Abstract
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 (PHAmax) 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 PHAmax 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 PHAmax was significantly positively correlated with the timing of PHAmax 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 PHAmax 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.
Highlights
Amphibians are experiencing a global biodiversity crisis derived from diseases, habitat destruction and altered ecosystems (Clulow, Harris & Mahony, 2015)
Pairwise comparisons showed that all groups, but Asiatic toad males lost body mass after the PHA assay (Fig. 1)
Sex differences of body mass before the assay were apparent for Asiatic toads, while Dark-spotted frogs and Mongolian toads did not differ
Summary
Amphibians are experiencing a global biodiversity crisis derived from diseases, habitat destruction and altered ecosystems (Clulow, Harris & Mahony, 2015). The assay consists of a subcutaneous injection of PHA that induces infiltration of leukocytes and where swelling is interpreted as an integrative immune response (Christe, Arlettaz & Vogel, 2000; Kennedy & Nager, 2006; Martin et al, 2006; Demas et al, 2011; Brock, Murdock & Martin, 2014) or as an inflammation in endotherms (Vinkler et al, 2012; Bílková, Vinklerová & Vinkler, 2015; Bílková et al, 2016) Inferences using this assay should be carefully evaluated because not always a greater immune response or inflammation means better defenses against infections or higher survival probabilities (Graham, Allen & Read, 2005; Vinkler et al, 2012). An objection is that the response to PHA of an organism might not be representative of the actual response of the same host to pathogens (reviewed by Graham, Allen & Read, 2005; Owen & Clayton, 2007; Brock, Murdock & Martin, 2014) or that energetic costs of mounting a response to PHA may vary in small mammals (Zhang, Qiu & Wang, 2011; Merlo et al, 2014) and birds (Ots et al, 2001; Martin, Scheuerlein & Wikelski, 2003; Lee, Martin & Wikelski, 2005; Nilsson, Granbom & Raberg, 2007)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
