Ontogenetic variation in composition and bioactivity of common adder ( Vipera berus ) venom revealed by genome-guided proteomics and in vitro functional assays

1 Ontogenetic shifts in diet are well documented in snakes and are increasingly linked to age-related venom variation. The common adder, Vipera berus , exhibits a dietary transition from predominantly ectothermic prey in its early life to increasingly incorporating endothermic prey as an adult. Here, we investigate whether this dietary shift is reflected in age-related changes in the venom composition and bioactivity of V. berus . Venoms from captive-bred V. berus from Germany were obtained and pooled across five age groups, from neonates to adults. Venom profiles were assessed by SDS-PAGE and genome-guided shotgun proteomics, with quantification based on normalized spectral abundance factors (NSAF) using a toxin-gene catalogue generated from a novel V. berus genome assembly. In parallel, we assayed general protease and PLA 2 activities, as well as FXa-, thrombin-, and plasmin-like activities, and cytotoxicity toward mammalian cell lines. We identified two distinct age-related venom phenotypes (ontotypes): an svMP/CTL-rich ontotype A (≤1 year) and an svSP/PLA 2 -rich ontotype B (≥2 years). Functionally, protease activity decreased with age, whereas thrombin-like, plasmin-like and PLA 2 activities, and cytotoxicity, increased. Our findings indicate an ontogenetic shift in composition and activities of V. berus venom that parallels dietary transitions and potentially reflect adaptation to differing prey physiologies.