Thermoregulation is a homeostatic process to maintain an organism’s internal temperature within a physiological range compatible with life. In ectotherms, body temperature fluctuates with that of the environment, with both physiological and behavioral responses employed to modify body temperature. Changing skin colour/reflectance and locomotor activity are both well-recognized temperature regulatory mechanisms, but little is known of the participating thermosensor/s. We find that Xenopus laevis tadpoles put in the cold exhibit a temperature-dependent and rapid melanosome aggregation in melanophores, which lightens the skin. To identify the cold-sensor, we focused on transient receptor potential (trp) channel genes from group 1 (TRPA, TRPM, TRPV and TRPC families). Of particular interest were TRPM family members, known as cold sensors. mRNAs for several TRPMs are present in Xenopus tails, and TRPM8 protein is present in skin melanophores. Temperature-induced melanosome aggregation is mimicked by the Trpm8 agonist menthol (WS12) and blocked by the Trpm8 antagonist PF05105679. The degree of skin lightening induced by cooling or the WS12 TRPM8 agonist correlates with the extent of alterations in locomotor performance. We propose that TRPM8 serves as a cool thermosensor in ectotherms that helps coordinate skin lightening and behavioural locomotor performance as adaptive thermoregulatory responses to cold.

Using Xenopus laevis as a model system, we study two responses in ectotherms that contribute to temperature regulation: 1) A skin coloration response where dorsal pigmentation of the tadpole is adjusted to the environmental temperature to change the light refractory conditions, and 2) A behavioural response where the locomotor performance of the organism aligns with the ambient temperature. We explore both the molecular thermosensors, and the relationship between skin colour change and the locomotor preference in response to an ethologically-relevant cold temperature (24 and 6 Celsius degrees). For skin pigmentation studies, pictures of the dorsal head were taken with identical conditions of light, exposure time and diaphragm aperture, and converted to binary white/black images using NIH ImageJ public domain software (see Bertolesi et all 2015; PCMR). Pigmentation level (arbitrary units) was automatically converted by the software. For locomotor performance, a tray containing 9-12 tadpoles set individually in 35 mm dishes were recorded for one minute, every 5 minute during a total time of 30 minutes. The free online software, Kinovea version 0.8.15, was used to track the movement (cm) of each tadpole.

Pigmentation Index (Figure 2; 3; 4 and 5). Physiological skin pigmentation indices were measured as described previously (Bertolesi et al 2015; PCMR). Briefly, pictures of the dorsal head of tadpoles were taken using a stereoscope (Stemi SV11; Carl Zeiss Canada, Ltd., Toronto, Canada) and a camera (Zeiss; Axiocam HRC), with identical conditions of light, exposure time and diaphragm aperture. Pictures were converted to binary white/black images using NIH ImageJ (U. S. National Institutes of Health, Bethesda, MD) public domain software. The density of positive pixels was measured, and the statistical significance (p < 0.05) between treatments was determined with GraphPad Prism 9 by using multiple ANOVA followed by Bonferroni's post hoc test or t-test.

Locomotor Performance: (Figure 5) Single tadpoles were set in a 35 mm dish containing MMR solution at 24 °C or 6 °C. 9-12 dishes on a tray were assessed concurrently for tadpole movement by using a Logitech C920S camera. Trays were kept in either the 24 °C or 6 °C incubator, with light shining from above. Both incubators (24 °C or 6 °C) were adjacent to the behaviour recording device, and trays with dishes were removed every 5 minutes (0-30 minutes) to record the movement for one minute. In this manner, the temperature of the MMR in each dish did not change during the 30 minutes of the study. At the end of the experiment, each tadpole was fixed for assessment of skin pigmentation, and the recordings were used to determine their locomotor performance. The free online software, Kinovea version 0.8.15, was used to track the movement of each tadpole.