Abstract
Amphibians are one of the most threatened groups of species, facing stressors ranging from habitat degradation and pollution to disease and overexploitation. Stress hormones (glucocorticoids, GCs) provide one quantitative metric of stress, and developing non-invasive methods for measuring GCs in amphibians would clarify how diverse environmental stressors impact individual health in this taxonomic group. Saliva is an advantageous matrix for quantifying GCs, as it is sampled less invasively than plasma while still detecting both baseline and acute elevation of GCs within a short timeframe. Little work has employed this method in amphibian species, and it has never been pharmacologically and biologically validated. Here, we conduct analytical, pharmacological and biological validation experiments for measuring salivary corticosterone in three amphibian species: the American bullfrog (Rana catesbeiana), the green frog (Rana clamitans) and the northern leopard frog (Rana pipiens). These species are faced with a broad range of environmental challenges, and in part of its range R. pipiens populations are currently in decline. In addition to demonstrating that this method can be reliably used in multiple amphibian species, we present an examination of intrinsic biological factors (sex, body condition) that may contribute to GC secretion, and a demonstration that saliva can be collected from free-living animals in the field to quantify corticosterone. Our findings suggest that saliva may be useful for less invasively quantifying GCs in many amphibian species.
Highlights
As one of the most threatened taxonomic groups, amphibians are currently faced with a broad range of stressors including habitat loss, disease, overexploitation and pollution (Hof et al, 2011; Vié et al, 2009)
In addition to demonstrating that this method can be reliably used in multiple amphibian species, we present an examination of intrinsic biological factors that may contribute to GC secretion, and a demonstration that saliva can be collected from free-living animals in the field to quantify corticosterone
Handling led to clear and significant increases in corticosterone for all species, with the expected lower magnitude increases than adrenocorticotrophic hormone (ACTH)-induced changes (Wilcoxon rank sum tests with false discovery rate adjusted P-values comparing corticosterone concentrations at 30 min for ACTH vs. handling challenges, R. catesbeiana: W = 87, P = 0.02; R. clamitans: W = 100, P = 0.01; R. pipiens: W = 57, P = 0.02; at 0 min all P-values testing for differences in salivary corticosterone between ACTH vs. handling challenges are >0.5; Table 2; Fig. 2)
Summary
As one of the most threatened taxonomic groups, amphibians are currently faced with a broad range of stressors including habitat loss, disease, overexploitation and pollution (Hof et al, 2011; Vié et al, 2009). There are often ties between GCs and individual or population health and fitness (Hing et al, 2016; Vitousek et al, 2018), and in a broad range of taxonomic groups, less invasive measures of GCs (e.g. feces, urine, saliva) have allowed for physiological monitoring of free-living populations while avoiding unnecessary. Despite the imperiled status of many amphibian species, few non-invasive methods for quantifying GCs have been validated in this group (Narayan, 2013)
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