Urea and glucose modulation during freezing exposure in three temperate frogs reveals specific targets in relation to climate.

Amphibian diversity is most prominent in the warm and humid tropical and subtropical regions across the globe. Nonetheless, amphibians also inhabit high-altitude tropical mountains and regions at medium and high latitudes, exposing them to subzero temperatures and requiring behavioural or physiological adaptations to endure freezing events. While freeze tolerance has been predominantly reported in high-latitude zones where species endure prolonged freezing (several weeks or months), less is known about mid-latitudes amphibians exposed to occasional subzero temperatures. In this study, we employed a controlled ecological protocol, subjecting three frog species from the Iberian Peninsula (Rana parvipalmata, Epidalea calamita, and Pelobates cultripes) to a 2-h exposure to temperatures of -2 °C to investigate the accumulation of urea and glucose as physiological mechanisms associated with survival at freezing temperatures. Our results revealed a moderate response in the production of cryoprotectant metabolites under experimental freezing conditions, particularly urea, with notable findings in R. parvipalmata and E. calamita and no response in P. cultripes. However, no significant alterations in glucose concentrations were observed in any of the studied frog species. This relatively weak freezing tolerance response differs from the strong response exhibited by amphibians inhabiting high latitudes and enduring prolonged freezing conditions, suggesting potential reliance on behavioural adaptations to cope with occasional freezing episodes.