014;Scudeler et al., 2016;Silva et al., 2016). The peritrophic matrix, which lines the lumen, is the first barrier against pathogens, and the midgut epithelium has detoxifying enzymes (Hegedus et al., 2009;Oliveira et al., 2019;Luz et al., 2022). ...
N,N-dimethylaniline and 1,2,5-trithiepane, present in the salivary glands of Podisus nigrispinus Dallas (Heteroptera: Pentatomidae), are toxic compounds which kill prey. The insecticidal activity and midgut cytotoxicity in Spodoptera frugiperda (J. E. Smith (Lepidoptera: Noctuidae) caterpillars fed on a diet with lethal concentrations of N,N-dimethylaniline and 1,2,5-trithiepane were evaluated. Midgut cell damage was evaluated with both light and transmission electron microscopy. The LC50 and LC90 of N,N-dimethylaniline were 0.611 and 0.818 μg L⁻¹, respectively, and for 1,2,5-trithiepane they were 0.671 and 0.885 μg L⁻¹, respectively. Vacuolization in the digestive and goblet cells occurred after 1 h of exposure in the midgut of the insects treated with either N,N-dimethylaniline and 1,2,5-trithiepane. Changes caused by N,N-dimethylaniline and 1,2,5-trithiepane in the midgut of S. frugiperda caterpillars may affect digestion and nutrient absorption with negative impacts on the insect's development and survival. The non-proteinaceous N,N-dimethylaniline and 1,2,5-trithiepane compounds have insecticidal effects, confirming the potential use on S. frugiperda caterpillars through oral administration.
... The roles of major detoxification genes such as glutathione S-transferase (GST), carboxylesterases (CarE), and cytochrome P450 monooxygenases in metabolic resistance in lepidopteran pests have been widely reported (Li et al., 2007;Eziah et al., 2009;Pauchet et al., 2010). In addition, several Bt-binding proteins have been documented in P. xylostella (Gahan et al., 2010;Tiewsiri and Wang, 2011;Zhu et al., 2011). Previous studies have focused on mutations primarily correlated with insecticide resistance Troczka et al., 2015). ...
Background: Diamondback moth (DBM), Plutella xylostella (L.), has developed resistance to many insecticides. The molecular mechanism of DBM resistance to Bt-G033A combined with chlorantraniliprole (CL) remains undefined.
Methods: In this study, field-resistant strains of Plutella xylostella to three pesticides, namely, Bacillus thuringiensis (Bt) toxin (Bt-G033A), CL, and a mixture of Bt + CL, were selected to evaluate the resistance level. Additionally, transcriptomic profiles of a susceptible (SS-DBM), field-resistant (FOH-DBM), Bt-resistant (Bt-DBM), CL-resistant (CL-DBM), and Bt + CL-resistant (BtC-DBM) strains were performed by comparative analysis to identify genes responsible for detoxification.
Results: The Bt-G033A was the most toxic chemical to all the DBM strains among the three insecticides. The comparative analysis identified 25,518 differentially expressed genes (DEGs) between pairs/combinations of strains. DEGs were enriched in pathways related to metabolic and catalytic activity and ABC transporter in resistant strains. In total, 17 metabolic resistance-related candidate genes were identified in resistance to Bt-G033A, CL, and Bt + CL by co-expression network analysis. Within candidate genes, the majority was upregulated in key genes including cytochrome P450, glutathione S-transferase (GST), carboxylesterase, and acetylcholinesterase in CL- and BtC-resistant strains. Furthermore, aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, trypsin, and ABC transporter genes were eminent as Bt-resistance-related genes. Expression patterns of key genes by the quantitative real-time PCR (qRT-PCR) proved the credibility of transcriptome data and suggest their association in the detoxification process.
Conclusion: To date, this study is the most comprehensive research presenting functional transcriptome analysis of DBM using Bt-G033A and CL combined insecticidal activity.
... McKenzie and Batterham (1994) Sodium channel DDT Pyrethroids Zhu et al. (2011b) TA B L E 7 Plant-derived compounds or extracts with potential for the control of H. armigera ...
Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is one of the most damaging insect pests globally, causing estimated global economic losses of over 3 billion US dollars annually. Crops most affected include cotton, tomato, soybean, grain crops such as corn and sorghum, chickpea and other pulses. Adults of this species possess strong migratory abilities (>2000 km), high fecundity and rapid reproductive rates; completing 4–6 generations per year in most cropping regions. Furthermore, the larvae are polyphagous, with a wide and diverse host range and possess the ability to enter diapause in order to survive adverse climatic conditions. At present, it is distributed across most of Oceania, Asia, Africa and southern Europe and has recently spread to South America. Various control measures have been trialled or proposed for the treatment of this pest, including synthetic insecticides, phytopesticides, microbial pesticides, macro-biocontrol agents (both parasitoids and predators) and the development of genetically modified crops (e.g. Bt cotton). Successful control necessitates the use of an integrated pest management (IPM) approach, wherein biological, chemical and physical control measures are combined for the greatest control efficacy.
... However, detoxification enzymes such as esterases, glutathione-S-transferases (GSTs) and cytochrome P450 monooxygenases (P450) are super families of enzymes that may be implicated in both insecticide tolerance and resistance (Li et al., 2007;Prasad & Roy, 2017). The epithelial cells in the insect midgut produce detoxification enzymes that facilitate oxidative detoxification, by metabolising active ingredients (a.i.s) and, sometimes, reducing penetration through the midgut wall (Zhu et al., 2011). ...
Results of dose response bioassays ' in vivo ' used to characterise the phenotypic response of pyrethroid resistant S. avenae in comparison to susceptible S. avenae, and two other cereal aphids, the rose-grain aphid ( Metopholophium dirhodum ) and the bird-cherry – oat aphid ( Rhopalosiphum padi ), are used to measure levels of pyrethroid resistance. Aphid pests on cereals in the British Isles are predominantly controlled by pyrethroid insecticides, especially since the implementation of the recent ban on neonicotinoid seed treatments on all outdoor crops. Resistance to pyrethroids has been detected in one of the main aphid pests, the grain aphid ( Sitobion avenae ), probably brought on by the sustained use of these pyrethroid sprays to control cereal aphids, which can transmit plant viruses, especially Barley Yellow Dwarf Virus (BYDV). The withdrawal of several insecticide compounds (e.g. pirimicarb, dimethoate, chlorpyrifos and the aforesaid neonicotinoids) for cereal aphid control will probably increase the selection pressure, leading to increased levels of resistance in S. avenae , and, potentially, the evolution of resistance in other cereal aphid species. In this article we present the results of dose response bioassays ' in vivo ' used to characterise the phenotypic response of pyrethroid resistant S. avenae in comparison to susceptible S. avenae, and two other cereal aphids, the rosegrain aphid ( Metopholophium dirhodum ) and the bird-cherry– oat aphid ( Rhopalosiphum padi ), in order to measure levels of pyrethroid resistance. At present, little is known about the extent of pyrethroid resistance in S. avenae beyond the UK and in other cereal aphids. It therefore becomes increasingly important to monitor these pests to inform crop management decisions in light of the recent loss of other insecticides. The unintended consequences of the rapid withdrawal of insecticides, together with a failure to prepare and install alternative products and control approaches in advance, will probably ultimately lead to the loss of effectiveness of insecticidal compounds like pyrethroids.
... Comparative Biochemistry and Physiology -Part D 30 (2019) 81-90 correlation of reduced activities of trypsin and chymotrypsin to the reduced conversion of protoxin to activated Cry toxin was established earlier (Pardo-Lopez et al., 2013;Adang et al., 2014;Tabashnik, 2015). Also relation of trypsin, chymotrypsin and proteinase/protease has been shown in the context of Bt resistance (Zhu et al., 2011). In a recent study, it was demonstrated that downregulation of trypsin gene is associated with development of Bt resistance (Yao et al., 2017). ...
... According to Cao et al. (2014), who established multiple regressions to predict overall fitness cost and resistance level with fitness costs, these LF-resistant strains may use a second phase of resistance. In this stage, resistance gene-encoding enzymes, such as digestive enzymes, hydrolase, detoxification enzymes, and catalytic enzymes are considered the most important factor to produce fitness cost (Rajagopal et al., 2009;Zhu et al., 2011;Guo et al., 2012;Cao et al., 2013;Lei et al., 2014;Liu et al., 2014;Wei et al., 2016a;Zhang et al., 2017). As a significant and universal phenomenon, we found in this study that a significant portion of DEGs were enriched predominantly in "catalytic activity, " "endopeptidase activity, " "aminopeptidase activity, " "serine-type endopeptidase activity, " "proteolysis, " "biological process, " "metabolic process, " "peptidase activity, " "metallopeptidase activity, " "serine-type peptidase activity, " "exopeptidase activity, " "serine hydrolase activity, " "hydrolase activity, " "protein metabolic process, " "acting on L-amino acid peptides, " "peptidase activity, " and "organic substance metabolic process" for all the resistant strains (Table 3; Figure S2). ...
Helicoverpa armigera can develop resistance to Bacillus thuringiensis (Bt), which threaten the long-term success of Bt crops. In the present study, RNAseq was employed to investigate the midgut genes response to strains with different levels of resistance (LF5, LF10, LF20, LF30, LF60, and LF120) in H. armigera. Results revealed that a series of differentially expressed unigenes (DEGs) were expressed significantly in resistant strains compared with the LF-susceptible strain. Nine trypsin genes, ALP2, were downregulated significantly in all the six resistant strains and further verified by qRT-PCR, indicating that these genes may be used as markers to monitor and manage pest resistance in transgenic crops. Most importantly, the differences in DEG functions in the different resistant strains revealed that different resistance mechanisms may develop during the evolution of resistance. The immune and detoxification processes appear to be associated with the low-level resistance (LF5 strain). Metabolic process-related macromolecules possibly lead to resistance to Cry1Ac in the LF10 and LF20 strains. The DEGs involved in the “proton-transporting V-type ATPase complex” and the “proton-transporting two-sector ATPase complex” were significantly expressed in the LF30 strain, probably causing resistance to Cry1Ac in the LF30 strain. The DEGs involved in binding and iron ion homeostasis appear to lead to high-level resistance in the LF60 and LF120 strains, respectively. The multiple genes and different pathways seem to be involved in Cry1Ac resistance depending on the levels of resistance. Although the mechanisms of resistance are very complex in H. armigera, a main pathway seemingly exists, which contributes to resistance in each level of resistant strain. Altogether, the findings in the current study provide a transcriptome-based foundation for identifying the functional genes involved in Cry1Ac resistance in H. armigera.
... Insect midgut plays an important role in detoxification of exogenic compounds such as insecticides, besides its biological and physiological functions. Within this organ, genes encoding detoxification enzymes are typically highly expressed (Valles et al., 1999;Feyereisen, 2006;Zhu et al., 2011;Wang et al., 2015). Similarly, fat body and Malpighian tubule are also important insect tissues involved in detoxification metabolism of insecticides (Daborn et al., 2007;Zhu et al., 2010). ...
Insect cytochrome P450 monooxygenases played an important role in detoxifying insecticides which potentially contributed to the metabolic resistance to insecticides. Bradysia odoriphaga, as a major pest of Chinese chive, was reported to be highly tolerant to neonicotinoid insecticides imidacloprid. In this study, a novel P450 gene, CYP6FV12, was cloned from B. odoriphaga. The full-length cDNA sequence of CYP6FV12 is 2520 bp long and its open reading frame (ORF) encodes 519 amino acids. Quantitative real-time PCR showed that the highest expression of CYP6FV12 was observed in fourth-instar larvae, which is 154.32-fold higher than that of eggs. Highest expression of CYP6FV12 was observed in the midgut, followed by fat body, which was 13.67 and 5.42-fold higher than that in cuticle, respectively. The expression of CYP6FV12 was significantly up-regulated in B. odoriphaga larvae after exposed to imidacloprid at the concentrations of 10, 30, 50, and 70 mg/L. Moreover, RNAi mediated silencing of CYP6FV12 increased mortality by 28.62 % when the fourth-instar larvae were treated with imidacloprid. This is the first systematic study on isolated P450s gene involved in imidacloprid resistance in B. odoriphaga and increased our understanding of the molecular mechanisms of insecticide detoxification in this pest insect.
... In the present study, it was observed that the vesicles were found in the cytoplasm (Ferreira et al., 1990;Aumüller et al., 1999), which was released by apocrine secretion in the lumen of the midgut (Ferreira et al., 2013). Thus, the increase of apocrine secretion in the midgut of P. nigrispinus fed with prey exposed to imidacloprid may be due to the release of enzymes to detoxification as observed in other insects (Yu and Hsu, 1993;Enayati et al., 2005;Zhu et al., 2011). ...
The selectivity of insecticides on natural enemies in pest control are an important strategy for Integrated Pest Management. However, insecticides can have side effects on non-target organisms such as natural enemies. This study evaluated the histological and cytological changes mediated by the sublethal concentration of the imidacloprid
insecticide on the midgut of non-target predator Podisus nigrispinus (Heteroptera: Pentatomidae), used in the biological control of pests. Imidacloprid was toxic for P. nigrispinus with LC50 =3.75 mg L−1 and survival of 51.8%. This sublethal concentration of imidacloprid causes histological alterations in the midgut epithelium and cytotoxic features were irregular border epithelium, cytoplasmic vacuolation, and apocrine secretions in the first 6 h after exposure with the insecticide. Apoptosis in the digestive cells occurs after 12 h of exposure in the midgut. These results suggest that imidacloprid may affect the digestive physiology of P. nigrispinus and compromise the effective predation of this insect a biological control agent. The associated use of this insecticide with the predator in pest control should be carefully evaluated.
... The AMG shows a more severe cellular degeneration probably because they come into contact with the contaminated food of the insecticide in the first stages of the digestion (Catae et al., 2014;Oliveira et al., 2014), and they consequently compromise the digestion, absorption and secretion of digestive enzymes (Terra, 1990;Fialho et al., 2012Fialho et al., , 2013Torres and Boyd, 2009). However, the increased secretion observed in the midgut lumen of P. nigrispinus can be attributed to the continued production of detoxifying enzymes that play a role in the metabolism of pesticides (Yu and Hsu, 1993;Enayati et al., 2005;Zhu et al., 2011). This seems to be supplanted by insecticide due to cell damage encountered. ...
Insecticides used in the agriculture and forestry have side effects on non-target organisms used as natural enemies. This study evaluated the histopathology and cytotoxicity of permethrin on the midgut of the non-target predatory bug, Podisus nigrispinus (Heteroptera: Pentatomidae) used in the biological control of pest insects. The toxicity and survival of this insect were determined using six concentrations of permethrin via ingestion. Histological and ultraestutural changes of the midgut of P. nigrispinus were analyzed after exposure to permethrin. The insecticide caused toxicity in P. nigrispinus with LC50 = 0.46 μg L-1 and survival of 47% after 72 h of exposure. The histological changes in the midgut were irregularly bordered epithelium, cytoplasmic vacuolization and apocrine secretions in the lumen after 6 h following exposure to the insecticide. Cytotoxic effects such as granules and vacuoles secreted into the lumen, presence of autophagosomes, and dilatation of infolds of the basal plasma membrane were observed in the three regions of the midgut. Cells of the midgut in apoptosis occurred after 12 h of exposure. Permethrin causes toxic effects, inhibits survival, and produces changes in the histology and cytology of the midgut in P. nigrispinus, suggesting that the cell stress induced by this insecticide can disrupt physiological processes such as digestion, compromising the potential of the predator as a biological control agent of pests. The low selectivity of permethrin to a non-target organism such as the predatory bug, P. nigrispinus indicates that the associated use of this insecticide in biological control should be better evaluated.
... P450 amino-acid sequences are extremely diverse, with levels of identity as low as 16%, but their structural fold has remained the same throughout evolution [66]. Of the cytochrome P450 superfamily, particularly the subclades CYP3, CYP4 and the mitochondrial clade are known for their role in detoxification processes in insects [18,67,68]. Among the 57 H. euphorbiae transcripts annotated as P450, 9 CYP3 and 4 CYP4 were found (with a mean length of 1290 bp). ...
Background
The European spurge hawkmoth, Hyles euphorbiae (Lepidoptera, Sphingidae), has been intensively studied as a model organism for insect chemical ecology, cold hardiness and evolution of species delineation. To understand species isolation mechanisms at a molecular level, this study aims at determining genetic factors underlying two adaptive ecological trait candidates, phorbol ester (TPA) detoxification and seasonal cold acclimation.
Method
A draft transcriptome of H. euphorbiae was generated using Illumina sequencing, providing the first genomic resource for the hawkmoth subfamily Macroglossinae. RNA expression levels in tissues of experimental TPA feeding larvae and cooled pupae was compared to levels in control larvae and pupae using 26 bp RNA sequence tag libraries (DeepSuperSAGE). Differential gene expression was assessed by homology searches of the tags in the transcriptome.
Results
In total, 389 and 605 differentially expressed transcripts for detoxification and cold hardiness, respectively, could be identified and annotated with proteins. The majority (22 of 28) of differentially expressed detox transcripts of the four ‘drug metabolism’ enzyme groups (cytochrome P450 (CYP), carboxylesterases (CES), glutathione S-transferases (GST) and lipases) are up-regulated. Triacylglycerol lipase was significantly over proportionally annotated among up-regulated detox transcripts. We record several up-regulated lipases, GSTe2, two CESs, CYP9A21, CYP6BD6 and CYP9A17 as candidate genes for further H. euphorbiae TPA detoxification analyses. Differential gene expression of the cold acclimation treatment is marked by metabolic depression with enriched Gene Ontology terms among down-regulated transcripts almost exclusively comprising metabolism, aerobic respiration and dissimilative functions. Down-regulated transcripts include energy expensive respiratory proteins like NADH dehydrogenase, cytochrome oxidase and ATP synthase. Gene expression patterns show shifts in carbohydrate metabolism towards cryoprotectant production. The Glycolysis enzymes, G1Pase, A1e, Gpi and an Akr isoform are up-regulated. Glycerol, an osmolyte which lowers the body liquid supercooling point, appears to be the predominant polyol cryoprotectant in H. euphorbiae diapause pupae. Several protein candidates involved in glucose, glycerol, myo-inositol and potentially sorbitol and trehalose synthesis were identified.
Conclusions
A majority of differently expressed transcripts unique for either detoxification or cold hardiness indicates highly specialized functional adaptation which may have evolved from general cell metabolism and stress response.The transcriptome and extracted candidate biomarkers provide a basis for further gene expression studies of physiological processes and adaptive traits in H. euphorbiae.
Electronic supplementary material
The online version of this article (10.1186/s12983-018-0252-2) contains supplementary material, which is available to authorized users.
... These roles are essential for environmental adaptation. In most insects, digestion occurs mainly in the midgut, where a large portion of the insect's digestive enzymes are produced and secreted, including proteases, lipases, and carbohydrases [1][2][3]. The insect midgut is also considered to be the centre of detoxification metabolism and stress response, which include three major interrelated pathways: oxidation-reduction, conjugation and hydrolysis [4, 5]. ...
The cockroach, Periplaneta americana, is an obnoxious and notorious pest of the world, with a strong ability to adapt to a variety of complex environments. However, the molecular mechanism of this adaptability is mostly unknown. In this study, the genes and microbiota composition associated with the adaptation mechanism were studied by analyzing the transcriptome and 16S rDNA pyrosequencing of the P. americana midgut, respectively. Midgut transcriptome analysis identified 82,905 unigenes, among which 64 genes putatively involved in digestion (11 genes), detoxification (37 genes) and oxidative stress response (16 genes) were found. Evaluation of gene expression following treatment with cycloxaprid further revealed that the selected genes (CYP6J1, CYP4C1, CYP6K1, Delta GST, alpha-amylase, beta-glucosidase and aminopeptidase) were upregulated at least 2.0-fold at the transcriptional level, and four genes were upregulated more than 10.0-fold. An interesting finding was that three digestive enzymes positively responded to cycloxaprid application. Tissue expression profiles further showed that most of the selected genes were midgut-biased, with the exception of CYP6K1. The midgut microbiota composition was obtained via 16S rDNA pyrosequencing and was found to be mainly dominated by organisms from the Firmicutes phylum, among which Clostridiales, Lactobacillales and Burkholderiales were the main orders which might assist the host in the food digestion or detoxification of noxious compounds. The preponderant species, Clostridium cellulovorans, was previously reported to degrade lignocellulose efficiently in insects. The abundance of genes involved in digestion, detoxification and response to oxidative stress, and the diversity of microbiota in the midgut might provide P. americana high capacity to adapt to complex environments.
... A large number of genes encoding trypsins and chymotrypsins have been identified in many lepidopteran insect species, such as Manduca sexta (Peterson et al., 1995), Choristoneura fumiferana (Wang et al., 1999;He et al., 2009), Plodia interpunctella (Zhu et al., 2000), Helicoverpa armigera (Mazumdar-Leighton et al., 2000), Helicoverpa zea (Mazumdar-Leighton and Broadway, 2001), Agrotis ipsilon (Mazumdar-Leighton and Broadway, 2001), Sesamia nonagrioides (Diaz-Mendoza et al., 2005), Spodoptera exigua (Herrero et al., 2005), Tineola bisselliella (Hughes & Vogler, 2006), Locusta migratoria manilensis (Wei et al., 2007), Bombyx mandarina (Arunkumar et al., 2008), B. mori (Zhao et al., 2010), S. litura (Zhang et al., 2010;Zhan et al., 2011), S. frugiperda (Rodriguez-Cabrera et al., 2010), Mamestra configurata (Erlandson et al., 2010), Ostrinia nubilalis (Coates et al., 2008;Yao et al., 2012), Heliothis virescens (Zhu et al., 2011, andPlutella xylostella (Xie et al., 2012;Shi et al., 2013). These identified proteases show the diversity of trypsin and chymotrypsin genes in insects, as well as differences and similarities between different insect species or within the same species. ...
Two cDNA sequences encoding a trypsin-like and a chymotrypsin-like serine protease (MsT and MsCT, GenBank accession Nos. KP730443 and KP730444, respectively) were cloned from midgut of oriental armyworm, Mythimna separata Walker. Multiple alignments revealed that the deduced amino acid sequences of MsT and MsCT contained a serine protease catalytic motif GDSGGPL and catalytic triads (His, Asp, and Ser). Analyses of tissue and developmental expression of MsT and MsCT showed that they were mainly expressed in midguts and could be detected in first to sixth instar larvae, prepupal and pupal stages. Expressions of both MsT and MsCT were downregulated after 24 h of starvation and upregulated by subsequent insect refeeding. MsT expression in response to 20-hydroxyecdysone (20E) was dose dependent and upregulated after 24 h. However, MsCT expression in response to 20E was downregulated compared with controls. MsCT, but not MsT, transcripts were upregulated after 24 h of Cry1Ac protoxin exposure. These results suggested that MsT was most likely involved in food protein digestion and molting in M. separata whereas MsCT was most likely involved in food protein digestion and Bacillus thuringiensis (Bt) protoxin activation. RNA interference indicated that MsT and MsCT expression levels decreased 76.7 and 86.2% after treated with MsT and MsCT dsRNA, respectively. This study showed that M. separata expressed midgut proteases in line with known lepidopteran counterparts and contributed valuable sequence resource information regarding insect proteases.
... Our current understanding of Bt toxin resistance in insects is generally associated with either altered binding of toxins to midgut receptors [25][26][27] or differences in post binding proteolytic processing of the toxin by the receptors [28,29]. Decreased binding of the toxins to its midgut receptors in a resistant strain might be due to the structural changes in the receptors or due to decreased expression of these specific receptors in the midgut. ...
Despite a number of recent reports of insect resistance to transgenic crops expressing insecticidal toxins from Bacillus thuringiensis (Bt), little is known about the mechanism of resistance to these toxins. The purpose of this study is to identify genes associated with the mechanism of Cry1F toxin resistance in European corn borer (Ostrinia nubilalis Hübner). For this, we compared the global transcriptomic response of laboratory selected resistant and susceptible O. nubilalis strain to Cry1F toxin. We further identified constitutive transcriptional differences between the two strains.
An O. nubilalis midgut transcriptome of 36,125 transcripts was assembled de novo from 106 million Illumina HiSeq and Roche 454 reads and used as a reference for estimation of differential gene expression analysis. Evaluation of gene expression profiles of midgut tissues from the Cry1F susceptible and resistant strains after toxin exposure identified a suite of genes that responded to the toxin in the susceptible strain (n = 1,654), but almost 20-fold fewer in the resistant strain (n = 84). A total of 5,455 midgut transcripts showed significant constitutive expression differences between Cry1F susceptible and resistant strains. Transcripts coding for previously identified Cry toxin receptors, cadherin and alkaline phosphatase and proteases were also differentially expressed in the midgut of the susceptible and resistant strains.
Our current study provides a valuable resource for further molecular characterization of Bt resistance and insect response to Cry1F toxin in O. nubilalis and other pest species.
... Thus, CCE metabolic resistance to organophosphates involves sequestering instead of hydrolysis of these xenobiotic compounds (for a review see Montella et al., 2012). Recently, phosphotriesterases have been found in some insects and expression studies revealed that this family of enzymes is implicated in insecticide resistance in the tobacco budworm (Zhu et al., 2011). Aryldialkylphosphatase is a triosephosphate isomerase (TIM) barrel metal ion-dependent enzyme that belongs to the amidohydrolase clan (Pfam CL0034). ...
... Specifically, the catalytic activity category in the molecular function domain was represented by 416 DEUs between ACB-BtS and ACB-AbR. Similar dominance of catalytic genes was also observed in the midgut transcriptome of D. saccharalis [54], H. virescens [55] and P. xylostlla [31]. However, the majority of these DEUs (85.8%) were down-regulated in ACB-AbR, unlike the discovery in P. xylostlla, in which majority of the DEUs were up-regulated in Cry1Ac resistant stain [31]. ...
Asian corn borer (ACB), Ostrinia furnacalis (Guenée), is the major insect pest of maize in China and countries of East and Southeast Asia, the Pacific and Australasia. ACB can develop strong resistance to the transgenic Bt maize expressing Cry1Ab, the most widely commercialized Bt maize worldwide. However, the molecular basis for the resistance mechanisms of ACB to Cry1Ab remained unclear. Two biological replicates of the transcriptome of Bt susceptible (ACB-BtS) and Cry1Ab resistant (ACB-AbR) strains of ACB were sequenced using Solexa/Illumina RNA-Seq technology to identify Cry1Ab resistance-relevant genes.
The numbers of unigenes for two biological replications were 63,032 and 53,710 for ACB-BtS and 57,770 and 54,468 for ACB-AbR. There were 35,723 annotated unigenes from ACB reads found by BLAST searching NCBI non-redundant, NCBI non-redundant nucleotide, Swiss-prot protein, Kyoto Encyclopedia of Genes and Genomes, Cluster of Orthologous Groups of proteins, and Gene Ontology databases. Based on the NOISeq method, 3,793 unigenes were judged to be differentially expressed between ACB-BtS and ACB-AbR. Cry1Ab resistance appeared to be associated with change in the transcription level of enzymes involved in growth regulation, detoxification and metabolic/catabolic process. Among previously described Bt toxin receptors, the differentially expressed unigenes associated with aminopeptidase N and chymotrypsin/trypsin were up-regulated in ACB-AbR. Whereas, other putative Cry receptors, cadherin-like protein, alkaline phosphatase, glycolipid, actin, V-type proton ATPase vatalytic, heat shock protein, were under-transcripted. Finally, GPI-anchor biosynthesis was found to be involved in the significantly enriched pathway, and all genes mapped to the pathway were substantially down-regulated in ACB-AbR.
To our knowledge, this is the first comparative transcriptome study to discover candidate genes involved in ACB Bt resistance. This study identified differentially expressed unigenes related to general Bt resistance in ACB. The assembled, annotated transcriptomes provides a valuable genomic resource for further understanding of the molecular basis of ACB Bt resistance mechanisms.
... Meanwhile, however, tissuespecific transcriptome data are providing early insights into an unusual diversity of genes for P450s and esterases in heliothine and spodopteran pests. Sequencing of over 1600 paralogous midgut transcripts in H. virescens identified 20 different P450s and nine esterases, 44 while a similar project on about 3000 unique midgut transcripts from H. armigera identified 40 paralogous esterases alone. 32,45 Trancriptomes of adult antennae (which also appear to include many detoxification genes) likewise reveal large numbers of P450s and esterases, 37 and 30, respectively, in S. littoralis. ...
The widely accepted paradigm for the development of insecticide resistance in field populations of insects is of selection for one or a very few genes of major effect. Limited genetic mapping data for organophosphate and pyrethroid resistance in heliothine and spodopteran pests generally agree with this paradigm. However other biochemical and transcriptomic data suggest a more complex set of changes in multiple P450 and esterase gene/enzyme systems in resistant strains of these species. We discuss possible explanations for this paradox, including the likely embedding of these genes in regulatory cascades and emerging evidence for their arrangement in large clusters of closely related genes. We conclude that there could indeed be an unusually large number of genetic options for evolving resistance in these species.
... Many lepidopteran insects have expansions of genes that encode trypsins and chymotrypsins. Lepidopteran gene expansions have been found in Helicoverpa zea [13], Manduca sexta [18], Choristoneura fumiferana [19], Plodia interpunctella [20], Helicoverpa armigera [21], Agrotis ipsilon [14,22], Sesamia nonagrioides [23], Tineola bisselliella [24], Bombyx mori [25], Spodoptera litura [26], Bombyx mandarina [27], S. frugiperda [28], Mamestra configurata [29], O. nubilalis [30], and Heliothis virescens [31]. Protease gene expansions are proposed to have occurred in response to the evolutionary selection pressure of plant inhibitors [32]. ...
Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and their homologs from the European corn borer (Ostrinia nubilalis) larval gut. Our analyses of the cDNA-deduced amino acid sequences indicated that 12 were putative trypsins, 12 were putative chymotrypsins, and the remaining 10 were trypsin and chymotrypsin homologs that lack one or more conserved residues of typical trypsins and chymotrypsins. Reverse transcription PCR analysis indicated that all genes were highly expressed in gut tissues, but one group of phylogenetically-related trypsin genes, OnTry-G2, was highly expressed in larval foregut and midgut, whereas another group, OnTry-G3, was highly expressed in the midgut and hindgut. Real-time quantitative PCR analysis indicated that several trypsin genes (OnTry5 and OnTry6) were significantly up-regulated in the gut of third-instar larvae after feeding on Cry1Ab protoxin from 2 to 24 h, whereas one trypsin (OnTry2) was down-regulated at all time points. Four chymotrypsin and chymotrypsin homolog genes (OnCTP2, OnCTP5, OnCTP12 and OnCTP13) were up-regulated at least 2-fold in the gut of the larvae after feeding on Cry1Ab protoxin for 24 h. Our data represent the first in-depth study of gut transcripts encoding expanded families of protease genes in O. nubilalis larvae and demonstrate differential expression of protease genes that may be related to Cry1Ab intoxication and/or resistance.
... Transcriptomics is an extremely useful approach for the identification of new genes and gene functions related to insecticide resistance [57]. DNA microarrays, one of the most powerful and versatile transcriptomic techniques, make it possible to compare expression profiles for hundreds or thousands of genes simultaneously, thereby linking the study of static genomes to dynamic proteomes [58]. ...
Background:
Insecticide resistance is one of the best examples of rapid micro-evolution found in nature. Since the development of the first synthetic insecticide in 1939, humans have invested considerable effort to stay ahead of resistance phenotypes that repeatedly develop in insects. Aphids are a group of insects that have become global pests in agriculture and frequently exhibit insecticide resistance. The green peach aphid, Myzus persicae, has developed resistance to at least seventy different synthetic compounds, and different insecticide resistance mechanisms have been reported worldwide.
Methodology/principal findings:
To further characterize this resistance, we analyzed genome-wide transcriptional responses in three genotypes of M. persicae, each exhibiting different resistance mechanisms, in response to an anti-cholinesterase insecticide. The sensitive genotype (exhibiting no resistance mechanism) responded to the insecticide by up-regulating 183 genes primarily ones related to energy metabolism, detoxifying enzymes, proteins of extracellular transport, peptidases and cuticular proteins. The second genotype (resistant through a kdr sodium channel mutation), up-regulated 17 genes coding for detoxifying enzymes, peptidase and cuticular proteins. Finally, a multiply resistant genotype (carrying kdr and a modified acetylcholinesterase), up-regulated only 7 genes, appears not to require induced insecticide detoxification, and instead down-regulated many genes.
Conclusions/significance:
This study suggests strongly that insecticide resistance in M. persicae is more complex that has been described, with the participation of a broad array of resistance mechanisms. The sensitive genotype exhibited the highest transcriptional plasticity, accounting for the wide range of potential adaptations to insecticides that this species can evolve. In contrast, the multiply resistant genotype exhibited a low transcriptional plasticity, even for the expression of genes encoding enzymes involved in insecticide detoxification. Our results emphasize the value of microarray studies to search for regulated genes in insects, but also highlights the many ways those different genotypes can assemble resistant phenotypes depending on the environmental pressure.
... es. Consequently, the transgenic expression of this bacterial enzyme confers OP resistance in both Drosophila melanogaster and Spodoptera frugiperda (Dumas et al. 1990, Benedict et al. 1994). Nevertheless, the extent to which the expression of native insect phosphotriesterase is associated with OP resistance in natural populations is not yet known. Zhu et al. (2011) have found that an enzyme homologous to a salmon phosphotriesterase was present in the transcriptional profile of the tobacco budworm and was implicated in the insecticide resistance of this organism. However, early esterase studies addressed alpha and beta-naphthyl acetate substrates and it is still not clear how many of these isozymes ...
The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.
... mRNA was purified from total RNA using NucleoTrap mRNA purification kit (BD Bioscience Clontech, Palo Alto, CA). The Creator Smart cDNA Library Construction Kit (BD Bioscience Clontech) was used for cDNA library construction, by following manufacturer's instructions and modified protocols described by Zhu et al. [14]. Approximately 1 mg mRNA was used for reverse transcription and cDNA library construction. ...
The tarnished plant bug has become increasingly resistant to organophosphates in recent years. To better understand acephate resistance mechanisms, biological, biochemical, and molecular experiments were systematically conducted with susceptible (LLS) and acephate-selected (LLR) strains. Selection of a field population with acephate significantly increased resistance ratio to 5.9-fold, coupled with a significant increase of esterase activities by 2-fold. Microarray analysis of 6,688 genes revealed 329 up- and 333 down-regulated (≥2-fold) genes in LLR. Six esterase, three P450, and one glutathione S-transferase genes were significantly up-regulated, and no such genes were down-regulated in LLR. All vitellogenin and eggshell protein genes were significantly down-regulated in LLR. Thirteen protease genes were significantly down-regulated and only 3 were up-regulated in LLR. More than twice the number of catalysis genes and more than 3.6-fold of metabolic genes were up-regulated, respectively, as compared to those down-regulated with the same molecular and biological functions. The large portion of metabolic or catalysis genes with significant up-regulations indicated a substantial increase of metabolic detoxification in LLR. Significant increase of acephate resistance, increases of esterase activities and gene expressions, and variable esterase sequences between LLS and LLR consistently demonstrated a major esterase-mediated resistance in LLR, which was functionally provable by abolishing the resistance with esterase inhibitors. In addition, significant elevation of P450 gene expression and reduced susceptibility to imidacloprid in LLR indicated a concurrent resistance risk that may impact other classes of insecticides. This study demonstrated the first association of down-regulation of reproductive- and digestive-related genes with resistance to conventional insecticides, suggesting potential fitness costs associated with resistance development. This study shed new light on the understanding of the molecular basis of insecticide resistance, and the information is highly valuable for development of chemical control guidelines and tactics to minimize resistance and cross-resistance risks.
... In most insects, food digestion largely occurs in the alimentary canal, in which most of the enzymes are produced and secreted, including protease, lipases, carboxylases, amylase, invertases, and maltases. Insect gut also produces a variety of detoxification enzymes which play important roles in adapting to an environment altered by endo and exogenic compounds (Zhu et al., 2011). There is, however, relatively little known about the factors controlling the release of digestive enzymes in insects. ...
Rhynocoris marginatus is a predominant and potential reduviid predator of many economically important pests in India. The venomous saliva (VS) was collected by milking method and diluted with HPLC grade water to prepare different concentrations (200, 400, 600, 800 and 1000ppm). The VS from R. marginatus was found to be toxic and the LD(50) of the VS in Spodoptera litura third instar were 768 and 929ppm at 48 and 96h for microinjection and oral toxicity studies, respectively. Level of hydrolase and detoxification enzymes significantly decreased in a dose-dependent manner after treating the host with VS for 96h. A decrease in carbohydrate (21%) and lipid (46%) contents and an increase in the protein content (50%) were prominent in the experimental category. The VS reduced the relative growth rate, approximate digestibility, efficiency of conversion of ingested and digested food of S. litura in the oral toxicity study. Salivary venom inhibits the haemocytes from aggregation and affects spreading behavior of haemocytes separated from the fifth stadium larvae of S. litura. The result showed that VS toxins caused mortality, changed the nutritional indices, and altered the levels of macromolecule quantity and digestive enzymes of S. litura. We concluded that the VS of R. marginatus is venomous to a prey species, S. litura.
Synthetic insecticides are the primary vector control method used globally. However, the widespread use of insecticides is a major cause of insecticide-resistance in mosquitoes. Hence, this study aimed at elucidating permethrin and temephos-resistant protein expression profiles in Ae. aegypti using quantitative proteomics. In this study, we evaluated the susceptibility of Ae. aegypti from Penang Island dengue hotspot and non-hotspot against 0.75% permethrin and 31.25 mg/l temephos using WHO bioassay method. Protein extracts from the mosquitoes were then analysed using LC-ESI-MS/MS for protein identification and quantification via label-free quantitative proteomics (LFQ). Next, Perseus 1.6.14.0 statistical software was used to perform differential protein expression analysis using ANOVA and Student's t-test. The t-test selected proteins with≥2.0-fold change (FC) and ≥2 unique peptides for gene expression validation via qPCR. Finally, STRING software was used for functional ontology enrichment and protein-protein interactions (PPI). The WHO bioassay showed resistance with 28% and 53% mortalities in adult mosquitoes exposed to permethrin from the hotspot and non-hotspot areas. Meanwhile, the susceptibility of Ae. aegypti larvae revealed high resistance to temephos in hotspot and non-hotspot regions with 80% and 91% mortalities. The LFQ analyses revealed 501 and 557 (q-value
Synthetic pesticides are extensively and injudiciously applied to control agriculture and household pests worldwide. Due to their high use, their toxic residues have enormously increased in the agroecosystem in the past several years. They have caused many severe threats to non-target organisms, including humans. Therefore, the complete removal of toxic compounds is gaining wide attention to protect the ecosystem and the diversity of living organisms. Several methods, such as physical, chemical and biological, are applied to degrade compounds, but as compared to other methods, biological methods are considered more efficient, fast, eco-friendly and less expensive. In particular, employing microbial species and their purified enzymes makes the degradation of toxic pollutants more accessible and converts them into non-toxic products by several metabolic pathways. The digestive tract of insects is usually known as a superior organ that provides a nutrient-rich environment to hundreds of microbial species that perform a pivotal role in various physiological and ecological functions. There is a direct relationship between pesticides and insect pests: pesticides reduce the growth of insect species and alter the phyla located in the gut microbiome. In comparison, the insect gut microbiota tries to degrade toxic compounds by changing their toxicity, increasing the production and regulation of a diverse range of enzymes. These enzymes breakdown into their derivatives, and microbial species utilize them as a sole source of carbon, sulfur and energy. The resistance of pesticides (carbamates, pyrethroids, organophosphates, organochlorines, and neonicotinoids) in insect species is developed by metabolic mechanisms, regulation of enzymes and the expression of various microbial detoxifying genes in insect guts. This review summarizes the toxic effects of agrochemicals on humans, animals, birds and beneficial arthropods. It explores the preferential role of insect gut microbial species in the degradation process and the resistance mechanism of several pesticides in insect species. Additionally, various metabolic pathways have been systematically discussed to better understand the degradation of xenobiotics by insect gut microbial species.
Combining the entomopathogenic nematodes (EPNs), Heterorhabditis beicherriana LF strain and Bacillus thuringiensis (Bt) HBF-18 strain is a practical strategy to manage the larvae of Holotrichia parallela Motschulsky. However, the intrinsic resistance mechanism between H. parallela larvae and the EPN-Bt combination is unknown. Herein, antioxidant enzymes [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] and detoxifying enzymes [carboxylesterase (CarE), glutathione S-transferase (GST), and acetylcholinesterase (AChE)] of H. parallela larvae showed an activation-inhibition trend throughout the EPN-Bt exposure time course. Eight potentially key antioxidant and detoxifying enzyme genes in response to EPN-Bt infection were identified from the midgut of H. parallela larvae through RNA-seq. Spatiotemporal analysis showed they were ubiquitously expressed in all development stages and tissues. After silencing CAT, CarE, and GST1, the enzyme activities were significantly decreased by 30.29%, 68.80%, and 34.63%, respectively. Meanwhile, the mortality of grubs was increased by 18.40%, 46.30%, and 42.59% after exposure to EPN-Bt for 1 day. The expression level change trends of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), cap ‘n’ collar isoform-C (CncC), kelch-like ECH-associated protein 1 (Keap1), and CarE were consistent when exposed to EPN-Bt. Furtherly, RNAi-mediated PI3K silencing showed a similar downregulated trend between PI3K/Akt/CncC and CarE. Furthermore, silencing PI3K rendered grubs more susceptible to EPN-Bt and accelerated symbiotic bacteria multiplication in grubs. Overall, these results suggest that PI3K/Akt/CncC pathway mediates the expression of CarE and participates in the resistance of H. parallela larvae to EPN-Bt infection.
Pink bollworm (PBW), Pectinophora gossypiella, is one of the most destructive insect
pests of cotton worldwide. In India, PBW has developed widespread resistance to
the Cry1Ac and Cry2Ab toxins of Bt Bollgard II cotton. To understand the molecular mechanism of Bt resistance, the midgut transcriptome of an Indian strain of Btsusceptible (PBW-BtS) and Bt-resistant (PBW-BtR) PBW larvae were sequenced
using Illumina platform to identify the genes and pathways associated with Bt resistance. A total of 31,764 unique unigenes were assembled from all the four samples,
and 1,741 unigenes were found to be differentially expressed between PBW-BtS and
PBW-BtR, out of which 1,024 unigenes were down-regulated and 717 were up-regulated. Down-regulated genes associated with already known Bt resistance genes in
different organisms included APN, ABCA, ABCG8 and cadherin. On the other hand,
unigenes related to metabolic resistance such as cytochrome P450, GST and carboxylesterase were up-regulated in PBW-BtR when compared with PBW-BtS. Moreover,
real-time quantitative results of randomly selected genes confirmed the transcriptome response. This is the first comparative transcriptome study to identify the candidate genes involved in Bt BG II resistance in pink bollworm. We believe that the
assembled and annotated transcriptome will serve as a valuable resource for future
studies to discern the molecular basis of PBW Bollgard II resistance
Bombyx mori is an important economic insect. However, the environmental pollution caused by the widespread use of neonicotinoid insecticides has significantly affected the safe production of sericulture. In this paper, we determined the LC50 of acetamiprid, a kind of neonicotinoid insecticides, to 5th instar silkworm larvae, examined its residues in hemolymph and midgut of silkworm after continuous exposure to low-dose of acetamiprid, and investigated the transcription level of detoxifying-related genes and the activity of detoxifying enzymes. The results showed that acetamiprid was highly toxic (24-h LC50, 1.50 mg/L) to silkworm larvae. After continuous exposure to low-dose of acetamiprid (0.15 mg/L), the acetamiprid residue concentrations in hemolymph and midgut were 0.90 and 0.58 μg/mg, respectively, at 48 h, but all decreased at 96 h. At 24 h of acetamiprid exposure, the transcription levels of CYP4M5 and CYP6AB4 and the P450 enzyme activity were significantly enhanced. However, the transcription levels of CarE and CarE-11 and the activity of CarE enzymes were both inhibited by acetamiprid exposure. After 24 h–72 h of acetamiprid exposure, the transcription levels of GSTe3 and GSTd1 were significantly up-regulated, and the GST enzyme activity was also significantly elevated from 48 h to 96 h. Furthermore, the expression levels of FoxO, CncC and Keap1, the key upstream genes of detoxification enzymes, showed a similar trend as the GST genes. These results indicated that acetamiprid was reduced in midgut and the expression of GSTs was upregulated may via FoxO/CncC/Keap1 signaling pathway, which plays a key role in detoxification responses.
Chemical spray on cotton is almost an exclusive method for control of tarnished plant bug (TPB, Lygus lineolaris). Frequent use of imidacloprid is a concern for neonicotinoid resistance in this key pest. Information of how and why TPB become less susceptible to imidacloprid is essential for effective monitoring and managing resistance.
Microarray analysis of 6,688 genes in imidacloprid-selected TPB (Im1500FF) revealed 955 up- and 1277 down-regulated (≥2-fold) genes in Im1500FF with 369 and 485 of them annotated. Five P450 and 9 esterase genes were significantly up-regulated, and only one esterase gene and no P450 genes were down-regulated. Other up-regulated genes include helicases, phosphodiesterases, ATPases, and kinases. Pathway analyses identified 65 up-regulated cDNAs, that encode 51 different enzymes involved in 62 different pathways, including P450 and esterase genes for drug and xenobiotic metabolisms. Sixty-four down-regulated cDNAs code only 17 enzymes that are associated with only 23 pathways mostly related to food digestions.
This study demonstrated a significant change of gene expression related to metabolic processes in imidacloprid-selected TPB, resulting in over-expression of P450 and esterase genes for potential excess detoxification and cross/multiple resistance development. The identification of these and other enzyme genes establishes a foundation to explore the complicity of potential imidacloprid resistance in TPB.
Extensive adoption of transgenic Bt corn in recent years for stalk borer control has increased risk of resistance evolution in the target pest populations. A Bt-resistant strain of the sugarcane borer, Diatraea saccharalis, was approximately 100-fold more tolerant to Cry1Ab toxin than the susceptible counterpart. To gain a better understanding of the molecular mechanisms of Bt resistance, the Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains of D. saccharalis were subjected to a microarray analysis.
Results showed that the expression levels of many genes were significantly different between the Cry1Ab-RR and Cry1Ab-SS strains. Microarray analysis of 7145 cDNAs revealed 384 differentially expressed genes. A total of 273 genes were significantly upregulated 2-51.6-fold, and 111 genes were significantly downregulated 2-22.6-fold in the Cry1Ab-RR strain. The upregulation of three potential resistance-related genes, coding for a glutathione S-transferase (GST), a chymotrypsin-like protease (CHY) and a lipase (LP), was confirmed using real-time PCR, indicating a reproducibility of the microarray data. Ontology analysis revealed that more than twice the number of metabolic-related genes were upregulated compared with downregulated genes with the same biological function. Up to 35.2% of the upregulated genes in the resistant strain were associated with catalytic activity, while only 9.5% of the downregulated genes were related to the same catalytic molecular function.
The large portion of metabolic- or catalytic-related genes with significant upregulations indicated a potential large increase in metabolic or catalytic activities in the Cry1Ab-RR strain. This cDNA microarray gene expression data could be used to characterize and identify new genes that may be associated with Bt resistance in D. saccharalis.
Insecticides derived from Bacillus thuringiensis have become important for pest management, but recently resistance has been reported from field populations of diamondback moth Plutella xylostella, and laboratory populations of a number of species of Lepidoptera, Coleoptera and Diptera have shown similar characteristics. In this context, the author examines laboratory selection for resistance, resistance risk assessment, variation among conspecific populations, mechanisms, cross-resistance, genetics, stability, fitness costs, and management. -P.J.Jarvis
This book contains 6 chapters focusing on the following topics: analysis of global pesticide resistance in arthropods; documentation of pesticide resistance in arthropods; the biochemical and molecular genetic basis of resistance to pesticides in arthropods; assessing the risk of the evolution of resistance to pesticides using spatially complex simulation models; pesticide and transgenic plant resistance management in the field; and the politics of resistance management.
Using quantitative polymerase chain reaction (QPCR), the relative transcriptional levels of cytochrome c oxidase subunit 3 (CO3) were studied in Aedes aegypti in response to treatment with acetone, permethrin, and fipronil. The transcriptional levels of CO3 were significantly (P < 0.05) higher in acetone-treated Ae. aegypti compared with that in untreated samples. Using ribosomal L24, heat shock protein (HSP), and actin as reference genes, relative transcription levels of CO3 in acetone-treated Ae. aegypti were 2.88 +/- 0.38-, 2.60 +/- 0.60-, and 3.24 +/- 0.70-fold higher, respectively, compared with that in untreated mosquitoes. Transcriptional levels of CO3 were induced significantly higher (6.54 +/- 1.22-, 4.62 +/- 0.74-, and 9.47 +/- 3.71-fold, respectively) by permethrin at LD10 compared with acetone (P < 0.05). Taken together, our results suggest that overexpression of CO3 is tightly regulated in Ae. aegypti in response to xenobiotic treatment.
Bacillus thuringiensis subsp. kurstaki HD-73 produces a crystal protein which is lethal to many lepidopteran larvae. The gene encoding this crystal protein has been isolated from a 75-kb plasmid and engineered into a recombinant Escherichia coli plasmid for analysis. The complete nucleotide sequences of the coding region and 387-bp 5' and 376-bp 3' to the coding region have been determined. The 3537-bp of the coding region specify a protein of Mr 133 330. The full-length gene and several 3' -truncated derivatives of the gene were examined in both E. coli and in an E. coli minicell-expression system to determine if the carboxy end of the protein is essential for toxicity. The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product.
Two Bacillus thuringiensis (Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs.
Bacillus thuringiensis (Bt) is a valuable source of insecticidal proteins for use in conventional sprayable formulations and in transgenic crops, and it is the most promising alternative to synthetic insecticides. However, evolution of resistance in insect populations is a serious threat to this technology. So far, only one insect species has evolved significant levels of resistance in the field, but laboratory selection experiments have shown the high potential of other species to evolve resistance against Bt. We have reviewed the current knowledge on the biochemical mechanisms and genetics of resistance to Bt products and insecticidal crystal proteins. The understanding of the biochemical and genetic basis of resistance to Bt can help design appropriate management tactics to delay or reduce the evolution of resistance in insect populations.
This review examines potential impacts of transgenic cultivars on insect population dynamics and evolution. Experience with classically bred, insecticidal cultivars has demonstrated that a solid understanding of both the target insect's ecology and the cultivar's performance under varied field conditions will be essential for predicting area-wide effects of transgenic cultivars on pest and natural enemy dynamics. This experience has also demonstrated the evolutionary capacity of pests for adaptive response to insecticidal traits in crops. Biochemical and genetic studies of insect adaptation to the Bacillus thuringiensis (Bt) toxins expressed by currently marketed transgenic cultivars indicate a high risk for rapid adaptation if these cultivars are misused. Theoretical and practical issues involved in implementing strategies to delay pest adaptation to insecticidal cultivars are reviewed. Emphasis is placed on examining the "high dose"/refuge strategy that has become the goal of industry and regulatory authorities.
Transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) were first commercialized in 1996 amid concern from some scientists, regulators and environmentalists that the widespread use of Bt crops would inevitably lead to resistance and the loss of a 'public good,' specifically, the susceptibility of insect pests to Bt proteins. Eight years later, Bt corn and cotton have been grown on a cumulative area >80 million ha worldwide. Despite dire predictions to the contrary, resistance to a Bt crop has yet to be documented, suggesting that resistance management strategies have been effective thus far. However, current strategies to delay resistance remain far from ideal. Eight years without resistance provides a timely opportunity for researchers, regulators and industry to reassess the risk of resistance and the most effective strategies to preserve Bt and other novel insect-resistant crops in development.
Glutathione-dependent degradation of parathion was studied in six strains of houseflies to find out whether it might be important as a cause of resistance. When supernatant fractions of high-speed centrifuged homogenates were fortified with glutathione and incubated with parathion, water-soluble products were formed. The rate of parathion detoxication was highest in a malathion-resistant strain (c. 4 μg parathion degraded per abdomen per hour), lowest in a susceptible strain, and intermediate in some other organophosphate-resistant strains. In one of the latter strains, E1, the gene for glutathione-dependent degradation is located on the second chromosome, closely linked with gene cm+. This is the same chromosome on which gene a for low ali-esterase activity and hydrolytic detoxication of paraoxon is located. It is not likely that the gene for glutathione-dependent degradation is identical with gene a, since it is also present in strain Nie which lacks gene a, and, therefore, the presence of a separate gene which is called gene g is postulated.Since the malathion-resistant strain was only 4-fold resistant to parathion, the glutathione-dependent degradation seems to confer only little resistance, at least to this insecticide. In three of the strains the products were identified. Three labeled products were formed from ethyl-labeled parathion: ethylglutathione, diethylphos-phorothionic acid, and desethylparathion.
Heliothis virescens (F.), tobacco budworms eggs were collected from 3 adjacent counties in North Carolina. A laboratory strain (YDK) was established from these eggs using precautions to avoid loss of genetic diversity. A subset of this laboratory strain (YHD2) was selected on artificial diet containing the Bacillus thuringiensis (Berliner) toxin, CryIA(c). In the, first 12 episodes of selection only moderate resistance (≍7- to 8-fold) was found. However, after 19 episodes of selection the strain had developed >500-fold resistance to the CryIA(c) toxin. Further selection lead to higher levels of resistance with the greatest resistance ratio recorded being ≍10,000-fold. The YHD2 strain was cross-resistant to CryIA(a), CryIA(b) and CryIF. We also found some resistance to CryIB, CryIC and CryIA, but the level of resistance to these toxins was more moderate. Reciprocal genetic crosses between the resistant and control strains indicated that resistance to CryIA(c) and to CryIA(b) was partially recessive, but that the low level of resistance to CryIA was more dominant. Progeny from backcrosses of F1 larvae to the resistant parent were placed on artificial diet containing a concentration of CryIA(b) that had previously been found to slow the growth of F1 larvae. When these larvae were weighed after 10 d, two clearly demarcated size classes were found in ≍1:1 ratios as is expected in backcrosses when a single locus (or a set of tightly linked loci) is coding for a major component of a recessive trait. Adults that developed from the larger size class of larvae were mated and their offspring inherited the ability to grow well on CryIA(b). As expected from the single locus model, one quarter of the offspring from matings of the small backcross larvae grew well on CryIA(b). Results of this selection experiment indicate that the initial frequency of this resistance trait could be approximately 10-3, but field tests will be needed to confirm this rough estimate.
A resistant strain of Phytoseiulus persimilis selected by methidathion pressure for several years metabolizes the [14C]methidathion faster than does the corresponding susceptible strain. The metabolism is for the main part glutathione dependent and gives the methidathion conjugate on glutathione as a first metabolite: S[5-methoxy-2-oxo-1,3,4-thiadiazol-3(2H)-yl]-l-glutathione. In addition, glutathione transferase with chlorodinitrobenzene as a substrate has a threefold lower Km in R strain than in S strain. Furthermore, this reaction is competitively inhibited by methidathion with a Ki which is threefold lower in R than in S strain. These results indicated that in this strain of P. persimilis resistance is due to an elevated detoxication of methidathion by a glutathione transferase. Other parameters known to be able to induce resistance in arthropods have been compared in resistant and sensitive strains. Esterase and monooxygenase activity measured with chromogenic substrates are the same in the two strains as is the level of acetylcholinesterase and its inhibition by methidathion oxon. No difference between the two strains has been found in the penetration kinetics measured with [14C]methidathion. These results indicated that glutathione transferase is the only mechanism which has been selected in P. persimilis, although other mechanisms are known to be involved in resistance to other insecticides in phytoseiid mites.
Kinetic parameters were measured for glutathione S-transferase, an enzyme important in metabolic resistance to insecticides, in one susceptible and two insecticide-resistant strains of the house fly (Musca domestica L.), and in untreated and chemically induced flies. Both resistant strains differed from the susceptible strain in apparent Km values for the enzyme, while only one differed in apparent Vmax. Two of the strains were inducible with phenobarbital; the third with 3-methylcholanthrene. Kinetic analysis indicated enzyme induction was associated with changes in Km rather than Vmax, and genetic experiments showed that most variation relating to Km and Vmax was controlled by chromosome II. Based on these results, both metabolic resistance and induction of enzyme activity were associated primarily with the production of different forms of glutathione S-transferase rather than more of the enzyme present in susceptible flies.
Potential resistance development to Bt cotton in certain lepidopterans has prompted research to develop strategies that will preserve this environmental-friendly biotechnology. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against lepidopteran pests and for expanding the spectrum of control for other insects. Interactions of Bt toxin from Bacillus thuringiensis and proteinase inhibitors were investigated by monitoring growth, development, and gut proteinase activities of the bollworm, Helicoverpa zea. Several proteinase inhibitors were combined with Bt protoxin Cry1Ac in artificial diet and fed to newly molted 3rd-instar bollworm larvae to determine effects on larval body weight and length, pupation progress, and mortality rate. Major midgut proteinase activities, including caseinase, tryptic, and chymotrypsin activities, were examined after treatment. A concentration of Bt at a level causing minimal mortality (<10%), was mixed with the following proteinase inhibitors: benzamidine, phenylmethylsulfonyl fluoride (PMSF), and N-α-tosyl-l-lysine chloromethyl ketone (TLCK). When compared with controls, the synergistic effect of Bt toxin and proteinase inhibitors caused significant decreases in mean larval weight and length over time. Midgut samples tested against the substrates azocasein, α-benzoyl-dl-arginine-p-nitroanilide (BApNA), and N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPFpNA) showed significant decreases in the protease activity of larvae fed Bt plus inhibitor versus control. Interaction of Bt and proteinase inhibitors significantly retarded larval growth and resulted in developmental delay and up to 20% mortality.
The metabolic enzymes of a multiresistant (i.e., carbamate, organophosphate, pyrethroid) population of tobacco budworm, Heliothis virescens (F.), were compared with those of a susceptible population. The resistant Hebert population, collected from areas where control failures with cypermethrin and thiodicarb had been reported, was selected in the laboratory with thiodicarb for seven generations. Resistance ratios as determined by topical bioassays were 91- and >150-fold to cypermethrin and thiodicarb, respectively. Resistance-associated increases in metabolism were found in fifth instar larvae for monooxygenases, carboxylester hydrolases, and glutathione transferases. Cytochrome P450 content was elevated in microsomes from gut (3.7-fold), fat body (4.4-fold), and carcass (4-fold). Monooxygenase activity varied substantially among the four substrates and three tissue sources examined. For three monooxygenase substrates (p-nitroanisole, benzo(a)pyrene, and benzphetamine) increases in metabolism varied from 3- to 33-fold. The greatest increases in metabolism were observed with methoxyresorufin, in which increases were observed in all three tissues (23-, 29-, and 58-fold in fat body, midgut, and carcass microsomes, respectively). Significant increases ranging from 3- to 5-fold were observed for two esterase substrates, while smaller differences of up to 2-fold were observed for a glutathione transferase substrate. The high metabolic activity of the resistant population suggested that homogenates of individual larvae might be used in resistance monitoring. The development of a monooxygenase-based microtiter plate assay using p-nitroanisole as substrate clearly discriminates between resistant and susceptible individuals in the third instar, suggesting that biochemical resistance monitoring can be carried out with field-collected individuals. The esterase substrate p-nitrophenyl acetate also may be a useful tool for resistance monitoring.
Bacterial glutathione transferases (GSTs) are part of a superfamily of enzymes that play a key role in cellular detoxification. GSTs are widely distributed in prokaryotes and are grouped into several classes. Bacterial GSTs are implicated in a variety of distinct processes such as the biodegradation of xenobiotics, protection against chemical and oxidative stresses and antimicrobial drug resistance. In addition to their role in detoxification, bacterial GSTs are also involved in a variety of distinct metabolic processes such as the biotransformation of dichloromethane, the degradation of lignin and atrazine, and the reductive dechlorination of pentachlorophenol. This review article summarizes the current status of knowledge regarding the functional and structural properties of bacterial GSTs.
Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E-P). The enzyme also catalyzes phosphoryl transfer reaction to various alcohols. Escherichia coli AP is a homodimer with 449 residues per monomer. It is a metalloenzyme with two Zn2+ and one Mg2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (Pi), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2.0 A resolution. Some parts of the molecular have been retraced, starting from the previous 2.8 A study. The active site has been modified substantially and is described in this paper. The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made. Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2.5 A resolution, and the phosphate-free native enzyme at 2.8 A resolution. At pH 7.5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E-P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E.P) form. Implication of these results for the catalytic mechanism of the enzyme is discussed. APs from other sources are believed to function in a similar manner.
Increased expression of the insect control protein genes of Bacillus thuringiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modified (3% nucleotide difference) and to fully modified (21% nucleotide difference) cryIA(b) and cryIA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified cryIA(b) gene had a 10-fold higher level of insect control protein and plants with the fully modified cryIA(b) had a 100-fold higher level of CryIA(b) protein compared with the wild-type gene. Similar results were obtained with the fully modified cryIA(c) gene in plants. Specific sequences of the partially modified cryIA(b) gene were analyzed for their ability to affect cryIA(b) gene expression in tobacco. The DNA sequence of a single region was identified as important to the improvement of plant expression of the cryIA(b) gene. The increased levels of cryIA(b) mRNA were not directly proportional to the increased levels of CryIA(b) protein in plants transformed with the modified cryIA(b) genes, indicating that the nucleotide sequence of these genes had an effect in improving their translational efficiency in plants.
Introduction Glyoxalase I Glyoxalase II Formaldehyde Dehydrogenase Glutathione S-Transferases Maleyl Isomerases Glutathione Peroxidase Glutathione Reductase γ-Glutamyl Transpeptidase
A trypsin-like enzyme purified from spruce budworm (Choristoneura fumiferana) gut juice has a molecular mass of 25 kDa and its pH activity profile indicates a pKa of 8. Sequence homology with bovine trypsin of the N-terminus and active site, and the ionization dependence for catalysis, reflect the typical trypsin-like activities measured. The action of this enzyme (designated CFT-1) is compared to the neat gut juice with regard to the proteolytic activation of the delta-endotoxin from Bacillus thuringiensis.
The P450 enzymes (mixed function oxidases, cytochrome P450 monooxygenases), a diverse class of enzymes found in virtually all insect tissues, fulfill many important tasks, from the synthesis and degradation of ecdysteroids and juvenile hormones to the metabolism of foreign chemicals of natural or synthetic origin. This diversity in function is achieved by a diversity in structure, as insect genomes probably carry about 100 P450 genes, sometimes arranged in clusters, and each coding for a different P450 enzyme. Both microsomal and mitochondrial P450s are present in insects and are best studied by heterologous expression of their cDNA and reconstitution of purified enzymes. P450 genes are under complex regulation, with induction playing a central role in the adaptation to plant chemicals and regulatory mutations playing a central role in insecticide resistance. Polymorphisms in induction or constitutive expression allow insects to scan their P450 gene repertoire for the appropriate response to chemical insults, and these evolutionary pressures in turn maintain P450 diversity.
Cry toxins form lytic pores in the insect midgut cells. The role of receptor interaction in the process of protoxin activation was analyzed. Incubation of Cry1Ab protoxin with a single chain antibody that mimics the cadherin-like receptor and treatment with Manduca sexta midgut juice or trypsin, resulted in toxin preparations with high pore-forming activity in vitro. This activity correlates with the formation of a 250 kDa oligomer that lacks the helix alpha-1 of domain I. The oligomer, in contrast with the 60 kDa monomer, was capable of membrane insertion as judged by 8-anilino-1-naphthalenesulfonate binding. Cry1Ab protoxin was also activated to a 250 kDa oligomer by incubation with brush border membrane vesicles, presumably by the action of a membrane-associated protease. Finally, a model where receptor binding allows the efficient cleavage of alpha-1 and formation of a pre-pore oligomeric structure that is efficient in pore formation, is presented.
Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gomez, I., Sanchez, J., Miranda, R., Bravo A., Soberon, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.
Many pest insect species are effectively controlled by Bacillus thuringiensis (Bt) Cry toxins delivered in plants and biopesticides. Since the insect midgut epithelium contains receptors and other molecules that determine Bt toxicity, characterization of these molecules is necessary for sustained usage of Bt toxins. Studies of Bt susceptible and resistant strains of Heliothis virescens have provided insights into resistance mechanisms and toxin receptors. For example, the first gene identified as involved in high levels of Cry1Ac resistance in H. virescens encodes a cadherin-like protein, a functional Cry1A receptor in Lepidoptera. This manuscript discusses the most updated information on the mode of action of Cry1A toxins obtained from the characterization of resistant mechanisms in H. virescens strains. Our studies are focused on biochemical and molecular comparison of a susceptible and three resistant H. virescens strains to identify alterations that correlate with toxin resistance. Following this approach we have been able to identify an alkaline phosphatase (HvALP) as a potential receptor and tested the utility of this protein as a marker for resistance to Cry1Ac. Comparison of brush border proteomes from susceptible and resistant larvae has allowed us to identify additional molecules directly involved in the toxicity process.
Xenobiotic resistance in insects has evolved predominantly by increasing the metabolic capability of detoxificative systems and/or reducing xenobiotic target site sensitivity. In contrast to the limited range of nucleotide changes that lead to target site insensitivity, many molecular mechanisms lead to enhancements in xenobiotic metabolism. The genomic changes that lead to amplification, overexpression, and coding sequence variation in the three major groups of genes encoding metabolic enzymes, i.e., cytochrome P450 monooxygenases (P450s), esterases, and glutathione-S-transferases (GSTs), are the focus of this review. A substantial number of the adaptive genomic changes associated with insecticide resistance that have been characterized to date are transposon mediated. Several lines of evidence suggest that P450 genes involved in insecticide resistance, and perhaps insecticide detoxification genes in general, may share an evolutionary association with genes involved in allelochemical metabolism. Differences in the selective regime imposed by allelochemicals and insecticides may account for the relative importance of regulatory or structural mutations in conferring resistance.
Voltage-gated sodium channels are essential for the generation and propagation of action potentials (i.e., electrical impulses) in excitable cells. Although most of our knowledge about sodium channels is derived from decades of studies of mammalian isoforms, research on insect sodium channels is revealing both common and unique aspects of sodium channel biology. In particular, our understanding of the molecular dynamics and pharmacology of insect sodium channels has advanced greatly in recent years, thanks to successful functional expression of insect sodium channels in Xenopus oocytes and intensive efforts to elucidate the molecular basis of insect resistance to insecticides that target sodium channels. In this review, I discuss recent literature on insect sodium channels with emphases on the prominent role of alternative splicing and RNA editing in the generation of functionally diverse sodium channels in insects and the current understanding of the interactions between insect sodium channels and insecticides.
Insect resistance management in GM crops: past, present, and future
- S L Bates
- J Z Zhao
- R T Roush
- A M Shelton
Bates, S.L., Zhao, J.Z., Roush, R.T., Shelton, A.M., 2005. Insect resistance management
in GM crops: past, present, and future. Nat. Biotech. 23, 57-62.
Mechanism of action of glutathione-dependent enzymes
- K T Douglas
Douglas, K.T., 1987. Mechanism of action of glutathione-dependent enzymes. Adv.
Enzymol. Relat. Areas Molec. Bio. 59, 103-167.