Development Of Helicoverpa Armigera Research Articles

20-Hydroxyecdysone counteracts insulin to promote programmed cell death by modifying phosphoglycerate kinase 1

BackgroundThe regulation of glycolysis and autophagy during feeding and metamorphosis in holometabolous insects is a complex process that is not yet fully understood. Insulin regulates glycolysis during the larval feeding stage, allowing the insects to grow and live. However, during metamorphosis, 20-hydroxyecdysone (20E) takes over and regulates programmed cell death (PCD) in larval tissues, leading to degradation and ultimately enabling the insects to transform into adults. The precise mechanism through which these seemingly contradictory processes are coordinated remains unclear and requires further research. To understand the coordination of glycolysis and autophagy during development, we focused our investigation on the role of 20E and insulin in the regulation of phosphoglycerate kinase 1 (PGK1). We examined the glycolytic substrates and products, PGK1 glycolytic activity, and the posttranslational modification of PGK1 during the development of Helicoverpa armigera from feeding to metamorphosis.ResultsOur findings suggest that the coordination of glycolysis and autophagy during holometabolous insect development is regulated by a balance between 20E and insulin signaling pathways. Glycolysis and PGK1 expression levels were decreased during metamorphosis under the regulation of 20E. Insulin promoted glycolysis and cell proliferation via PGK1 phosphorylation, while 20E dephosphorylated PGK1 via phosphatase and tensin homolog (PTEN) to repress glycolysis. The phosphorylation of PGK1 at Y194 by insulin and its subsequent promotion of glycolysis and cell proliferation were important for tissue growth and differentiation during the feeding stage. However, during metamorphosis, the acetylation of PGK1 by 20E was key in initiating PCD. Knockdown of phosphorylated PGK1 by RNA interference (RNAi) at the feeding stage led to glycolysis suppression and small pupae. Insulin via histone deacetylase 3 (HDAC3) deacetylated PGK1, whereas 20E via acetyltransferase arrest-defective protein 1 (ARD1) induced PGK1 acetylation at K386 to stimulate PCD. Knockdown of acetylated-PGK1 by RNAi at the metamorphic stages led to PCD repression and delayed pupation.ConclusionsThe posttranslational modification of PGK1 determines its functions in cell proliferation and PCD. Insulin and 20E counteractively regulate PGK1 phosphorylation and acetylation to give it dual functions in cell proliferation and PCD.

Influence of temperature on Survival and Development of Helicoverpa armigera (Hubner)

Influence of temperature on Survival and Development of Helicoverpa armigera (Hubner)

In vivo developmental studies of Helicoverpa armigera and in silico molecular interactions with trypsin reveal the bio-insecticidal potential of trypsin inhibitor (SSTI) isolated from Solanum surattense

We report the impact of gut protease inhibition on the development of Helicoverpa armigera by trypsin inhibitor and the use of molecular modeling to understand the mechanism of trypsin inhibition. Larvae of H. armigera fed on an artificial diet containing 150 and 300 μg/ml SSTI showed a negative impact on the insects' development in terms of mean larval weight, larval fatality, survival rate, and nutritional indices. Prominent physical abnormalities like curled wings, malformed appendages, and small body size were observed during the development. Gene expression studies revealed down regulation in trypsin (HaTry 1, 2, 3, 4, 6, 8) and chymotrypsin (HaChy 1, 2, 3, 4) genes of the larval gut upon treatment of SSTI. Homology modeling has been used to build the three-dimensional structure of SSTI, which showed β-sheets having a stable canonical inhibitory loop (CIL) with conserved lysine residue. Molecular docking studies showed the strong binding of SSTI at the active site of trypsin. Molecular dynamic (MD) simulation revealed the stable interactions of the rigid CIL of SSTI at the active site of trypsin, leading to its destabilization. Conserved lysine63 of the P1 site in SSTI forms a strong hydrogen bonding network with residues Asp189 and Ser190 of trypsin.

Discovery of ZQ-8, a Novel Starting Point To Develop Inhibitors against the Potent Molecular Target Chitinase.

In a previous study, we found that compound ZQ-8 had a strong impact on the growth and development of Helicoverpa armigera. However, the mechanism and target of ZQ-8 are not clear, which makes it difficult to optimize the structure of this compound. In this study, the preliminary mode of action of ZQ-8 was studied through RNA sequencing and molecular docking. We also analyzed the underlying mechanisms from the aspect of ZQ-8 with respect to chitinase. The results showed that ZQ-8 mainly affects chitinase activity in the epidermis of H. armigera. ZQ-8 can competitively combine with chitinase 2 and endochitinase to form a relatively stable complex of ZQ-8, resulting in the failure of chitinase to degrade chitin. These findings indicate that the epidermis of H. armigera was identified as the action site of ZQ-8 and chitinase 2 and endochitinase were potential targets.

Survival and Development of <i>Helicoverpa Armigera</i> (Hubner) On Public Sector <i>BT</i> Cotton Hybrids

Laboratory experiment was conducted to find out the survival and development of Helicoverpa armigera (Hubner) on public sector Bt cotton hybrids (NHH-44 BG II, PKV Hy-2 BG II, PDKV-JKAL-116 BG II, G. COT-10 Hy. BG II and G. COT-08 Hy. BG II). Instarwise observations were documented on leaves, squares, young bolls and mature bolls at 60-80, 90-110, 120-140 and 150-170 days old crop, respectively. The results revealed that the early instars of H. armigera exhibited higher larval mortality than the later instars. The surviving larvae revealed adverse effect on many parameters viz., larval and pupal weight, % pupation and adult emergence indicating low growth and survival indices.

Insulin-like Growth Factor 2 Promotes Tissue-Specific Cell Growth, Proliferation and Survival during Development of Helicoverpa armigera.

During development, cells constantly undergo fate choices by differentiating, proliferating, and dying as part of tissue remodeling. However, we only begin to understand the mechanisms of these different fate choices. Here, we took the lepidopteran insect Helicoverpa armigera, the cotton bollworm, as a model to reveal that insulin-like growth factor 2 (IGF-2-like) prevented cell death by promoting cell growth and proliferation. Tissue remodeling occurs during insect metamorphosis from larva to adult under regulation by 20-hydroxyecdysone (20E), a steroid hormone. An unknown insulin-like peptide in the genome of H. armigera was identified as IGF-2-like by sequence analysis using human IGFs. The expression of Igf-2-like was upregulated by 20E. IGF-2-like was localized in the imaginal midgut during tissue remodeling, but not in larval midgut that located nearby. IGF-2-like spread through the fat body during fat body remodeling. Cell proliferation was detected in the imaginal midgut and some fat body cells expressing IGF-2-like. Apoptosis was detected in the larval midgut and some fat body cells that did not express IGF-2-like, suggesting the IGF-2-like was required for cell survival, and IGF-2-like and apoptosis were exclusive, pointing to a survival requirement. Knockdown of Igf-2-like resulted in repression of growth and proliferation of the imaginal midgut and fat body. Our results suggested that IGF-2-like promotes cell growth and proliferation in imaginal tissues, promoting cell death avoidance and survival of imaginal cells during tissue remodeling. It will be interesting to determine whether the mechanism of action of steroid hormones on insulin growth factors is conserved in other species.

The effect of group IV chitinase, HaCHT4, on the chitin content of the peritrophic matrix (PM) during larval growth and development of Helicoverpa armigera.

Extensive research has been conducted on insect chitinases. However, little is known about the function of chitinase in the regulation of the surface structure of the peritrophic matrix (PM) in larval midguts. The aim of this study was to analyze the effect of HaCHT4 on the chitin content and surface structure of the PM during larval growth and development of Helicoverpa armigera. The expression level of HaCHT4 was lower and the chitin content was higher in the early stages of fourth to sixth instar larvae, but they were reversed in the corresponding late stages. The correlation coefficient between the expression level of HaCHT4 and the chitin content was -0.585 (P < 0.05), with a higher negative correlation of -0.934 for the fourth instar (P < 0.01). Scanning electron microscopy (SEM) showed that the surface structure of PM was multi-laminated with small pores in the early stages of fourth to sixth instar larvae, and more and bigger pores in the late stages. Low expression of HaCHT4 caused by RNA interference (RNAi) resulted in the increase of chitin content in the PM, and the surface structure of PM became multilayered with smaller pore size in the late stage of fourth instar larvae. Also, induction of HaCHT4 by application of 2-tridecanone (2-TD), decreased the chitin content of PM, caused larger pores to form and lots of food bolus to attach to the PM surface, and also increased the larval susceptibility to chlorantraniliprole. These results provided strong evidence that HaCHT4 plays an important role by regulating the chitin content of the PM and its surface structure, thereby affecting the sensitivity of H. armigera to chlorantraniliprole.

The Effect of Glucosinolates on the Growth and Development of Helicoverpa armigera Larvae and the Expression of Midgut Sulfatase Genes

The plant–pest interaction and its mechanisms are a novel research direction for pest control. They provide molecular targets for developing new pesticides and targeted control measures to control insect herbivores. Glucosinolate is a large family of secondary substances found in cruciferous plants that are harmful to herbivorous insects. Specialist herbivores have developed specific anti-defense genes and detoxifying mechanisms against glucosinolate from the host plant, but how generalist herbivores respond to glucosinolate at the molecular level is unknown. In this study, we investigated the effects of different glucosinolate concentrations on the growth and development of Helicoverpa armigera. Moreover, the expression of sulfatase genes (HaSulfs) was also checked following exposure to glucosinolate concentrations. The developmental duration of larvae and pre-pupa of H. armigera was significantly increased by 14.79–25.03% after feeding glucosinolate compared to the control. Quantitative Real-Time PCR (RT-qPCR) was carried out to analyze the expression of HaSulf family genes in the midgut of fifth instar larvae of H. armigera. The results showed that the upregulated expression patterns of HaSulf family genes were diversified after feeding at different concentrations. The expression level of HaSulf was detected with the HaSulf antibody. Only the glucosinolate-fed larvae had a visible target band and were mainly distributed in the midgut wall. Taken together, glucosinolate can significantly affect the growth and development of H. armigera larvae. It can induce the expression of HaSulf in the midgut of H. armigera at gene and protein levels. This study could be useful to understand the development of plant-derived insecticides resistance in H. armigera.

New insights on the effects of spinosad on the development of Helicoverpa armigera

Helicoverpa armigera (cotton bollworm) is one of the most destructive pests worldwide. Due to resistance to Bacillus thuringiensis and conventional insecticides, an effective management strategy to control this pest is urgently needed. Spinosad, a natural pesticide, is considered an alternative; however, the mechanism underlying the developmental effects of sublethal spinosad exposure remains elusive. In this study, the mechanism was examined using an insect model of H. armigera. Results confirmed that exposure to sublethal spinosad led to reduced larval wet weight, delayed larval developmental period, caused difficulty in molting, and deformed pupae. Further investigation demonstrated that exposure to sublethal spinosad caused a significant decrease in 20E titer and increase in JH titer, thereby leading to the discordance between 20E and JH titers, and consequently alteration in the expression levels of HR3 and Kr-h1. These results suggested that sublethal spinosad caused hormonal disorders in larvae, which directly affect insect development. Our study serves as a reference and basis for the toxicity evaluation of spinosad on molting and pupation in insect metamorphosis, which may contribute to identifying targets for effective control of cotton bollworm.

Balancing nutrients in a toxic environment: the challenge of eating.

Insect herbivores can regulate their food intake by mixing food sources with different nutrient content, but face the resulting challenge of ingesting various plant secondary metabolites. How insects deal with toxins in a complex nutrient environment is unclear. Here we investigated the influence of a classic plant secondary metabolite, allyl glucosinolate (sinigrin), and its hydrolyzed product allyl isothiocyanate (AITC), on the development of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) when fed on diets with different protein-to-carbohydrate (p : c) ratios. We also examined the effects of these toxins on larval biochemistry, by chemically analyzing the frass produced by insects feeding on the different diets. As expected, AITC had a greater negative effect than sinigrin on H. armigera life-history traits. However, AITC at low concentration appeared to have a positive effect on some traits. Both sinigrin and AITC-induced detoxification activity in the gut, and the reaction was related to diet protein concentration. High-protein diets can provide the required free amino acid, especially cysteine, needed for the detoxification process. The nutrient content of the diet influences how plant secondary metabolites are handled, and the use of artificial diets in experiments investigating the metabolic fate of plant secondary compounds needs to be carefully evaluated.

Molecular stacking of two codon-modified genes encoding Bt insecticidal proteins, Cry1AcF and Cry2Aa for management of resistance development in Helicoverpa armigera

Molecular stacking of two codon-modified genes encoding Bt insecticidal proteins, Cry1AcF and Cry2Aa for management of resistance development in Helicoverpa armigera

Self-assembled nano-vesicles based on mPEG-NH2 modified carboxymethyl chitosan-graft-eleostearic acid conjugates for delivery of spinosad for Helicoverpa armigera

In this work, carboxymethyl chitosan-graft-eleostearic acid (CMCS-g-EA) was synthesized via the amide reaction between the amino groups of carboxymethyl chitosan and the carboxyl group of eleostearic acid, and then mPEG-NH2 was grafted to CMCS-g-EA to prepare amphiphilic polymers (mPEG-CMCS-g-EA). The chemical structures of the above conjugates were characterized by FT-IR and 1H NMR. Both CMCS-g-EA and mPEG-CMCS-g-EA based nano-vesicles were prepared by ultrasonic self-assembly method and they exhibited a low critical aggregation concentration (CAC) of 14.97 μg/mL, 16.82 μg/mL, respectively. The spinosad-loaded mPEG-CMCS-g-EA nano-vesicles (SSD@mPEG-CMCS-g-EA NVs) were spherical in shape with an average diameter of 502.8 nm and the zeta potential of −25.60 mV. The encapsulation efficiency (EE) and drug loading content (LC) of SSD@mPEG-CMCS-g-EA nano-vesicles were 42.00%, 23.07%, respectively. In vitro release revealed that the SSD@mPEG-CMCS-g-EA nano-vesicles exhibited a sustained and pH-responsive drug release property, and could significantly enhance the photostability of spinosad. Furthermore, the toxicological tests demonstrated that the SSD@mPEG-CMCS-g-EA nano-vesicles could efficiently inhibit the growth and development of Helicoverpa armigera. These results indicated that the SSD@mPEG-CMCS-g-EA nano-vesicles were highly potential for the treatment of Helicoverpa armigera.

A potent chitin-hydrolyzing enzyme from Myrothecium verrucaria affects growth and development of Helicoverpa armigera and plant fungal pathogens

Chitin, a crucial structural and functional component of insects and fungi, serves as a target for pest management by utilizing novel chitinases. Here, we report the biocontrol potential of recombinant Myrothecium verrucaria endochitinase (rMvEChi) against insect pest and fungal pathogens. A complete ORF of MvEChi (1185 bp) was cloned and heterologously expressed in Escherichia coli. Structure based sequence alignment of MvEChi revealed the presence of conserved domains SXGG and DXXDXDXE specific for GH-18 family, involved in substrate binding and catalysis, respectively. rMvEChi (46.6 kDa) showed optimum pH and temperature as 7.0 and 30 °C, respectively. Furthermore, rMvEChi remained stable within the pH range of 6.0 to 8.0 and up to 40 °C. rMvEChi exhibited kcat/Km values of 129.83 × 103 [(g/L)−1 s−1] towards 4MU chitotrioside. Hydrolysis of chitooligosaccharides with various degrees of polymerization (DP) using rMvEChi indicated the release of DP2 as main end product with order of reaction as DP6 > DP5 > DP4 > DP3. Bioassay of rMvEChi against Helicoverpa armigera displayed potent anti-feedant activity and induced mortality. In vitro antifungal activity against plant pathogenic fungi (Ustilago maydis and Bipolaris sorokiniana) exhibited significant inhibition of mycelium growth. These results suggest that MvEChi has significant potential in enzyme-based pest and pathogen management.

The microRNA pathway core genes are differentially expressed during the development of Helicoverpa armigera and contribute in the insect's development

MicroRNAs (miRNAs) are small non-coding RNAs (18–25 nt) that are produced by all animals and plants as well as some viruses. Their roles have been revealed in many physiological processes including development, cancer, immunity, apoptosis and, host–microbe interactions through post-transcriptional regulation of gene expression. In this study, we predicted, characterized and transcriptionally analyzed the core miRNA pathway genes in Helicoverpa armigera. Our results showed that the canonical miRNA biogenesis pathway genes including Pasha, Drosha, Loquacious, Exportin-5, Dicer-1 and Argonaute-1 are differentially expressed in different tissues and during the development of this insect. Considering the essential role of Dicer-1 in this pathway, we used RNA interference to silence the expression of this gene in H. armigera. Silencing of Dicer-1 decreased the levels of cellular miRNAs, let-7 and miR-184. Together, our results showed that the miRNA pathway functions during the development of H. armigera, and silencing of Dicer-1 resulted in the miRNA pathway blockage and depletion of the miRNA contents leading to mortalities in the immature stage and abnormalities in the mature stage. Blockage of this pathway can therefore be considered in future attempts for interrupting/suppressing populations of this important crop pest.

Integrated Transcriptomic and Proteomic Analyses Suggest the Participation of Endogenous Protease Inhibitors in the Regulation of Protease Gene Expression in Helicoverpa armigera

Insects adapt to plant protease inhibitors (PIs) present in their diet by differentially regulating multiple digestive proteases. However, mechanisms regulating protease gene expression in insects are largely enigmatic. Ingestion of multi-domain recombinant Capsicum annuum protease inhibitor-7 (CanPI-7) arrests growth and development of Helicoverpa armigera (Lepidoptera: Noctuidae). Using de novo RNA sequencing and proteomic analysis, we examined the response of H. armigera larvae fed on recombinant CanPI-7 at different time intervals. Here, we present evidence supporting a dynamic transition in H. armigera protease expression on CanPI-7 feeding with general down-regulation of protease genes at early time points (0.5 to 6 h) and significant up-regulation of specific trypsin, chymotrypsin and aminopeptidase genes at later time points (12 to 48 h). Further, coexpression of H. armigera endogenous PIs with several digestive protease genes were apparent. In addition to the differential expression of endogenous H. armigera PIs, we also observed a distinct novel isoform of endogenous PI in CanPI-7 fed H. armigera larvae. Based on present and earlier studies, we propose potential mechanism of protease regulation in H. armigera and subsequent adaptation strategy to cope with anti-nutritional components of plants.

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

Effect of Cholesterol, Stigmasterol and Sitosterol in Artificial Diet on Survival and Development of Helicoverpa Armigera

Effect of Cholesterol, Stigmasterol and Sitosterol in Artificial Diet on Survival and Development of Helicoverpa Armigera

Effect of biosynthesized Silver nanoparticles on growth and development of Helicoverpa armigera (Lepidoptera: Noctuidae): Interaction with midgut protease

We investigated the effect of Silver nanoparticles (AgNPs) on the gut protease activity of insecticide resistant gram caterpillar, Helicoverpa armigera. The leaf extracts of Peepal tree, Ficus religiosa (FR) and banyan tree, Ficus benghalensis (FB) mediated biogenic AgNPs were synthesized to modulate the function of gut protease activity in H. armigera (Ha). Bioassay with FR and FB AgNPs significantly reduced both larval weight and survival rate of H. armigera. The FR and FB AgNPs inhibited the Ha-Gut protease activity by 50 and 70% at 100μg concentration respectively. The FR and FB Silver nanoparticles were interacted and binds with high affinity with protease. The inhibition studies on Ha-gut protease activity may contribute towards developing new IRM (Insecticide Resistant Management) strategies against H. armigera to overcome insecticidal resistance issues.

Data of in vitro synthesized dsRNAs on growth and development of Helicoverpa armigera.

The data presented in this article is related to the research article “RNAi of selected candidate genes interrupts growth and development of Helicoverpa armigera” (Chikate et al., 2016) [1]. RNA interference (RNAi) is emerging as a potent insect pest control strategy over current methods and their resistance by pest. In this study we tested 15 different in vitro synthesized dsRNAs for gene silencing in Helicoverpa armigera. These dsRNAs were specific against H. armigera enzymes/proteins such as proteases like trypsins (HaTry2, 3, 4 and 6), chymotrypsin (HaChy4) and cysteine proteases such as cathepsin (HaCATHL); glutathione S-transferases (HaGST1a, 6 and 8); esterases (HaAce4, HaJHE); catalase (HaCAT); super-oxide-dismutase (HaCu/ZnSOD); fatty acid binding protein (HaFabp) and chitin deacetylase (HaCda5b). These dsRNAs were fed to second instar larvae at an optimized dose (60 µg/day) for 3 days separately. Effects of dsRNA feeding were observed in terms of larval mass gain, percentage mortality and phenotypic abnormalities in later developmental stages of H. armigera. These findings might provide potential new candidates for designing sequence-specific dsRNA as pesticide in crop protection.

RNAi of selected candidate genes interrupts growth and development of Helicoverpa armigera

Helicoverpa armigera is one of the major crop pests and is less amenable to current pest control approaches. RNA interference (RNAi) is emerging as a potent arsenal for the insect pest control over current methods. Here, we examined the effect on growth and development in H. armigera by targeting various enzymes/proteins such as proteases like trypsins (HaTry2, 3, 4 and 6), chymotrypsin (HaChy4) and cysteine protease like cathepsin (HaCATHL); glutathione S-transferases (HaGST1a, 6 and 8); esterases (HaAce4, HaJHE); catalase (HaCAT); super-oxide-dismutase (HaCu/ZnSOD); fatty acid binding protein (HaFabp) and chitin deacetylase (HaCda5b) through dsRNA approach. Significant downregulation of cognate mRNA expression and reduced activity of trypsin and GST-like enzyme were evident upon feeding candidate dsRNAs to the larvae. Among these, the highest mortality was observed in HaAce4 dsRNA fed larvae followed by HaJHE; HaCAT; HaCuZnSOD; HaFabp and HaTry3 whereas remaining ones showed relatively lower mortality. Furthermore, the dsRNA fed larvae showed significant reduction in the larval mass and abnormalities at the different stages of H. armigera development compared to their control diets. For example, malformed larvae, pupae and moth at a dose of 60μg/day were evident in high number of individual insects fed on dsRNA containing diets. Moreover, the growth and development of insects and moths were retarded in dsRNA fed larvae. These findings might provide potential new candidates for designing effective dsRNA as pesticide in crop protection.