Rapid Adaptation and Interspecific Introgression in the North American Crop Pest Helicoverpa zea.

Insect crop pests threaten global food security. This threat is amplified through the spread of nonnative species and through adaptation of native pests to control measures. Adaptations such as pesticide resistance can result from selection on variation within a population, or through gene flow from another population. We investigate these processes in an economically important noctuid crop pest, Helicoverpa zea, which has evolved resistance to a wide range of pesticides. Its sister species Helicoverpa armigera, first detected as an invasive species in Brazil in 2013, introduced the pyrethroid-resistance gene CYP337B3 to South American H. zea via adaptive introgression. To understand whether this could contribute to pesticide resistance in North America, we sequenced 237 H. zea genomes across 10 sample sites. We report H. armigera introgression into the North American H. zea population. Two individuals sampled in Texas in 2019 carry H. armigera haplotypes in a 4 Mbp region containing CYP337B3. Next, we identify signatures of selection in the panmictic population of nonadmixed H. zea, identifying a selective sweep at a second cytochrome P450 gene: CYP333B3. We estimate that its derived allele conferred a ∼5% fitness advantage and show that this estimate explains independently observed rare nonsynonymous CYP333B3 mutations approaching fixation over a ∼20-year period. We also detect putative signatures of selection at a kinesin gene associated with Bt resistance. Overall, we document two mechanisms of rapid adaptation: the introduction of fitness-enhancing alleles through interspecific introgression, and selection on intraspecific variation.

The novel function of an orphan pheromone receptor reveals the sensory specializations of two potential distinct types of sex pheromones in noctuid moth.

Sex pheromones play crucial role in mating behavior of moths, involving intricate recognition mechanisms. While insect chemical biology has extensively studied type I pheromones, type II pheromones remain largely unexplored. This study focused on Helicoverpa armigera, a representative species of noctuid moth, aiming to reassess its sex pheromone composition. Our research unveiled two previously unidentified candidate type II sex pheromones-3Z,6Z,9Z-21:H and 3Z,6Z,9Z-23:H-in H. armigera. Furthermore, we identified HarmOR11 as an orphan pheromone receptor of 3Z,6Z,9Z-21:H. Through AlphaFold2 structural prediction, molecular docking, and molecular dynamics simulations, we elucidated the structural basis and key residues governing the sensory nuances of both type I and type II pheromone receptors, particularly HarmOR11 and HarmOR13. This study not only reveals the presence and recognition of candidate type II pheromones in a noctuid moth, but also establishes a comprehensive structural framework for PRs, contributing to the understanding of connections between evolutionary adaptations and the emergence of new pheromone types.

FoxO induces pupal diapause by decreasing TGFß signaling.

Diapause is a form of dormancy used widely by insects to survive adverse seasons. Previous studies have demonstrated that forkhead box O (FoxO) is activated during pupal diapause initiation in the moth Helicoverpa armigera. However, it is unclear how FoxO induces diapause. Here, we show that knockout of FoxO causes H. armigera diapause-destined pupae to channel into nondiapause, indicating that FoxO is a master regulator that induces insect diapause. FoxO activates the ubiquitin-proteasome system (UPS) by promoting ubiquitin c (Ubc) expression via directly binding to the Ubc promoter. Activated UPS decreases transforming growth factor beta (TGFß) receptor signaling via ubiquitination to block developmental signaling to induce diapause. This study significantly advances the understanding of insect diapause by uncovering the detailed molecular mechanism of FoxO.

Activation of protein arginine methyltransferase 1 and subsequent extension of moth lifespan is effected by the ROS/JNK/CREB signaling axis.

Previous studies have demonstrated that high physiological levels of reactive oxygen species induce pupal diapause and extend lifespan in the moth Helicoverpa armigera. This has been shown to occur via protein arginine methyltransferase 1 (PRMT1) blockade of Akt-mediated phosphorylation of the transcription factor FoxO, after which activated FoxO promotes the initiation of diapause. However, it is unclear how PRMT1 is activated upstream of FoxO activity. Here, we show that high reactive oxygen species levels in the brains of H. armigera diapause-destined pupae activate the expression of c-Jun N-terminal kinase, which subsequently activates the transcription factor cAMP-response element binding protein. We show that cAMP-response element binding protein then directly binds to the PRMT1 promoter and upregulates its expression to prevent Akt-mediated FoxO phosphorylation and downstream FoxO nuclear localization. This novel finding that c-Jun N-terminal kinase promotes FoxO nuclear localization in a PRMT1-dependent manner to regulate pupal diapause reveals a complex regulatory mechanism in extending the healthspan of H. armigera.

Non-CG DNA methylation marks the transition from pupa to adult in Helicoverpa armigera.

DNA methylation in insects is generally low in abundance, and its role is not well understood. It is often localised in protein coding regions and associated with the expression of 'housekeeping' genes. Few studies have explored DNA methylation dynamics during lifecycle stage transitions in holometabolous (metamorphosing) insects. Using targeted mass spectrometry, we have found a significant difference in global DNA methylation levels between larvae, pupae and adults of Helicoverpa armigera (Lepidoptera: Noctuidae) Hübner, a polyphagous pest of agricultural importance. Whole-genome bisulfite sequencing confirmed these observations and pointed to non-CG context being the primary explanation for the difference observed between pupa and adult. Non-CG methylation was enriched in genes specific to various signalling pathways (Hippo signalling, Hedgehog signalling and mitogen-activated protein kinase (MAPK) signalling) and ATP-dependent chromatin remodelling. Understanding the function of this epigenetic mark could be a target in future studies focusing on integrated pest management.

Earthworms Enhance Crop Resistance to Insects Under Microplastic Stress by Mobilizing Physical and Chemical Defenses.

To assess the ecological risk of microplastics (MPs) in agricultural systems, it is critical to simultaneously focus on MP-mediated single-organism response and different trophic-level organism interaction. Herein, we placed earthworms in soils contaminated with different concentrations (0.02% and 0.2% w/w) of polyethylene (PE) and polypropylene (PP) MPs to investigate the effect of earthworms on tomato against Helicoverpa armigera (H. armigera) under MPs stress. We found that earthworms alleviated the inhibitory effects of MPs stress on tomato growth and disrupted H. armigera growth. Compared to individual MPs exposure, earthworm incorporation significantly increased the silicon and lignin content in herbivore-damaged tomato leaves by 19.1% and 57.6%, respectively. Metabolites involved in chemical defense (chlorogenic acid) and phytohormones (jasmonic acid) were also activated by earthworm incorporation. Furthermore, earthworms effectively reduced oxidative damage induced by H. armigera via promoting antioxidant metabolism. Overall, our results suggest that utilizing earthworms to regulate above- and below-ground interactions could be a promising strategy for promoting green agriculture.

Tyramine-Mediated Hyperactivity Modulates the Dietary Habits in Helicoverpa armigera.

Helicoverpa armigera exhibits extensive variability in feeding habits and food selection. Neuronal regulation of H. armigera feeding behavior is primarily influenced by biogenic amines such as Tyramine (TA) and Octopamine (OA). The molecular responses of H. armigera to dietary challenges in the presence of TA or OA have yet to be studied. This investigation dissects the impact of OA and TA on H. armigera feeding choices and behaviors under non-host nutritional stress. It has been observed that feeding behavior remains unaltered during the exogenous administration of OA and TA through an artificial diet (AD). Ingestion of higher OA or TA concentrations leads to increased mortality. OA and TA treatment in combination with host and non-host diets results in the induction of feeding and higher locomotion toward food, particularly in the case of TA treatment. Increased expression of markers, prominin-like, and tachykinin-related peptide receptor-like transcripts further assessed increased locomotion activity. Insects subjected to a non-host diet with TA treatment exhibited increased feeding and overexpression of the feeding indicator, the Neuropeptide F receptor, and the feeding regulator, Sulfakinin, compared with other conditions. Expression of sensation and biogenic amine synthesis genesis elevated in insects fed a non-host diet in combination with OA or TA. Metabolomics analysis revealed a decreased concentration of the feeding behavior elicitor, dopamine, in insects fed a non-host diet containing TA. This work highlights the complex interplay between biogenic amine functions during dietary stress and suggests the role of tyramine in feeding promotion under stressed conditions.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

Influence of shifting thermal regimes on tomato fruit borer, Helicoverpa armigera (Hubner) in the Eastern Himalaya: implications for pest management strategies.

Climate change, particularly temperature fluctuations, profoundly impacts pest populations. This study focuses on the tomato, a crucial commercial crop in the Eastern Himalayan Region of India. The study examined the impact of varying thermal regimes on tomato fruit borers. Field experiments were conducted at three locations, with altitudes ranging from < 500 to > 1500 m. At lower altitudes, fruit borer incidence commenced earlier (5th - 18th March) and peaked higher (1.47 ± 0.34 to 1.73 ± 0.37 larvae/plant), causing more damage (26-29%) as compared to the highest location (~ 9%). The generalized linear mixed model (GLMM) analysis indicated that maximum temperature had significant positive impacts on the H. armigera incidence and fruit damage. Climatic datasets indicate an increase in the temperature of the region during the tomato growing season, thereby increasing the risk of fruit borer impact. As an adaptation option, we evaluated eight different tomato varieties/genotypes and studied biochemical parameters to understand their tolerance. Results showed a strong positive association of fruit borer incidence with total soluble solids whereas negative association with acidity. Cherry tomato (7.62%) and MT-2 (10.04%) had relatively lower fruit damage; MT-3 (50.92 t/ha) and MT-2 (50.57 t/ha) consistently yielded the highest across all locations. Hence, the selection of appropriate genotypes and the development of varieties with suitable characteristics hold the key to fruit borer management. This insight is crucial for developing effective pest management strategies and ensuring sustainable agricultural practices in the region.

Heterologous expression, biochemical characterization and prospects for insecticide biosensing potential of carboxylesterase Ha006a from Helicoverpa armigera.

Enzymes have attracted considerable scientific attention for their crucial role in detoxifying a wide range of harmful compounds. In today's global context, the extensive use of insecticides has emerged as a significant threat to the environment, sparking substantial concern. Insects, including economically important pests like Helicoverpa armigera, have developed resistance to conventional pest control methods through enzymes like carboxyl/cholinesterases. This study specifically focuses on a notable carboxyl/cholinesterase enzyme from Helicoverpa armigera (Ha006a), with the goal of harnessing its potential to combat environmental toxins. A total of six insecticides belonging to two different classes displayed varying inhibitory responses towards Ha006a, thereby rendering it effective in detoxifying a broader spectrum of insecticides. The significance of this research lies in discovering the bioremediation property of Ha006a, as it hydrolyzes synthetic pyrethroids (fenvalerate, λ-cyhalothrin and deltamethrin) and sequesters organophosphate (paraoxon ethyl, profenofos, and chlorpyrifos) insecticides. Additionally, the interaction studies between organophosphate insecticides and Ha006a helped in the fabrication of a novel electroanalytical sensor using a modified carbon paste electrode (MCPE). This sensor boasts impressive sensitivity, with detection limits of 0.019 µM, 0.15 µM, and 0.025 µM for paraoxon ethyl, profenofos, and chlorpyrifos, respectively. This study provides a comprehensive biochemical and biophysical characterization of the purified esterase Ha006a, showcasing its potential to remediate different classes of insecticides.

Dissecting the manipulation of lufenuron on chitin synthesis in Helicoverpa armigera.

Lufenuron, a benzoylurea chitin synthesis inhibitor, is effective against many insect pests. However, the insecticidal activity of lufenuron has not been completely elucidated, nor has its disturbing effect on chitin synthesis genes. In this study, bioassay results demonstrated an outstanding toxicity of lufenuron against Helicoverpa armigera larvae. The treated larvae died from abortive molting and metamorphosis defects, and severe separation of epidermis and subcutaneous tissues was observed. Treatment of 3rd- and 4th-instar larvae with LC25 lufenuron significantly extended the duration of larval and pupal stage, reduced the rates of pupation and emergence, and adversely affected pupal weight. Besides, lufenuron can severely reduce chitin content in larval integument, and the lufenuron-treated larvae showed reduced trehalose content in their hemolymph. Further analysis using RNA sequencing revealed that five chitin synthesis genes were down-regulated, whereas the expressions of two chitin degradation genes were significantly enhanced. Knockdown of chitin synthase 1 (HaCHS1), uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (HaUAP), phosphoacetyl glucosamine mutase (HaPGM), and glucosamine 6-phosphate N-acetyl-transferase (HaGNPAT) in H. armigera led to significant increase in larval susceptibilities to LC25 lufenuron by 75.48%, 65.00%, 68.42% and 28.00%, respectively. Our findings therefore revealed the adverse effects of sublethal doses of lufenuron on the development of H. armigera larvae, elucidated the perturbations on chitin metabolism, and proved that the combination of RNAi and lufenuron would improve the control effect of this pest.

Identification of insect cuticular protein genes LCP17 and SgAbd5 from Helicoverpa armigera and evaluation their roles in fenvalerate resistance.

Insect cuticular protein (ICP) plays an important role in insect growth and development. However, research on the role of ICP in insecticide resistance is very limited. In this study, insect cuticular protein genes LCP17 and SgAbd5 were cloned and characterized in Helicoverpa armigera based on previous transcriptome data. The functions of LCP17 and SgAbd5 genes in fenvalerate resistance were assessed by RNA interference (RNAi), and their response to fenvalerate was further detected. The results showed that LCP17 and SgAbd5 were overexpressed in the fenvalerate-resistant strain comparing with a susceptible strain. The open reading frames of LCP17 and SgAbd5 genes were 423 bp and 369 bp, encoding 141 and 123 amino acids, respectively. LCP17 and SgAbd5 genes were highly expressed in the larval stage, but less expressed in the adult and pupal stages. The expression level of LCP17 and SgAbd5 genes increased significantly after fenvalerate treatment at 24 h. When the cotton bollworms larvae were exposed to fenvalerate at LD50 level, RNAi-mediated silencing of LCP17 and SgAbd5 genes increased the mortality from 50.68% to 68.67% and 63.89%, respectively; the mortality increased to even higher level, which was 73.61%, when these two genes were co-silenced. Moreover, silencing of these two genes caused the cuticle lamellar structure to become loose, which led to increased penetration of fenvalerate into the larvae. The results suggested that LCP17 and SgAbd5 may be involved in the resistance of cotton bollworm to fenvalerate, and LCP17 and SgAbd5 could serve as potential targets for H. armigera control.

Functional study on candidate regulators mediating the expression of CYP6B7 induced by fenvalerate in a susceptible strain of Helicoverpa armigera.

Transcription factors play an important role in regulating the expression of detoxification genes (e.g. P450s) that confer insecticide resistance. Our previous study identified a series of candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may be related to fenvalerate-induced expression of CYP6B7 in a field HDTJ strain of H. armigera. Whether these CAPs can mediate the transcript of CYP6B7 induced by fenvalerate in a susceptible HDS strain of H. armigera remains unknown. Further study showed that the expression levels of multiple CAPs were significantly induced by fenvalerate in HDS strain. Knockdown of CAP19 [fatty acid synthase-like (FAS)], CAP22 [polysaccharide biosynthesis domain-containing protein 1 (PBDC1)], CAP24 [5-formyltetrahydrofolate cycloligase (5-FCL)], CAP30 [peptidoglycan recognition protein LB-like (PGRP)] and CAP33 [NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 (NDUFA11)] resulted in significant inhibition of CYP6B7 and some other P450 genes expression; meanwhile, the sensitivity of HDS strain larvae to fenvalerate was significantly increased. In addition, PBDC1, PGRP and NDUFA11, either alone or in combination, could significantly enhance the activity of CYP6B7 promoter in HDS strain, as well as the expression level of CYP6B7 gene in Sf9 cells line. These results suggested that PBDC1, PGRP and NDUFA11 may be involved in the transcript regulation of key detoxifying genes in response to fenvalerate in HDS strain of H. armigera.

The vital hormone 20-hydroxyecdysone controls ATP production by upregulating binding of trehalase 1 with ATP synthase subunit α in Helicoverpa armigera.

Trehalose is the major "blood sugar" of insects and it plays a crucial role in energy supply and as a stress protectant. The hydrolysis of trehalose occurs only under the enzymatic control of trehalase (Treh), which plays important roles in growth and development, energy supply, chitin biosynthesis, and abiotic stress responses. Previous reports have revealed that the vital hormone 20-hydroxyecdysone (20E) regulates Treh, but the detailed mechanism underlying 20E regulating Treh remains unclear. In this study, we investigated the function of HaTreh1 in Helicoverpa armigera larvae. The results showed that the transcript levels and enzymatic activity of HaTreh1 were elevated during molting and metamorphosis stages in the epidermis, midgut, and fat body, and that 20E upregulated the transcript levels of HaTreh1 through the classical nuclear receptor complex EcR-B1/USP1. HaTreh1 is a mitochondria protein. We also found that knockdown of HaTreh1 in the fifth- or sixth-instar larvae resulted in weight loss and increased mortality. Yeast two-hybrid, coimmunoprecipitation, and glutathione-S-transferase (GST) pull-down experiments demonstrated that HaTreh1 bound with ATP synthase subunit alpha (HaATPs-α) and that this binding increased under 20E treatment. In addition, 20E enhanced the transcript level of HaATPs-α and ATP content. Finally, the knockdown of HaTreh1 or HaATPs-α decreased the induction effect of 20E on ATP content. Altogether, these findings demonstrate that 20E controls ATP production by up-regulating the binding of HaTreh1 to HaATPs-α in H. armigera.

Cadherin Is a Binding Protein but Not a Functional Receptor of Bacillus thuringiensis Cry2Ab in Helicoverpa armigera.

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

A highly accumulated secretory protein from cotton bollworm interacts with basic helix-loop-helix transcription factors to dampen plant defense.

Caterpillar oral secretion (OS) contains active molecules that modulate plant defense signaling. We isolated an effector-like protein (Highly Accumulated Secretory Protein 1, HAS1) from cotton bollworm (Helicoverpa armigera) that is the most highly accumulated secretory protein of the nondigestive components in OS and belongs to venom R-like protein. Elimination of HAS1 by plant-mediated RNA interference reduced the suppression of OS on the defense response in plants. Plants expressing HAS1 are more susceptible to insect herbivory accompanied by the reduced expressions of multiple defense genes. HAS1 binds to the basic helix-loop-helix (bHLH) transcription factors, including GoPGF involved in pigmented gland formation and defense compounds biosynthesis in cotton and MYC3/MYC4 the main regulators in jasmonate (JA) signaling in Arabidopsis. The binding activity is required for HAS1 to inhibit the activation of bHLHs on plant defense gene expressions. Together with our previous study that another venom R-like protein HARP1 in cotton bollworm OS blocks JA signaling by interacting with JASMONATE-ZIM-domain repressors, we conclude that the venom R-like proteins in OS interfere with plant defense in a dual suppression manner. Considering the venom proteins in parasitic wasp assault the immune system of its host animal, our investigation reveals their conserved function in carnivorous and herbivorous insects.

Identification and analysis of toxins in novel Bacillus thuringiensis strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera (Lep.: Noctuidae).

Despite the successful application of toxins from Bacillus thuringiensis as biological control agents against pests, pests are showing resistance against an increasing number of Bacillus thuringiensis toxins due to evolution; thus, new toxins with higher toxicity and broad-spectrum activity against insects are being increasingly identified. To find new toxins, whole genome sequencing of the novel B. thuringiensis strain Bt S3076-1 was performed, and ten predicted toxic genes were identified in this study, including six cry genes, two tpp genes, one cyt gene and one vip gene, among which six were novel toxins. Subsequently, SDS‒PAGE analysis showed that the major proteins at the spore maturation stage were approximately 120 kDa, 70 kDa, 67 kDa, 60 kDa and 40 kDa, while active proteins after trypsin digestion (approximately 70 kDa and 40 kDa) exhibited LC50 values of 149.64 µg/g and 441.47 µg/g against Spodoptera frugiperda and Helicoverpa armigera larvae, respectively. Furthermore, pathological observation results showed that the peritrophic membrane of Spodoptera frugiperda and Helicoverpa armigera larvae was degraded. These findings will provide an experimental reference for further research on the insecticidal activity, toxicity spectrum and synergism of these toxins in Bt S3076-1.

Effects of different insecticides on transcripts of key genes in CncC pathway and detoxification genes in Helicoverpa armigera.

The CncC pathway regulates the expression of multiple detoxification genes and contributes to the detoxification and antioxidation in insects. Many studies have focused on the impacts of plant allelochemicals on the CncC pathway, whereas studies on the effects of pesticides on key genes involved in this pathway are very limited. In this study, the effects of different types of commonly used insecticides on the transcripts of CncC, Keap1, and Maf and multiple detoxification genes of Helicoverpa armigera were evaluated using real-time quantitative polymerase chain reaction. The results showed that 8 insecticides (bifenthrin, λ-cyhalothrin, chlorantraniliprole, cyantraniliprole, spinosad, indoxacarb, chlorfenapyr, tolfenpyrad, and thiacloprid) significantly induced the expression of CncC and 4 insecticides (cypermethrin, acetamiprid, thiacloprid, and indoxacarb) suppressed the expression of Keap1 both at 24 h and 48 h; meanwhile, the expression levels of Maf were induced by 5 insecticides (fenvalerate, chlorantraniliprole, cyantraniliprole, lufenuron, and tolfenpyrad) at 24 h or 48 h. Multiple detoxification genes, especially cytochrome P450s genes, showed different up-regulation after bifenthrin, λ-cyhalothrin, chlorantraniliprole, cyantraniliprole, indoxacarb, and spinosad treatment for 48 h. Our results suggest that the CncC pathway and detoxification genes can be activated by different insecticides in H. armigera. These results establish a foundation for further studies on the relationship between the CncC pathway and the detoxification genes in H. armigera.

Mechanisms of feeding cessation in Helicoverpa armigera larvae exposed to Bacillus thuringiensis Cry1Ac toxin.

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been applied in sprayable formulations and expressed in transgenic crops for the control of pests in the field. When exposed to Bt proteins insect larvae display feeding cessation, yet the mechanism for this phenomenon remains unknown. In this study, we investigated the feeding behavior and underlying mechanisms of cotton bollworm (Helicoverpa armigera) larvae after exposure to the Cry1Ac protein from Bt. Three H. armigera strains were studied: the susceptible SCD strain, the C2/3-KO strain with HaABCC2 and HaABCC3 knocked out and high-level resistance to Cry1Ac (>15,000-fold), and the SCD-KI strain with a T92C point mutation in tetraspanin (HaTSPAN1) and medium-level resistance to Cry1Ac (125-fold). When determining the percentage of insects that continued feeding after various exposure times to Cry1Ac, we observed quick cessation of feeding in larvae from the susceptible SCD strain, whereas larvae from the C2/3-KO strain did not display feeding cessation. In contrast, larvae from the SCD-KI strain rapidly recovered from the initial feeding cessation. Histopathological analyses and qRT-PCR in midguts of SCD larvae after Cry1Ac exposure detected serious epithelial damage and significantly reduced expression of the neuropeptide F gene (NPF) and its potential receptor gene NPFR, which are reported to promote insect feeding. Neither epithelial damage nor altered NPF and NPFR expression appeared in midguts of C2/3-KO larvae after Cry1Ac treatment. The same treatment in SCD-KI larvae resulted in milder epithelial damage and subsequent repair, and a decrease followed by an initial increase in NPF and NPFR expression. These results demonstrate that the feeding cessation response to Cry1Ac in cotton bollworm larvae is closely associated with midgut epithelial damage and downregulation of NPF and NPFR expression. This information provides clues to the mechanism of feeding cessation in response to Bt intoxication and contributes to the mode of action of the Cry1Ac toxin in target pests.

Helicoverpa armigera GATAe transcriptional factor regulates the expression of Bacillus thuringiensis Cry1Ac receptor gene ABCC2 by its interplay with additional transcription factors.

Helicoverpa armigera is a worldwide pest that has been efficiently controlled by transgenic plants expressing Bt Cry toxins. To exert toxicity, Cry toxins bind to different receptors located in larval midgut cells. Previously, we reported that GATA transcription factor GATAe activates the expression of multiple H. armigera Cry1Ac receptors in different insect cell lines. Here, the mechanism involved in GATAe regulation of HaABCC2 gene expression, a key receptor of Cry1Ac, was analyzed. HaGATAe gene silencing by RNAi in H. armigera larvae confirmed the activation role of HaGATAe on the expression of HaABCC2 in the midgut. The contribution of all potential GATAe-binding sites was analyzed by site-directed mutagenesis using Hi5 cells expressing a reporter gene under regulation of different modified HaABCC2 promoters. DNA pull-down assays revealed that GATAe bound to different predicted GATA-binding sites and mutations of the different GATAe-binding sites identified two binding sites responsible for the promoter activity. The binding site B9, which is located near the transcription initiator site, has a major contribution on HaABCC2 expression. Also, DNA pull-down assays revealed that all other members of GATA TF family in H. armigera, besides GATAe, HaGATAa, HaGATAb, HaGATAc and HaGATAd also bound to the HaABCC2 promoter and decreased the GATAe dependent promoter activity. Finally, the potential participation in the regulation of HaABCC2 promoter of several TFs other than GATA TFs expressed in the midgut cells was analyzed. HaHR3 inhibited the GATAe dependent activity of the HaABCC2 promoter, while two other midgut-related TFs, HaCDX and HaSox21, also bound to the HaABCC2 promoter region and increased the GATAe dependent promoter activity. All these data showed that GATAe induces HaABCC2 expression by binding to HaGATAe binding sites in the promoter region and that additional TFs participate in modulating the HaGATAe-driven expression of HaABCC2.