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

1Ontogenetic 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 PLA2 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/PLA2-rich ontotype B ([≥]2 years). Functionally, protease activity decreased with age, whereas thrombin-like, plasmin-like and PLA2 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.