Many animals communicate by rapidly (within minutes or seconds) changing their body coloration. We know, however, little about the physiology of this behaviour. Here we study how catecholaminergic hormones regulate rapid colour change in explosive breeding toads (Duttaphrynus melanostictus), where large groups of males gather and quickly change their colour from brown to bright yellow during reproduction. We find that both epinephrine (EP) and/or norepinephrine (NE) cause the toads’ skin to become yellow in minutes, even in the absence of social and environmental cues associated with explosive breeding. We hypothesize that natural selection drives the evolution of rapid colour change by co-opting the functional effects of catecholaminergic action. If so, then hormones involved in “fight or flight” responsivity may mechanistically facilitate the emergence of dynamic visual signals that mediate communication in a sexual context.

On three separate experimental sessions, we examined the effect of EP and NE treatment on skin colouration. In each session, we randomly assigned 15 males to three different treatment groups (EP, NE, or control; n=5/group). We allowed four weeks to pass between sessions, as this time exceeds an adequate period that ensures all catecholamines have cleared from the toads’ circulation. Each experimental session consisted of two parts: (1) priming with human Chorionic Gonadotropin (hCG) and (2) treatment with different catecholamines or saline solution (control). Before part 1, we measured the toads’ size (SVL, mm), weight (g) and back reflectance (baseline 1). Subsequently, we primed each toad with a single injection of 30 µl hCG (Sigma-Aldrich CG10-1VL; 20 IU/g bodyweight in saline solution) and placed them in their respective Fauna box (20x19x20 cm) for a period of five hours. hCG is a common exogenous reproductive hormone, widely used to induce reproduction and breeding behaviour in amphibians. To verify the impact of hCG injections on body colouration, we visually checked the individual's body colouration hourly. Pigmentation changes of the toads between the general brown colouration and their conspicuous nuptial bright yellow are highly noticeable to human observers and easy to assess. After five hours, we again performed reflectance measurements of all individuals (baseline 2) followed by the respective EP, NE or saline treatments via 50 µl intraperitoneal injection in the dorsal lymph sac. The test groups were administrated a dose of i) 50 µl EP (Sigma-Aldrich E4250; 1.8 µg/g bodyweight), ii) 50 µl NE (Sigma-Aldrich A7256; 0.4 µg/g bodyweight), or iii) 50 µl saline (0.9% NaCl) as a control. Drug dosages were chosen according to the weight of the species based on prior studies in fish and amphibians. In all experiments, we minimized handling time to less than 5 min to reduce unintended stress effects on circulating levels of catecholamines. Back colouration was measured 30, 60, 120, 180 min and 12 hours after treatment with a spectrometer (JAZ series; Ocean Optics, Dunedin, USA) according to a standardized protocol. We used Avicol v6 software (31) to extract brightness, hue, and chroma measures – the essential parameters to describe colouration from reflectance spectra. Significant effects of our treatments on the colour variables do not necessarily imply that these effects can be perceived by conspecific rivals or mating partners. Thus, we additionally quantified whether spectral differences could be detectable by toads using a colour vision model. We thereby examined the ability of toads to discriminate among untreated females (n=15), control group males, and EP- and NE-treated males, 30 minutes after the treatment (for detailed information see supplementary material of publication).