Integrated Transcriptomic and Physiological Analyses Reveal Disruption of Digestive Homeostasis Induced by Protease Inhibitors in Anticarsia gemmatalis Caterpillars

Abstract

ABSTRACT Protease inhibitors (PIs) are promising bioactive compounds for sustainable pest management because they interfere with the digestive processes of herbivorous insects. However, the physiological mechanisms underlying insect adaptation to natural and synthetic PIs remain poorly understood. Here, we investigated the midgut responses of Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae) to the soybean Kunitz trypsin inhibitor (SKTI) and the synthetic tripeptide GORE‐2 using integrated transcriptomic, biochemical, and histological approaches, complemented by an exploratory metatranscriptomic analysis of putative gut‐associated microbial transcripts. RNA‐seq analysis identified 2542 unique differentially expressed genes (FDR‐adjusted p < 0.05, |log2FC | ≥ 1), including extensive modulation of digestive proteases and redox‐related pathways. SKTI induced a compensatory response characterized by the up‐regulation of serine proteases, antioxidant enzymes, and detoxification‐associated pathways. In contrast, GORE‐2 promoted broader repression of digestive proteases, including chymotrypsin‐ and trypsin‐related transcripts and matrix metalloproteinases, together with a distinct antioxidant response. Biochemical assays demonstrated increased CAT, GST, POX, and SOD activities under both treatments. Compared with SKTI, GORE‐2 induced lower POX, GST, and SOD activities, whereas CAT activity did not differ significantly between the two treatments. Histological analyses demonstrated notable epithelial disruption, vacuolization, and disorganization of the striated border in the larval midgut, with lesion scores being significantly elevated following GORE‐2 exposure. Exploratory analysis of putative gut‐associated microbial transcripts recovered from whole‐midgut RNA‐seq revealed taxonomically diverse microbial signals associated with carbon metabolism, proteolysis, and detoxification. However, no treatment‐associated differential expression was detected among the putative bacterial transcripts. These findings indicate that GORE‐2 induced broader disruption of digestive and protective midgut functions than SKTI, with weaker antioxidant enzyme induction and more pronounced histological injury, consistent with reduced physiological compensation in A. gemmatalis larvae. These results underscore the potential of synthetic peptide‐based protease inhibitors to interfere with adaptive digestive processes in pest insects of agricultural importance.