Assay validation of saliva glucocorticoids in Columbia Spotted Frogs and effects of handling and marking

Abstract/Summary

Non-invasive methods are important to conservation physiology to reduce negative effects on the species being studied. Glucocorticoid (GC) hormones are often used to assess health of individuals, but collection methods can be invasive. Many amphibians are imperiled worldwide, and saliva is a non- or semi-invasive matrix to measure GCs that has been partially validated for only four amphibian species. Validation ensures that assays are reliable and can detect changes in saliva corticosterone (sCORT) following exposure to stressors, but it is also necessary to ensure sCORT concentrations are correlated with plasma concentrations. To help validate the use of saliva in assessing CORT responses in amphibians, we captured uniquely marked Columbia Spotted Frogs (Rana luteiventris) on sequential days and collected baseline and stress-induced (after handling) samples. For a subset of individuals, we collected and quantified CORT in both saliva and blood samples, which have not been compared for amphibians. We tested several aspects of CORT responses and, by collecting across separate days, measured repeatability of CORT responses across days. We also evaluated whether methods common to amphibian conservation, such as handling alone or handling, clipping a toe, and tagging elevated sCORT. Similar to previous studies, we show that sCORT is reliable concerning parallelism, recovery, precision, and sensitivity. Saliva CORT was weakly correlated with plasma CORT (R2 = 0.21), and we detected elevations in sCORT after handling, demonstrating biological validation. Toe-clipping and tagging did not increase sCORT over handling alone, but repeated handling elevated sCORT for ~72 hours. However, sCORT responses were highly variable and repeatability was low within individuals and among capture sessions, contrary to previous studies with urinary and waterborne CORT. Saliva CORT is a semi-invasive and rapid technique that could be useful to assess effects of anthropogenic change, and conservation efforts, but will require careful study design and future validation.

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