Abstract: One of the major challenges in amphibian ecological immunology is to screen available immunological parameters suitable for different life history stages of amphibian species, including tadpoles, juveniles, and adults. Phytohemagglutinin (PHA) is a kind of mitogen, which is commonly used to predict the strength of integrated immune response in vertebrates according to the maximal response value to PHA (PHAmax) and its occurrence time. However, it was employed relatively later in amphibian species. Based on the complex life history of amphibians and the components of their immune system, we introduced the advantages and disadvantages of responsiveness to PHA as an immunological parameter, available injection positions and recommended doses, as well as calculation method, histological and hematological mechanisms for increased thickness of skin tissue. We addressed the relationship between PHA response and immunocompetence in amphibians, including the influence of species identity, gender, developmental stage, microplastics, water level, and temperature on its response pattern and swelling peak in juvenile and adult individuals. Compared to the diversity of amphibians, the application range of responsiveness to PHA as an immunological parameter is extremely limited and needs to be rapidly expanded. For some specific species, the injection dose of PHA solution, the measurement interval setting of swelling response, the optimization of the sampling measurement time point, and the histological basis induced by PHA, as well as suitability for field studies, require further studies. Optimizing the experimental protocols of responsiveness to PHA, then forming a unified methodology within and across amphibian species, are key to introducing this immunological parameter into the field of wild amphibian ecology.

Key words: amphibian, responsiveness to phytohemagglutinin, ecological immunology, life history, tadpole