Identifying individual age and size is crucial in venom research to understand potential phenotypic changes. When studying venom ontogeny, juveniles and adults are often determined by size at sexual maturity. However, in gape-limited predators such as snakes, venom shifts may occur earlier, coinciding with an increase in jaw size that allows the intake of larger prey. This study explored venom variation in Vipera ammodytes along the snout-vent length (SVL), linking these changes to dietary shifts and reproductive status. A total of 57 venom samples from two populations were analysed using chip electrophoresis (CE) and MALDI-TOF mass spectrometry. Individuals were categorised into three groups: immature individuals feeding on lizards (<300 mm SVL), immature individuals feeding on lizards but start including mammals in their diet (between 300 and 440 mm), and mature individuals feeding on mammals and lizards (>440 mm). Significant venom composition changes around 300 mm SVL were observed, aligning with a dietary shift marked by increased mammalian prey intake and preceding sexual maturity. This finding highlights the need to use SVL as a metric for accurate venom analysis along ontogeny. Our results indicate that when pooling venom samples is necessary, allocating individuals to age categories should be based on dietary shifts rather than sexual maturity. Additionally, CE and MALDI-TOF MS proved effective in detecting ontogenetic venom changes, offering a fast and affordable approach to venom profiling. This study emphasises the importance of integrating natural history data in venom studies to better understand the ecological and evolutionary drivers of venom adaptation.
