Comprehensive Analysis of Cathepsin Genes in Hemiptera: Functional Characterization of the Venomous Cathepsin B from Sycanus bifidus

Cathepsins represent a crucial group of protein enzymes involved in insect metabolism. Within the Hemiptera order, comprising a diverse array of predatory, blood-feeding, and herbivorous species, the understanding of cathepsin types and their roles as venom components in predatory bugs remains limited. This investigation systematically identified cathepsin genes present in Hemiptera genomes, highlighting a prevalence of cathepsin B and L, with cathepsin D exhibiting a higher gene count in the Heteroptera suborder. Examining the predatory assassin bug <i>Sycanus bifidus</i>, eight cathepsin genes were notably expressed in its venom glands, with the SbCAB2 gene from the cathepsin B subfamily demonstrating the highest expression in the posterior main gland, indicating its significance as a venom component. Subsequent expression and purification of the recombinant SbCAB2 protein revealed heightened hydrolytic activity (0.91 U/mg protein) compared to extracts from the anterior main gland, accessory gland, and gut. Functional assays demonstrated that SbCAB2, at lower doses (0.625-2.5 μg), can impede phenoloxidase activity in <i>Tenebrio molitor</i> pupal hemolymph, with a 2.5 μg dose inhibiting 86.5% of this activity, thereby preventing hemolymph melanization. Conversely, a higher dose of 10 μg led to effects akin to human placental cathepsin B, promoting melanization in <i>T. molitor</i> pupal hemolymph. These findings lay the foundation for further exploration of the adaptive evolution of cathepsin genes in Hemiptera and offer crucial insights into the functional role of venomous cathepsins in predatory bugs.