Synthetic Peptide Inhibition of Trypsin‐Like Proteases in Spodoptera frugiperda (Lepidoptera: Noctuidae): Evaluating the Influence of Gut Microbiota

Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) is a major agricultural pest whose control has been increasingly challenged by resistance to conventional insecticides. Synthetic protease inhibitors represent promising alternatives; however, the potential modulation of their efficacy by gut microbiota remains poorly understood. Here, we evaluated two rationally designed synthetic tripeptides, GORE1 and GORE2, as inhibitors of trypsin-like digestive proteases and investigated whether gut microbiota disruption alters their inhibitory performance in S. frugiperda. Enzymatic assays revealed competitive inhibition patterns, with Ki values of 1.41 mM for GORE1 and 0.49 mM for GORE2. Most treatments increased apparent KM values, indicating reduced substrate affinity, whereas GORE2 consistently showed lower KM values. Despite stronger in vitro affinity (lower Ki), GORE2 did not produce greater biological impairment than GORE1. Microbiota imbalance induced by antibiotic treatment did not significantly alter larval survival or enzymatic inhibition patterns but modulated specific developmental responses, particularly prolonged larval development and reduced body mass under GORE1 exposure. Nutritional indices (ECI and ECD) were significantly reduced in peptide-treated larvae, supporting impaired protein metabolism. These findings demonstrate that synthetic peptides effectively inhibit trypsin-like proteases in S. frugiperda and negatively affect larval nutritional performance. Although microbiota disruption did not modify survival or primary enzymatic inhibition, microbiota-mediated compensatory mechanisms cannot be excluded. Future integrative studies combining microbiome profiling and metabolic analyses will be essential to resolve host-enzyme-microbiota interactions under digestive inhibition.