DOI: 10.5061/dryad.6q573n61f
title: TRPM8 thermosensation in ectotherms mediates both skin colour and locomotor performance responses to cold temperature

description: 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.
description: 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 <strong>pigmentation
 studies</strong>, 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
 <strong>locomotor performance</strong>, 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 &lt; 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.