Increased radiation from Chernobyl decreases the expression of red colouration in natural populations of bank voles (Myodes glareolus)

Pheomelanin is a pink to red version of melanin pigment deposited in skin and hair. Due to its bright colour, pheomelanin plays a crucial function in signalling, in particular sexual signalling. However, production of pheomelanin, as opposed to its dark alternative, eumelanin, bears costs in terms of consumption of antioxidants important for protection of DNA against naturally produced reactive oxidative species. Therefore, decreased expression of pheomelanin is expected in organisms exposed to severe oxidative stress such as that caused by exposure to chronic ionizing radiation. We tested if variable exposure to radiation among natural populations of bank voles Myodes glareolus in Chernobyl affected expression of red colouration in their dorsal fur. The relative redness of dorsal fur was positively correlated with weight, but also negatively correlated with the level of background radiation. These results suggest that the development of the natural red colouration in adult bank voles is affected by ionizing background radiation, and potentially causing elevated levels of oxidative stress. Reduced production of pheomelanin allows more antioxidants to mitigate the oxidative stress caused by radiation. However, changing natural animal colouration for physiological reasons can have ecological costs, if e.g. it causes mismatch with habitat colouration and conspicuousness for predators.

C olouration in animals may be adaptive and have variable functions, from concealment and communication to regulation of physiological processes 1 . Experimental selection on colouration has been demonstrated 2,3 , and it has been argued that it can cause rapid evolutionary change 4,5 . In mammals colouration mainly depends on deposition of two forms of melanin, red to pink pheomelanin and black to brown eumelanin, during growth of hair. Regulation of expression of two antagonistic genes (Melanocortin-1 receptor and Agouti signalling protein) determines the proportion of expression in the same melanocytes of either the darker or the lighter form 6 . However, production of either of the two melanin forms carry variable costs that can affect expression levels, particularly under stressful condition 7 . Despite of its potential function 8,9 during synthesis of pheomelanin substantial amounts of the most important intracellular antioxidant, glutathione (c-glutamylcysteinyl-glycine), is consumed. Consequently this antioxidant is not available for neutralizing free radicals, accumulation of which can cause oxidative stress, DNA damage and development of diseases 10 . Production of the alternative to pheomelanin, eumelanin, does not carry such a cost, and, therefore, down-regulation of red colour expression may be a useful mechanism for animals exposed to chronic stressors that cause release of free radicals 7,11 .
Chronic exposure to ionizing radiation can cause oxidative stress, deterioration of phenotypes, and death, affecting the ecology and evolution of organisms 12 . It has been shown that birds can adapt to chronic and elevated ionizing radiation, where birds exposed to higher doses of radiation have evolved lowered expression of pheomelanin 13 . Such an effect has never been detected in mammals, although they are exposed to higher and more chronic doses of external ionizing radiation than birds as they live closer to or in (e.g. rodents and shrews) media that accumulate radioactive particles, such as soil. To estimate expression level of pheomelanin we measured OPEN SUBJECT AREAS: RISK FACTORS EVOLUTIONARY ECOLOGY relative redness of colouration 7 of dorsal fur in wild rodents, bank voles Myodes (5 Clethrionomys) glareolus (Schreiber, 1780), exposed to variable levels of ionizing radiation in Chernobyl (Fig. 1). We expected to find a negative association between the level of dorsal fur redness and the level of ionizing radiation of soil if the animals' expression of pheomelanin changes either due to adaptation or phenotypic plasticity.
The level of radiation of the soil in the locations where animals were trapped varied from 0.01 to 34.69 mSv/h (with median and mean of 0.37 and 2.75). Overall colour luminosity and reflectance of red colour varied from 21 and 28 to 53 and 65 of 8-bit RGB scale (with medians and means of 35.3, 44.6, 35.6 and 45.1, respectively), with relative red colour varying from 0.38 to 0.51 (with median and mean of 0.46). Animal weight varied from 11.9 to 35.1 g (with median and mean of 23.5 and 23.4). Repeatability (coefficient of intraclass correlation 14 ) of colour measures estimated from repeated calculations of 21 specimens were significant for all traits (overall luminosity: t 5 0.93, p , 0.001; reflectance of red colour: t 5 0.88, p , 0.001; relative red colour: t 5 0.44, p 5 0.0196). Also repeatability of spectroscopy measures of relative fur irradiance, conducted to validate photographic analyses, was significant (t 5 0.51, , 0.001), and spectroscopy measures were significantly correlated with both the reflectance of red (r 5 0.40, t 5 3.01, df 5 49, p 5 0.004) and the overall luminosity (r 5 0.34, t 5 2.57, df 5 49, p 5 0.013) calculated from photographs.

Discussion
In this study we showed that expression of the relative red colour of dorsal fur in a common rodent depended on the weight of specimens, but also on the level of ionizing radiation of the habitat in Chernobyl where the animals were trapped (Fig. 1). The results based on the photographic method used here (Fig. 2), were validated and corroborated with a spectrophotometric analysis on a smaller sub-sample (Fig. 3).
While dorsal fur colouration might depend on habitat type 14,15 and affect animal fitness 2,5 , here we did not find an association between animal colouration and vegetation coverage (i.e. descriptions of habitat type in the forest ecosystem). Rather we found that the relative redness of dorsal fur was a function of animal size, which is a surrogate of age, suggesting that bigger, and older, animals express   brighter colouration (Table 1). This is expected as adult colouration in this species is reddish while pups are born greyish 16 . At the same time the effect of animal size and the relative amount of red colour of dorsal fur was significantly explained by radioactive contamination in the trapping area measured here as ionizing radiation ( Fig. 2A,  Fig. 3). The size of animals and habitat contamination were not correlated with each other. But as the effect of radiation on colouration (Fig. 2a) was opposite in direction to the effect of body size (Fig. 2b), it suggests that ionizing radiation compromises the expression of adults' natural fur colouration and/or there might be selection for larger size in more contaminated areas. The ecological significance of the decrease of relative red colouration in Chernobyl bank vole populations is speculative as there are no data showing the importance of this character for performance in this species. In other rodent species dorsal colouration is often cryptic and conspicuous animals are more easily found by predators 2 . Nevertheless, it is likely that rodents experiencing conditions of chronic oxidative stress, such as around nuclear disaster sites (e.g. Chernobyl and Fukushima), can down-regulate their expression of pheomelanin, or alternatively, have evolved adaptive responses similar to those observed for birds 13 whereby low expression levels of pheomelanin, is associated with mitigation of oxidative stress.

Methods
Study animals and study area. Bank voles were captured with Ugglan Special 2 live traps (Grahnab, Sweden), with sunflower seeds and potato as bait, during 2011 (June), 2013 (September) and 2014 (June) from 68 locations in the Chernobyl region of Ukraine (Fig. 1). At each location 8-20 traps were placed, with an inter-trap distance of 10 m. Traps were set for two nights. In 2011, 2013, and 2014, 51 (17 females, 34 males), 11 (5 females, 6 males) and 127 voles (53 females, 74 males) were captured, respectively. Vegetation cover (%) was estimated within 1 m radius from each trap. The vegetation cover was estimated in three layers: forest litter (0-50 cm), bushes (0.5-2 m) and canopy (above 2 m). Animals captured in 2011 and 2013 were sacrificed by cervical dislocation and stored at 220uC. Animals captured in 2014 were released back to their original trapping locations. All animals were weighed (Mettler Toledo XS204, to the nearest 0.1 g). All procedures were performed in accordance with relevant guidelines and regulations. The study was approved by the Finnish Ethical Committee (license numbers: ESLH-2008-04660/Ym-23 and ESLH-2009-09663/Ym-23).
Measurements of background radiation. Hand-held dosimeters (Inspector, SE International, Inc, Summertown, TN, USA) calibrated to measure Sieverts (Sv) were used to measure soil radiation at the exact trapping sites of each animal. Radiation at capture sites varied from 0.01 to 133.00 mSv/hour, although no animals were trapped in sites with radiation levels above 34.69 mSv/hour.   colour and white balance grey card. The original large RAW photographs were white balanced with 250 pixels of white standard in GIMP 2.6.12 (www.gimp.org) and saved in high resolution (3900 3 2600 pixels) TIFF format. Colouration was estimated from a square-shaped area (300 3 300 pixels) of a dorsal part of the skin in Hyper-Utility 2 (www.fujifilm.com). Reflectance of red, blue and green were measured as Red-Green-Blue (RGB, 8-bit) standard values (from 0 to 255) from which relative red colour (red/ (red1blue1green) of the dorsal fur was estimated 14 . In order to estimate repeatability of colour measurements, we calculated the intraclass correlation coefficient (t) for 21 individual samples that were reanalysed without prior knowledge of identity or colouration during the second measurements.
To validate the above method, reflectance spectroscopy was used to measure relative irradiance of red colour in fur of dorsal skin specimens. Light was provided by an Ocean Optics PX-2 light-source (Ocean Optics, Dunedin, FL, USA). Relative irradiance was measured with a MayaPro 2000 spectrometer (Spectrecology, Jasper, GA, USA) and analysed with the SpectraSuite software (Ocean Optics, Dunedin, FL, USA). Prior to measurements and after each tenth measurement the setup was recalibrated against a Labsphere SRS-99-010 reference surface (Labsphere, North Sutton, NH, USA). The light source and sensor were aligned at an angle to measure irradiance at a distance of 10 cm from the fur. Relative irradiance was measured at 600 nm with a 200 ms integration time. From each dorsal skin sample three measures were taken along the medial line. The average of these measures was used for analyses. Furthermore, 19 animals were re-analysed on three consecutive days to estimate repeatability.
Statistical analyses. As the distribution of soil radiation was right skewed all analyses were performed on log 10 transformed values of continuous variables. Statistical significance of effects explaining variation in relative redness [mean luminosity of red/(mean luminosity of red 1 mean luminosity of green 1 mean luminosity of blue)] of dorsal fur was tested with models that included soil radiation, animal weight (continuous predictors), sex (fixed effect), interactions between continuous predictors and sex, and random effects of population of origin (68 levels) and year (3 levels). Vegetation cover and interactions with sex were omitted from the final model as they were not significant. R Project packages ''lm4'', for generalized linear mixed model, and ''LMERConvenienceFunctions'', for log likelihood ratio test were used.