DOI: 10.5061/dryad.dbrv15f4b
title: Evidence that catecholaminergic systems mediate dynamic colour change during explosive breeding events in toads

description: 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.
description: 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 <em>i</em>) 50 µl EP
 (Sigma-Aldrich E4250;
 1.8 µg/g bodyweight),
 <em>ii</em>) 50 µl NE (Sigma-Aldrich A7256; 0.4 µg/g bodyweight), or <em>iii</em>) 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).