Identification and biophysical characterization of potential phytochemical inhibitors of carboxyl/choline esterase from Helicoverpa armigera for advancing integrated pest management strategies.

In the realm of disease vectors and agricultural pest management, insecticides play a crucial role in preserving global health and ensuring food security. The pervasive use, particularly of organophosphates (OPs), has given rise to a substantial challenge in the form of insecticide resistance. Carboxylesterases emerge as key contributors to OP resistance, owing to their ability to sequester or hydrolyze these chemicals. Consequently, carboxylesterase enzymes become attractive targets for the development of novel insecticides. Inhibiting these enzymes holds the potential to restore the efficacy of OPs against which resistance has developed. This study aimed to screen the FooDB library to identify potent inhibitory compounds targeting carboxylesterase, Ha006a from the agricultural pest Helicoverpa armigera. The ultimate objective is to develop effective interventions for pest control. The compounds with the highest scores underwent evaluation through docking studies and pharmacophore analysis. Among them, four phytochemicals-donepezil, protopine, 3',4',5,7-tetramethoxyflavone, and piperine-demonstrated favorable binding affinity. The Ha006a-ligand complexes were subsequently validated through molecular dynamics simulations. Biochemical analysis, encompassing determination of IC50 values, complemented by analysis of thermostability through Differential Scanning Calorimetry and interaction kinetics through Isothermal Titration Calorimetry was conducted. This study comprehensively characterizes Ha006a-ligand complexes through bioinformatics, biochemical, and biophysical methods. This investigation highlights 3',4',5,7-tetramethoxyflavone as the most effective inhibitor, suggesting its potential for synergistic testing with OPs. Consequently, these inhibitors offer a promising solution to OP resistance and address environmental concerns associated with excessive insecticide usage, enabling a significant reduction in their overuse.

Knocking Out of UDP-Glycosyltransferase Gene UGT2B10 via CRISPR/Cas9 in Helicoverpa armigera Reveals Its Function in Detoxification of Insecticides.

The role of insect UDP-glycosyltransferases (UGTs) in the detoxification of insecticides has rarely been reported. A UGT gene UGT2B10 was previously found overexpressed in a fenvalerate-resistant strain of Helicoverpa armigera. Herein, UGT2B10 was cloned, and its involvement in insecticide detoxification was investigated. UGT2B10 was highly expressed in the larvae, mainly in the fat body and midgut. Treatment with UGT inhibitors 5-nitrouracil and sulfinpyrazone significantly enhanced the fenvalerate toxicity. Knocking down UGT2B10 by RNAi significantly increased the larvae mortality by 17.89%. UGT2B10 was further knocked out by CRISPR/Cas9, and a homozygous strain (HD-dUGT2B10) with a C-base deletion at exon 2 was obtained. The sensitivity of HD-dUGT2B10 to fenvalerate, deltamethrin, cyantraniliprole, acetamiprid, and lufenuron increased significantly, with sensitivity index increased 2.523-, 2.544-, 2.250-, 2.473-, and 3.556-fold, respectively. These results suggested that UGT2B10 was involved in the detoxification of H. armigera to insecticides mentioned above, shedding light upon further understanding of the detoxification mechanisms of insecticides by insect UGTs.

Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

Cloning of three epsilon-class glutathione S-transferase genes from Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) and their response to tannic acid.

Micromelalopha troglodyta (Graeser) is an important pest of poplar in China, and glutathione S-transferase (GST) is an important detoxifying enzyme in M. troglodyta. In this paper, three full-length GST genes from M. troglodyta were cloned and identified. These GST genes all belonged to the epsilon class (MtGSTe1, MtGSTe2, and MtGSTe3). Furthermore, the expression of these three MtGSTe genes in different tissues, including midguts and fat bodies, and the MtGSTe expression in association with different concentrations of tannic acid, including 0.001, 0.01, 0.1, 1, and 10 mg ml-1, were analysed in detail. The results showed that the expression levels of MtGSTe1, MtGSTe2, and MtGSTe3 were all the highest in the fourth instar larvae; the expression levels of MtGSTe1 and MtGSTe3 were the highest in fat bodies, while the expression level of MtGSTe2 was the highest in midguts. Furthermore, the expression of MtGSTe mRNA was induced by tannic acid in M. troglodyta. These studies were helpful to clarify the interaction between plant secondary substances and herbivorous insects at a deep level and provided a theoretical foundation for controlling M. troglodyta.

Biodegradation of Free Gossypol by Helicoverpa armigera Carboxylesterase Expressed in Pichia pastoris.

Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial degradation of gossypol produces metabolites that reduce feed quality. We adopted an enzymatic method to degrade free gossypol safely and effectively. We cloned the gene cce001a encoding carboxylesterase (CarE) into pPICZαA and transformed it into Pichia pastoris GS115. The target protein was successfully obtained, and CarE CCE001a could effectively degrade free gossypol with a degradation rate of 89%. When esterase was added, the exposed toxic groups of gossypol reacted with different amino acids and amines to form bound gossypol, generating substances with (M + H) m/z ratios of 560.15, 600.25, and 713.46. The molecular formula was C27H28O13, C34H36N2O6, and C47H59N3O3. The observed instability of the hydroxyl groups caused the substitution and shedding of the group, forming a substance with m/z of 488.26 and molecular formula C31H36O5. These properties render the CarE CCE001a a valid candidate for the detoxification of cotton meal. Furthermore, the findings help elucidate the degradation process of gossypol in vitro.

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.

Serine protease SP7 cleaves prophenoloxidase and is regulated by two serpins in Ostrinia furnacalis melanization.

Melanization is an innate immune response in insects to defend against the invading pathogens and parasites. During melanization, prophenoloxidase (PPO) requires proteolytic activation by its upstream prophenoloxidase-activating protease (PAP). We here cloned a full-length cDNA for a serine protease, named as SP7, from Ostrinia furnacalis. The open reading frame of SP7 encodes 421-amino acid residue protein with a 19-residue signal peptide. qRT-PCR analysis showed that SP7 mRNA levels were significantly upregulated upon exposure to microbial infection. Recombinant SP7 zymogen was activated by serine protease SP2. The active SP7 could cleave O. furnacalis PPOs including PPO2, PPO1b and PPO3. Additionally, active SP7 could form covalent complexes with serine protease inhibitor serpin-3 and serpin-4. The activity of SP7 in cleaving a colorimetric substrate IEARpNA or O. furnacalis PPOs was efficiently blocked by either serpin-3 or serpin-4. Our work thus revealed that SP7 and SP2 partially constituted a PPO activation cascade in which SP7 was activated by SP2 and then likely worked as a PAP. SP7 was effectively regulated by serpin-3 and serpin-4. The results would allow further advances in the understanding of melanization mechanisms in O. furnacalis.

Functional expression of recombinant hybrid enzymes composed of bacterial and insect's chitinase domains in E. coli.

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect's domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one's leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other's chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi's LCL connected with BtChi's ChBD (MbLCL-BtChBD) and BtChi's LCL connected with MbChi's ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.

Contribution of phenoloxidase activation mechanism to Bt insecticidal protein resistance in Asian corn borer.

Phenoloxidase (PO) is a crucial enzyme in the Arthropods melanization process, in which synthesized melanin rapidly acts at the site of injury and infection. In this study, we observed significant changes in humoral and cellular responses after exposing susceptible and resistant strains to a sub-lethal concentration of Cry1Ah toxin. Based on STRING v 11.0 computational protein-protein interaction analysis, we selected seven immune genes namely Prophenoloxidase PPO1b, PP03, Serpin-3, Serpin-5, Beta-1,3-glucan recognition protein, Immulectin-3 and Serine protease SP105 reported in Asian corn borer. Quantitative real-time PCR gene expression studies showed Cry1Ah resistant strain had higher expression of PPO1b, PP03, Serpin-3, Beta-1,3-glucan recognition protein, Immulectin-3 and Serine protease SP105 genes in midgut and hemocyte samples. This study also investigated and found that the level of prophenoloxidation (proPO) activity in Cry1Ah resistant strains was significantly higher than susceptible strains. Cry1Ah toxin significantly increased the resistant strain's immune responses, the difference was observed through assays of bacterial agglutination and phagocytosis. Additionally, immune response induced by Cry1Ah toxin influences the microbiome composition associated with the host system. These parameters seem to explain the contribution of PO/PO regulating proteins render the host to resist the Cry1Ah toxin.

Effect of Tannic Acid on Nutrition and Activities of Detoxification Enzymes and Acetylcholinesterase of the Fall Webworm (Lepidoptera: Arctiidae).

Plant tannins, polyphenolic plant secondary metabolites are involved in important chemical defense processes in plants. In this study, tannic acid was used as the standard of plant tannins to determine the effects on nutritional indices and activities of glutathione S-transferases (GSTs), cytochrome P450 monooxygenase (CYP450), carboxylesterase (CarE), and acetylcholinesterase (AChE) in fourth-instar larvae of Hyphantria cunea (Drury) by feeding on an artificial diet containing tannic acid under different treatments. We found that tannic acid significantly affected the digestive capacity and food utilization rate of H. cunea larvae. A tannic acid concentration of less than 2.0% promoted feeding and the utilization of undesirable food by H. cunea larvae, while inhibitory effects were observed at high concentrations (>2.5%). Tannic acid had a significant effect on the activity of detoxification enzymes and AChE in H. cunea larvae in concentration-dependent and time-dependent manners (P < 0.05). These results provide new insights into the potential mechanisms underlying detoxification in H. cunea larvae against tannic acid in host plants.

Fabrication of smart stimuli-responsive mesoporous organosilica nano-vehicles for targeted pesticide delivery.

It is highly desirable to construct stimuli-responsive nanocarriers for improving pesticides targeting and preventing the pesticides premature release. In this work, a novel redox and α-amylase dual stimuli-responsive pesticide delivery system was established by bonding functionalized starch with biodegradable disulfide-bond-bridged mesoporous silica nanoparticles which loaded with avermectin (avermectin@MSNs-ss-starch nanoparticles). The results demonstrated that the loading capacity of avermectin@MSNs-ss-starch nanoparticles for avermectin was approximately 9.3 %. The starch attached covalently on the mesoporous silica nanoparticles could protect avermectin from photodegradation and prevent premature release of active ingredient. Meanwhile, the coated starch and disulfide-bridged structure of nanoparticles could be decomposed and consequently release of the avermectin on demand when nanoparticles were metabolized by glutathione and α-amylase in insects. The bioactivity survey confirmed that avermectin@MSNs-ss-starch nanoparticles had a longer duration in controlling Plutella xylostella larvae compared to avermectin emulsifiable concentrate. In consideration of the superior insecticidal activity and free of toxic organic solvent, this target-specific pesticide release system has promising potential in pest management.

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars.

Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showed major activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography-mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.

Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling.

Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP-AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP)1-4. 2',3'-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2',3'-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5-7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2',3'-cGAMP-degrading enzymes. Poxins cleave 2',3'-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2',3'-cGAMP degradation through metal-independent cleavage of the 3'-5' bond, converting 2',3'-cGAMP into linear Gp[2'-5']Ap[3']. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2',3'-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2',3'-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.

Parameters of oxidative stress, cholinesterase activity, Cd bioaccumulation in the brain and midgut of Lymantria dispar (Lepidoptera: Lymantriidae) caterpillars from unpolluted and polluted forests.

Cadmium (Cd) can display a variety of different effects on living organisms. The objectives of the present study were to investigate Cd bioaccumulation and differences in parameters of oxidative stress (activities of the enzymes: superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase, glutathione S-transferase, and amounts of non-enzymatic free sulfhydryl groups and total glutathione) and cholinesterase activity in larval brain and midgut tissues of the polyphagous forest insect Lymantria dispar collected from unpolluted and polluted oak forests. Fourth instar L. dispar caterpillars from the unpolluted forest had higher body mass but accumulated more Cd in comparison to caterpillars from the polluted forest. In both populations the midgut was more sensitive than the brain to the prooxidative effects of Cd. Enzyme activities and amounts of non-enzymatic parameters tended to be greater in midgut tissues than in the brain, except for cholinesterase activity. Parameters of oxidative stress had higher values in caterpillar tissues from the polluted than from the unpolluted oak forest. The observed differences between the two natural populations point to the importance of knowing the history of population exposure to environmental pollution when monitoring forest ecosystems.

Green light and light stress in moth: influence on antioxidant enzymes in the oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae).

Recently, light traps equipped with light-emitting diodes (LEDs) have been widely applied for controlling nocturnal pest moths. The oriental armyworm, known as Mythimna separata Walker, is an important insect pest in eastern Asia. The present study aimed to evaluate an influence of green light irradiation on antioxidant enzymes and light stress in M. separata adults. We determined total antioxidant capacity (T-AOC) and an activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione-S-transferase (GST) in the moth bodies according to an exposure time (0 to 180 min) of a green LED light. By our results, we suggested that the green (520 nm) LED light irradiation could induce a weak oxidative stress in M. separata adult moths, and the moths under the green light could restore the damage caused by this light stress. Additionally, we proposed that the adaptive ability to the light stress varied between the moths of different sexes. Our results may provide a theoretical and scientific basis for elucidating a reason of the phototactic behavior of nocturnal moths, including M. separata adults.

Identification of Key Residues Associated with the Interaction between Plutella xylostella Sigma-Class Glutathione S-Transferase and the Inhibitor S-Hexyl Glutathione.

Glutathione S-transferases (GSTs) are important detoxification enzymes involved in the development of metabolic resistance in Plutella xylostella. Uncovering the interactions between representative PxGSTs and the inhibitor S-hexyl glutathione (GTX), helps in the development of effective PxGST inhibitors for resistance management. As the PxGST most severely inhibited by GTX, PxGSTσ (sigma-class PxGST) adopts the canonical fold of insect GSTs. The formation of the PxGSTσ-GTX complex is mainly driven by H-bond and hydrophobic interactions derived from the side chains of favorable residues. Of the residues composing the active site of PxGSTσ, Lys43 and Arg99 are two hot spots, first reported in the binding of GSH derivatives to GSTs. Such differences indicate the metabolism discrimination of different insect GSTs. Unfavorable interactions between the PxGSTσ active site and GTX are depicted as well. The research guides the discovery and optimization of PxGSTσ inhibitors.

Yeast Surface Display of Antheraea pernyi Lysozyme Revealed α-Helical Antibacterial Peptides in Its N-Terminal Domain.

The present study investigated a novel lysozyme ApLyz from the Chinese oak silkmoth, Antheraea pernyi, for its active expression with N- or C-terminus fused to the yeast cell surface, and the antimicrobial activities of the corresponding expressed lysozymes were evaluated. The bactericidal activity of C-terminal fusion of ApLyz surpassed that of the N-terminal fusion, which revealed the implication of an N-terminal stretch of ApLyz in the bactericidal function based on the structural mobility of this region. Two N-terminal peptides of ApLyz (residues 1-15 and 1-32), which primarily consist of amphiphilic α-helices, exerted similar bactericidal efficacy and had a strong preference for the Gram-negative strains. Further investigation revealed that the N-terminal peptides are membrane-targeting peptides causing cell permeabilization and also possess nonmembrane disturbing bactericidal mechanism. Overall, in addition to the key findings of novel bactericidal peptides from silkmoth lysozyme, this work laid the foundation for future improvement of ApLyz by protein engineering.

Molecular characterization of a typical 2-Cys thioredoxin peroxidase from the Asiatic rice borer Chilo suppressalis and its role in oxidative stress.

In insects, thioredoxin peroxidase (TPX) plays an important role in protecting against oxidative damage. However, studies on the molecular characteristics of TPXs in the Asiatic rice borer, Chilo suppressalis, are limited. In this work, a cDNA sequence (CsTpx3) encoding a TPX was identified from C. suppressalis. The deduced CsTPX3 protein shares high sequence identity and two positionally conserved cysteines with orthologs from other insect species, and was classified as a typical 2-Cys TPX. CsTpx3 was expressed most highly during the fifth-instar larval stage, and transcripts were most abundant in the midgut. Recombinant CsTPX3 protein expressed in Escherichia coli displayed the expected peroxidase activity by removing H2 O2 . Furthermore, CsTPX3 protected DNA from oxidative damage, and E. coli cells overexpressing CsTPX3 exhibited long-term resistance to oxidative stress. Exposure to various oxidative stressors, such as cold (8°C), heat (35°C), bacteria (E. coli), and two insecticides (chlorpyrifos and lambda-cyhalothrin), significantly upregulated transcription of CsTpx3. However, exposure to abamectin had no such effect. Our results provide valuable information for future studies on the antioxidant mechanism in this insect species.

Tripeptides derived from reactive centre loop of potato type II protease inhibitors preferentially inhibit midgut proteases of Helicoverpa armigera.

Potato type II protease inhibitors (Pin-II PIs) impede the growth of lepidopteran insects by inhibiting serine protease-like enzymes in the larval gut. The three amino acid reactive centre loop (RCL) of these proteinaceous inhibitors is crucial for protease binding and is conserved across the Pin-II family. However, the molecular mechanism and inhibitory potential of the RCL tripeptides in isolation of the native protein has remained elusive. In this study, six peptides corresponding to the RCLs of the predominant Pin-II PIs were identified, synthesized and evaluated for in vitro and in vivo inhibitory activity against serine proteases of the polyphagous insect, Helicoverpa armigera. RCL peptides with sequences PRN, PRY and TRE were found to be potent inhibitors that adversely affected the growth and development of H. armigera. The binding mechanism and differential affinity of the RCL peptides with serine proteases was delineated by crystal structures of complexes of the RCL peptides with trypsin. Residues P1 and P2 of the inhibitors play a crucial role in the interaction and specificity of these inhibitors. Important features of RCL peptides like higher inhibition of insect proteases, enhanced efficacy at alkaline gut pH, longer retention and high stability in insect gut make them suitable molecules for the development of sustainable pest management strategies for crop protection.

Double-Stranded RNA-Mediated Suppression of Trypsin-Like Serine Protease (t-SP) Triggers Over-Expression of Another t-SP Isoform in Helicoverpa armigera.

High diversity of digestive proteases is considered to be the key factor in the evolution of polyphagy in Helicoverpa armigera. Serine proteases (SPs) contribute ~85% of the dietary protein digestion in H. armigera. We investigated the dynamics of SP regulation in the polyphagous pest, H. armigera using RNA interference (RNAi). HaTry1, an isoform of SP, expressed irrespective of the composition of the diet, and its expression levels were directly proportional to the larval growth rate. Therefore, HaTry1 was silenced by delivering 10 and 20 µg concentrations of double-stranded RNA through semi-synthetic diet. This led to a drastic reduction in the target gene transcript levels that manifested in a significant reduction in the larval weight initially, but the larvae recovered in later stages despite continuous dsRNA treatment. This was probably due to the compensatory effect by over-expression of HaTry13 (31-folds), another isoform of SP. Phylogenetic analysis of H. armigera SPs revealed that the over-expressed isoform was closely related to the target gene as compared to the other tested isoforms. Further, silencing of both the isoforms (HaTry1 and HaTry13) caused the highest reduction in the larval weight and there was no larval growth recovery. These findings provide a new evidence of the existence of compensatory effect to overcome the effect of silencing individual gene with RNAi. Hence, the study emphasizes the need for simultaneous silencing of multiple isoforms.