Seasonal oestradiol dynamics and male responses to female chemical cues in Salamandrina perspicillata

Chemical communication plays a central role in reproductive interactions, yet the extent to which female endocrine state is reflected in chemical cues and used by males remains poorly understood in most amphibians. We investigated seasonal variation in urinary 17 beta-oestradiol (E2) and its association with male responses to female chemical cues in the Italian endemic salamander Salamandrina perspicillata. Using non-invasive urine sampling, we quantified female E2 across the reproductive cycle and conducted two Y-maze assays to assess male responses to female odours. Female urinary E2 showed marked seasonality, peaking in late winter during vitellogenesis but preceding oviposition, consistent with a dissociated reproductive cycle. In an orientation assay (female cue vs. control), male responses varied with female E2 levels, with lower-E2 females eliciting more male approaches. However, in a forced-choice assay between two females differing in E2, males did not show a consistent preference. These results indicate that oestradiol-related variation in female chemical cues may influence male orientation behaviour without acting as a standalone determinant of mate choice. To our knowledge, this is the first study linking female urinary E2 to male behavioural responses to female chemical cues in urodeles. More broadly, our results support a context-dependent view of mate assessment based on multicomponent chemical phenotypes. Finally, the successful application of minimally invasive urinary hormone monitoring provides a useful tool for studying endocrine dynamics in small-bodied amphibians, with potential relevance for understanding reproductive ecology, particularly in environments affected by endocrine-disrupting chemicals.