Plant defence mechanisms can reduce the digestive enzyme activity of insect pests. The aim of this study was to determine the relationship between the production of proteinase inhibitors, lipoxygenase and polyphenol oxidase activity in Coffea arabica (Catuai IAC 15) plants, and the digestive enzyme activity in the pest Leucoptera coffeella (Lepidoptera: Lyonetiidae) after feeding on the plant. The production of proteinase inhibitors was evaluated with L‐BApNA as a substrate. We studied lipoxygenase activity with linoleic acid and polyphenol oxidase activity with catechol substrates, in coffee plants damaged (T1) and not damaged (T2) by L. coffeella. L. coffeella digestive enzyme activity was verified by trypsin‐like (substrate l‐BApNA and l‐TAME), chymotrypsin‐like (BTpNA and ATEE), cysteine proteases (l‐BApNA) and total protease (azocasein). Proteinase inhibitor production and lipoxygenase and polyphenol oxidase activity in C. arabica increases (P ≤ 0.05) with L. coffeella damage. Our results provide important information that these enzymatic activities may play a role in plant defence processes in C. arabica. Trypsin‐like activity increases, whereas chymotrypsin‐like and cysteine protease activity decrease in the midgut of L. coffeella, which acts as a defence mechanism.

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It is observed that among the three peptides analyzed, the Gor 5 presented lower inhibition constant and therefore, the most potent among the tested ones. The predominance of hydrophobic residues in the region of the secondary site of the enzymes favored the interaction of the peptide. After characterization by three different types of graphs profiles, it is possible to verify that the inhibition model of the trypsin-like enzymes for the tested peptides is of the linear competitive type, in the concentration range of inhibitors and substrates analyzed. However, by the graphing profiles it is observed that the inhibition occurred due to the interaction of the peptides at the secondary site S2' in the hydrophobic cavity of the enzymes analyzed.

The acquired resistance by insects and the harmful environmental effects of chemical pesticides have encouraged the search of new tools for proper pest management. Among them, the use of protease inhibitors (PIs) obtained from plants has gained interest because they are a natural system against herbivory, are organic molecules with higher specificity and have the potential to cause less damage to nature. The aim of this work was to characterise the inhibitory potential of the proteins ApTI (Adenanthera pavonina trypsin inhibitor) and ILGA (Inga laurina trypsin inhibitor) on the digestive trypsins of Spodoptera cosmioides through molecular docking, enzymatic kinetics and biological survival analyses. The docking between trypsins and inhibitors was performed using the program CLUSPRO; the inhibitory constant Ki and the inhibition type were determined through chromogenic assays. In order to analyse survival, several concentrations of ApTI and ILTI inhibitors were included in the artificial diet of neonatal larvae. In this study, we determined that ILTI binds to the active site of the trypsins with a specificity similar to its natural substrate, whereas APTI showed that the inhibitor reactive site is not in contact with the trypsins catalytic site. The ILTI and APTI inhibitors were characterized as competitive and uncompetitive tight‐binding inhibitors, respectively. The survival curves obtained using Kaplan–Meier estimators indicated that the lowest percentage survival (20%) for all inhibitors tested was obtained using 1.0% doses at a development time of less than 20 days. We concluded that ILTI and APTI present biotechnological potential as agents against phytophagous Lepidoptera insects, inhibiting trypsins through tight‐binding inhibition, with competitive and non‐competitive mechanisms, respectively. The effect of ApTI and ILTI on the development of S. cosmioides larvae is shown to be toxic.

The economic loss in soybean crops caused by the Lepidoptera insects has encouraged the search for new strategies to control this pest, which are currently based on synthetic insecticides. This paper evaluated the ability of ApTI (Adenanthera pavonina trypsin inhibitor) to inhibit trypsin-like proteins from Anticarsia gemmatalis by docking, molecular dynamics, and enzymatic and survival assay. The docking and molecular dynamic simulation between trypsin and ApTI were performed using the program CLUSPRO and NAMD, respectively. The inhibitory constant K i and the inhibition type were determined through chromogenic assays. The survival assay of neonatal larvae under treatment with artificial diet supplemented with ApTI was also performed. The ApTI binding site was predicted to block substrate access to trypsin due to four interactions with the enzyme, producing a complex with a surface area of 1,183.7 Å 2 .The kinetic analysis revealed a noncompetitive tightbinding mechanism. The survival curves obtained using Kaplan-Meier estimators indicated that the highest larvae mortality was 60%, using 1.2 mg of ApTI per 100 ml of artificial diet. The in vitro, in vivo, and in silico studies demonstrated that ApTI is a strong noncompetitive inhibitor of trypsin with biotechnological potential for the control of A. gemmatalis insect. K E Y W O R D S binding, insects, Kunitz inhibitor, molecular docking, noncompetitive, trypsin enzyme

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Proteases present in the salivary glands of phytosuccivorous insects are responsible for the initial breakdown of dietary protein into the amino acids necessary for insect growth and development. Here, we characterised the total proteases and trypsin‐like activities in the salivary glands of the Neotropical brown stink bug, Euschistus heros (F.) and used in silico and biochemical approaches to determine the interactions between trypsin‐like enzymes and the soybean Kunitz trypsin inhibitor (SKTI). Additionally, we evaluated whether these enzyme activities were affected in individuals resistant to the actions of the insecticide imidacloprid. We used an originally field‐collected strain to select individuals resistant to imidacloprid alone (ImiGoiasRes) or synergised with piperonyl butoxide (ImiPBORes). A laboratory‐susceptible (ImiSusc) strain was also used to select piperonyl butoxide synergised imidacloprid‐resistant (ImiPBOSel) individuals. Our results revealed that pH and temperature significantly affected the specific trypsin‐like activities in imidacloprid‐susceptible individuals. Significant alterations in the total protease activities were recorded for field‐ (ImiGoiasRes) and laboratory‐selected imidacloprid‐resistant individuals (ImiPBOSel and ImiPBORes). Our computational docking analysis demonstrated that the molecular interactions and the presence of the SKTI inhibitor (0.5 mg mL−1) could reduce up to 40% of the trypsin‐like activities of imidacloprid‐susceptible individuals. However, all imidacloprid‐resistant strains exhibited significantly less trypsin‐like activities and exhibited lower hydrolysing velocity (VMAX) and affinity (KM) rates, and the prior exposure to SKTI only synergised the imidacloprid toxicity for individuals of ImiFieldRes strain. Collectively, our findings demonstrate, for the first time, insecticide resistance detrimentally affected the activities of proteolytic enzymes in phytosuccivorous stink bug pests.

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