Hidden in Asopinae venom: a proteo-transcriptomic characterization reveals novel venom protein families in the predatory stink bug, Picromerus lewisi.

Venoms of predatory heteropterans represent complex cocktails with diverse biological functions. However, studies on their composition and evolutionary background remain limited, particularly within the subfamily Asopinae. This study employed an integrated proteomic and transcriptomic approach to characterize the venom of the predatory stink bug Picromerus lewisi, aiming to address gaps in understanding venom diversity and specialization. Four venom samples were collected: secreted pure venom harvested via harassment (VSH), and homogenates prepared from three dissected venom gland compartments: anterior main gland (AMG), posterior main gland (PMG), and accessory gland (AG). Integrated proteo-transcriptomic analysis identified 472 putative venom proteins, with trypsins (12.7%) being the most abundant family. Notably, 28.0% (132 proteins) were classified as unknown secreted proteins, highlighting substantial unexplored functional diversity. Transcript abundance analysis indicated that the PMG and AMG were the major sites of venom protein transcription, with trypsins and unknown families as major gland-specific components. Clustering analysis identified several previously uncharacterized venom protein families that were detected only in P. lewisi within the comparative datasets. Microinjection-based toxicity assays revealed that all four venom samples induced high lethality in 2nd-instar larvae of Spodoptera litura. Moreover, distinct lethal phenotypes were observed: VSH and AMG homogenate treatments were associated with larval liquefaction and decay, whereas PMG homogenate induced edema and AG homogenate caused marked reductions in mobility accompanied by body contraction. This integrated proteo-transcriptomic analysis characterized the venom of P. lewisi and revealed abundant uncharacterized proteins, thereby not only expanding our knowledge of venom diversity but also providing a useful basis for the discovery of novel insecticidal toxins.