Although the importance of intestinal hydrolases is recognized, there is little information on the intestinal proteome of lepidopterans such as Anticarsia gemmatalis. Thus, we carried out the proteomic analysis of the A. gemmatalis intestine to characterize the proteases by LC/MS. We examined the interactions of proteins identified with protease inhibitors (PI) using molecular docking. We found 54 expressed antigens for intestinal protease, suggesting multiple important isoforms. The hydrolytic arsenal featured allows for a more comprehensive understanding of insect feeding. The docking analysis showed that the soybean PI (SKTI) could bind efficiently with the trypsin sequences and, therefore, insect resistance does not seem to involve changing the sequences of the PI binding site. In addition, a SERPIN was identified and the interaction analysis showed the inhibitor binding site is in contact with the catalytic site of trypsin, possibly acting as a regulator. In addition, this SERPIN and the identified PI sequences can be targets for the control of proteolytic activity in the caterpillar intestine and serve as a support for the rational design of a molecule with greater stability, less prone to cleavage by proteases and viable for the control of insect pests such as A. gemmatalis.

New approaches are needed to reduce risks to the environment and natural enemies and to avoid or delay the onset of insecticide resistance. The use of insecticides based on proteinase inhibitors of hemolymph is an alternative for the control of Lepidoptera pests primarily by having low toxicity and short persistence in the environment. Thus, in this study, we describe the purification process and identification of protease inhibitors from hemolymph Anticarsia gemmatalis and their activities against trypsin enzymes. Furthermore, the three-dimensional (3D) structure of the inhibitor and binding mode to trypsin enzymes was determined, and the stability of the inhibitory activity in several pHs and temperature values was evaluated. The inhibitor was characterized as a serpin family inhibitor and named A. gemmatalis hemolymph serpin inhibitor (AHSI), with an approximate mass of 38 ± 2 kDa, highly stable to temperature and pH variations, and with inhibitory capacity on bovine trypsin and gut trypsin of A. gemmatalis demonstrated by calculated K i values and affinity energy through molecular docking, being a reversible competitive inhibitor that binds to the active site of

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