Ontogenetic variation in Milos viper (Macrovipera schweizeri) venom composition and biochemical activity

Ontogenetic shifts in diet are common in venomous snakes, with juveniles and adults often feeding on different prey types. These dietary changes are frequently paralleled by age-related variation in venom composition, likely reflecting adaptive optimisation for prey with different physiologies, particularly ectothermic versus endothermic organisms. Here, we investigated age-dependent differences in venom composition and biochemical activity in the Milos viper (Macrovipera schweizeri), a medically relevant venomous snake species that shifts from a lizard-based to an avian-based diet during growth. Genome-guided shotgun proteomics, supported by RP-HPLC and SDS-PAGE profiling, identified 12 toxin families in both juvenile and adult venoms, with four components detected exclusively in adults. Juvenile venom was dominated by snake venom metalloproteinases (SVMPs; 45% vs. 22% in adult venom), whereas adult venom contained a higher proportion of C-type lectins and C-type lectin-related proteins (CTLs; 40% vs. 25% in juvenile venom). Other toxin families, including phospholipase A₂ (PLA₂), serine protease (SVSP), and cysteine-rich secretory protein (CRISP), showed less pronounced differences. In vitro assays revealed potent proteolytic activity in both venoms, with juveniles showing higher activity and exceeding the positive control at the highest concentrations (25 and 50 μg/ml). Juvenile venom also exhibited higher overall PLA₂ activity than adult venom. Both venoms induced marked cytotoxicity in MDCK II and BHK cells, but only at the highest concentration tested (25 μg/ml). Our results reveal a clear ontogenetic shift in the composition of M. schweizeri venom, consistent with the species' dietary transition and potentially relevant for the subjugation of prey with differing physiologies.