Alarm cue properties and euthanasia impact on tadpole antipredator behaviour in Rana temporaria and Bufo bufo

Chemical cues are crucial for aquatic organisms to assess predation risks, impacting their antipredator mechanisms. Tadpoles, recognized as valuable model organisms, use conspecific alarm cues to modulate their behaviours. However, ethical considerations in chemical communication research often necessitate the use of chemical anaesthetics, potentially disrupting ecologically relevant processes and complicating result interpretation. Furthermore, our understanding of alarm cue characteristics across most anuran species and their potential interactions with chemical anaesthetics remains limited or unexplored. In this study, we examined euthanasia and extract processing techniques on tadpole behaviour in the common toad, Bufo bufo, and common frog, Rana temporaria, to achieve three objectives: investigate alarm cue properties, assess the impact of euthanasia methods on alarm cue effectiveness, and explore anaesthetic interference mechanisms using tricaine and l -arginine. Tadpoles were exposed to various chemical stimuli, including alarm cues extracted via mechanical, hypothermal and chemical euthanasia methods, as well as differently processed crude extracts (formerly frozen, boiled, aged or protein-digested). Additionally, tadpoles were exposed to varying concentrations of l -arginine and tricaine, both individually and in combination. Our results revealed consistent species-specific responses, with R. temporaria tadpoles exhibiting reduced activity in response to alarm cues, while B. bufo tadpoles displayed no behavioural changes. Moreover, we observed significant differences among methods of euthanasia. Tadpoles of R. temporaria responded to processed extracts and l -arginine, indicating the presence of amino acids but not proteinaceous compounds in alarm cues. Finally, divergent species’ responses to l -arginine and tricaine suggest interference within individuals rather than alarm cues. This study enhances our understanding of alarm cue characteristics in tadpoles and underscores the potential species-specific impact of chemical anaesthetics on behavioural responses in aquatic organisms. It underscores the necessity for further research into interference mechanisms and alternative euthanasia methods while emphasizing the importance of comparative studies in comprehending chemical communication in aquatic ecosystems. • Responses to alarm cues and l -arginine are consistent but species specific. • Nonproteinaceous alarm cues in R. temporaria are temperature and time stable. • Method of euthanasia impacted alarm cue efficiency in our study set-up. • Responses to subeffective tricaine doses are species specific. • Tricaine interferes with an individual's response capability rather than alarm cues.