Are baseline and short-term corticosterone stress responses in free-living amphibians repeatable?

Amphibians respond to environmental stressors by secreting corticosterone, a stress hormone which promotes physiological and behavioral responses. Capture handling can be used to stimulate physiological stress response in amphibians. The use of single blood sampling and presentation of mean data often limits the quantification of within and between individual variation in baseline and short-term corticosterone stress responses in amphibians. It is important for studies of amphibian physiological ecology to determine whether baseline and short-term corticosterone stress responses are consistent or not. We quantified repeatability (r), a statistical measure of consistency, in baseline and short-term corticosterone stress responses to a standard capture and handling stress protocol in free-living adult male cane toads (Rhinella marina). Corticosterone metabolite concentrations were measured entirely non-invasively in male toad urine samples via an enzyme-immunoassay. During the first sampling occasion, urine samples were collected manually from individual male toads (n=20) immediately upon field capture. Toads were handled for 5min then transferred to plastic bags (constituting a mild stressor), and urine samples were collected hourly over 8h in the field. The toads were resampled for baseline (0h) urine corticosterone with hourly urine sampling over 8h (for quantification of the stress induced corticosterone) at 14 day intervals on three consecutive occasions. Within and between sample variations in urinary corticosterone metabolite concentrations were also quantified. All toads expressed a corticosterone stress response over 8h to our standard capture and handling stress protocol. Variations both within and between toads was higher for corrected integrated corticosterone concentrations than corticosterone concentrations at baseline, 3 or 6h. Baseline urinary corticosterone metabolite concentration of the male toads was highly repeatable (r=0.877) together with high statistical repeatabilities for 3h (r=0.695), 6h (r=0.428) and 8h (r=0.775) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r=0.807; r=0.743 respectively). This study highlights that baseline and short-term corticosterone stress responses are repeatable in free-living amphibians. Future studies should utilize this non-invasive tool to explore repeatability among seasons and across years, and determine its functional significance in relation to behavioral ecology and reproduction in amphibians generally.