Does the Glucocorticoid Stress Response Make Toads More Toxic? An Experimental Study on the Regulation of Bufadienolide Toxin Synthesis

Abstract

Chemical defense is a crucial component of fitness in many organisms, yet the physiological regulation of defensive toxin synthesis is poorly understood, especially in vertebrates. Bufadienolides, the main defensive compounds of toads, are toxic to many predators and other natural enemies, and their synthesis can be upregulated by stressors, including predation risk, high conspecific density, and pollutants. Thus, higher toxin content may be the consequence of a general endocrine stress response in toads. Therefore, we hypothesized that bufadienolide synthesis may be stimulated by elevated levels of corticosterone (CORT), the main glucocorticoid hormone of amphibians, or by upstream regulators that stimulate CORT production. To test these alternatives, we treated common toad tadpoles with exogenous CORT (exoCORT) or metyrapone (MTP, a CORT-synthesis inhibitor that stimulates upstream regulators of CORT by negative feedback) in the presence or absence of predation cues for 2 or 6 days, and subsequently measured their CORT release rates and bufadienolide content. We found that CORT release rates were elevated by exoCORT, and to a lesser extent also by MTP, regardless of treatment length. Bufadienolide content was significantly decreased by treatment with exoCORT for 6 days but was unaffected by exposure to exoCORT for 2 days or to MTP for either 6 or 2 days. The presence or absence of predation cues affected neither CORT release rate nor bufadienolide content. Our results suggest that changes in bufadienolide synthesis in response to environmental challenges are not driven by CORT but may rather be regulated by upstream hormones of the stress response.

Fig. 1

Schematic representation of the HPI axis and potential physiological regulatory pathways of bufadienolide synthesis in common toads. Green arrows indicate upregulation, whereas a red arrow indicates negative feedback. Abbreviations: CRF, corticotropin-releasing factor; ACTH, adrenocorticotropic hormone; CORT, corticosterone; H1, hypothesis 1; H2, hypothesis 2. Photo of a juvenile toad by Nikolett Ujhegyi, used with permission.

Fig. 2

Experimental design applied to containers of tadpoles during the last week of the experiment (treatment period), beginning with day 15. (a) treatment length 6 days, (b) treatment length 2 days. Beakers represent changing the tadpoles’ rearing water with water containing exogenous chemicals (CORT, MTP) or the control solution. Perch represent the addition of reconstituted soft water (control tadpoles) or water from the fish's tank + tadpole homogenate. Icons of beakers used in the figure are licensed by Biorender.

Fig. 3

Effects of treatments (exoCORT: exogenous corticosterone, MTP: metyrapone) on corticosterone (CORT) release rate (a) and mass-corrected total bufadienolide content (mcTBQ, b) of toad tadpoles. Error bars represent estimated means and 84% confidence intervals (CI) calculated from linear mixed-effects models (N = 283). Nonoverlapping CIs indicate significant differences between groups after correction for false discovery rate. Letters above the error bars represent pairwise comparisons; groups not sharing any letter differ significantly (P < 0.05). An asterisk depicts a marginally non-significant difference (P = 0.071) between groups treated with exoCORT and MTP.