A venom serpin from the assassin bug Sycanus croceovittatus exhibiting inhibitory effects on melanization, development, and insecticidal activity towards its prey

Abstract

Serine protease inhibitors (SPIs) have been identified as main common components in the venom of the predatory bugs, while their functional roles remain unexplored. In this study, we identified 35 SPI genes belonging to three subfamilies of serpin, canonical SPI, and A2M in genome of the assassin bug, Sycanus croceovittatus. The amino acid sequences of these SPI genes reveal conserved functional regions, albeit with mutations or deletions at certain active site residues. Transcriptomic and qPCR analyses of gene expression patterns in various tissues across developmental stages indicate that most SPI genes exhibit high expression levels in venom apparatus, suggesting their role as venom proteins. Notably, the ScSPI5 gene from the serpin class was found to be most abundantly expressed in all three distinct venom glands, indicating its significant role as a venomous protein. Functional characterization demonstrated that this venom serpin effectively inhibits trypsin activity in vitro and suppresses phenoloxidase activity, thereby blocking hemolymph melanization in preys, including Spodoptera frugiperda, Achelura yunnanensis, and Tenebrio molitor. When ingested, it reduces the larval and pupal weight of the fall armyworm by impeding trypsin activity in the midgut. Upon injection, ScSPI5 exhibits a dose-dependent insecticidal effect against T. molitor, with an LD₅₀ of 5.6 ± 1.1 μg/g. These findings elucidate the specific functions of SPIs in the venom of predatory bugs, enhancing our understanding of their predation efficiency, and highlighting the potential application of venomous SPIs as protease inhibitors in pest management strategies.