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Phytohemagglutinin (PHA)-induced skin swelling response is widely used as a rough surrogate of integrative cell-mediated and innate immunity across multiple vertebrate taxa due to its simplification and feasibility. However, little is known whether there are sex and interspecific differences of immune responsiveness to PHA in ectotherms, especially for anurans. Therefore, we studied sex and species differences of PHA response in three anurans, Asiatic toads ( Bufo gargarizans ), Dark-spotted frogs ( Pelophylax nigromaculatus ) and Mongolian toads ( Pseudepidalea raddei ), captured in northern regions of Anhui Province (China). Footpad thickness was measured prior to (0 h) and after (6, 12, 24, 48 and 72 h) a PHA injection and normalized against saline injection in the opposite footpad. Body mass was recorded at the beginning (0 h) and end of each assay (72 h). Results showed effects of PHA assay, sex and taxa on body mass. Relative maximum swelling response (PHA max ) ranged from 18.58–29.75%, 9.77 to 20.56% and 21.97 to 31.78% and its occurrence over time was apparent 10.6–19.72 h , 7.74–14.01 h and 17.39–23.94 h postinjection for Asiatic toads, Dark-spotted frogs and Mongolian toads, respectively. Finally, the magnitude or timing of PHA max  in Dark-spotted frogs was significantly thinner and faster than in Mongolian toads, and Asiatic toads had an in-between value, not different from the other two species. The magnitude of PHA max  was significantly positively correlated with the timing of PHA max  considering individuals altogether, but not when analyzed within species. Our results indicate that male and female anuran species respond similarly to PHA antigen stimulation, but the magnitude and timing of PHA max  is species-specific. Briefly, we provide new evidence for the suitability of PHA assay in non-model anuran species with different body sizes, and exhort the need to further investigate the nature of PHA assay at the hematological and histological levels in order to extend its application in ecoimmunological studies of amphibians.

Immune responsiveness to phytohemagglutinin displays species but not sex differences in three anuran species