Melanophore metachrosis response in amphibian tadpoles: Effect of background colour, light and temperature

Manuscript Click here to access/download;Manuscript;Crypsis Short note EJRR-RM.docx This is an Accepted Manuscript of an article published by Brill in Amphibia - Reptilia 1 on 17 Sep 2020, available at: https://doi.org/10.1163/15685381-bja10032 It is deposited under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/bync-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way 2 3 4 Melanophore metachrosis response in amphibian tadpoles: 5 effect of background colour, light and temperature. 6 Rodríguez-Rodríguez, Eduardo José1; Beltrán, Juan Francisco1; Márquez, Rafael2 7 1Grupo evolución, ecología y conservación de vertebrados. Departamento de Zoología. 8 Facultad de Biología. Universidad de Sevilla. 9 2Fonoteca Zoológica, Departamento de Biodiversidad y Biología Evolutiva, Museo 10 Nacional de Ciencias Naturales-CSIC, Madrid, Spain. 8 9 10 11 Corresponding author: Rodríguez-Rodríguez, EJ. E-mail: [email protected] 12 13 ORCID id: 14 EJRR:https://orcid.org/0000-0002-1170-0788 15 JFB: https://orcid.org/0000-0002-0077-575X 16 RM: https://orcid.org/0000-0002-2070-860X 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Running title: Melanophore metachrosis response in tadpoles 35 Melanophore metachrosis response in amphibian tadpoles: 36 effect of background colour, light and temperature. 37 38 Key words: behavioral, melanosome, melanin, phenotypic plasticity, thermoregulation. 39 Summary 40 The developmental and biochemical mechanisms of colour change through 41 chromatophore metachrosis in amphibian tadpoles are relatively well studied, but the 42 environmental factors driving colour change remain unclear. A cryptic response to background 43 colour in order to reduce predation is an intuitively valid explanation, however, other 44 hypotheses need to be explored. In this study, we aimed to investigate the environmental 45 factors driving the melanophore metachrosis process in Alytes dickhilleni tadpoles. First, we 46 tested the response to two backgrounds with clearly distinct reflectance: black and white. The 47 proportion of dark tadpoles became significantly higher when they were located on the black 48 background, and pale tadpole proportion was dominant on the white background, as expected 49 from the crypsis hypothesis. Secondly, we added two new factors, temperature and 50 photoperiod, maintaining the background variation. Our results suggest that lower 51 temperatures, and short photoperiods were significantly driving a change to dark colouration in 52 tadpoles, possibly allowing a more efficient thermoregulation, and in consequence, 53 development and growth. Next we tested whether dark and pale tadpoles selected backgrounds 54 that matched their colouration (black and white background), and found no evidence for 55 behavioral selection. The apparent response in colour change to background appears to be 56 mediated by the background reflectance of light, that there does not seem to be behavioral 57 selection of matching background by the tadpoles, and therefore it suggests that color change 58 is more likely to be a physiological response with thermoregulatory implications. 59 60 Short note. 61 Information on environmental factors affecting the colouration of amphibian tadpoles 62 is scarce and incomplete. This information is better developed, including genetics and evolution, 63 in several model organisms like birds or fishes. We thus need a better understanding of 64 ontogeny, diversity and functions of colouration in amphibian tadpoles (Thibaudeau & Altig, 65 2012). Excluding descriptive notes, studies about the functions, including crypsis, 66 thermoregulation, ultraviolet protection, and modification of social interactions of colouration 67 of amphibian larvae, are rare (Caldwell et al., 1982) 68 Amphibians, unlike other vertebrates (eg. mammals), have melanophores (containing 69 melanin that produce browns and blacks), xanthophores (containing purine and pteridine 70 pigments), and iridophores (containing crystalline purines). These cells arise from some trunk 71 neural-crest cells that migrate dorsolaterally during development (Olsson & Löfberg, 1992). In 72 amphibians, the pigment is located in organelles called melanosomes, pterinosomes, and 73 reflecting platelets, found in each of these cell types respectively (Frost et al., 1984). These 74 organelles have the ability of being rearranged within the cells, allowing metachrosis (i.e., 75 change colour and pattern) (Thibaudeau et al., 2012). 76 Melanophores are mainly responsible of pigment patterns observed during the first 77 larval stages (Parichy, 1996). A larger quantity of organelles per cell or pigment per organelle 78 results in a higher intensity of black in melanophores. In addition, melanosomes can aggregate 79 within the cell body or disperse radially by means of microtubules (Nascimento et al., 2003) 80 when stimulated by melanocyte stimulating hormone, epinephrine or melatonin (Odenthal et 81 al., 1996). 82 Excluding studies of biochemistry and cellular mechanism of pigmentation, there is not 83 enough information to understand the influence of colouration on behavioral ecology of 84 tadpoles. Behavioral consequences of ontogenic colour changes are known in only a few cases 85 (Nilsson et al., 2013). Some forms of sociality can often be involved in colouration, promoting 86 the behavioral changes. Several studies show that tadpoles can preferentially recognize and 87 associate with others of their own species, using colour for conspecific recognition (Blaustein, 88 1998; Foster & McDiarmid, 1982). 89 Sometimes colour differences among tadpoles of the same species in adjacent ponds 90 are obvious and until recently, these differences were assumed to be caused by light regimes, 91 turbidity and tannin content (Oshima, 2001). The inducible colour response varies among 92 individuals, and variations can also be mediated by the presence of predators and competitors, 93 tadpoles with modified colours proving to have a selective advantage in predatory encounters 94 (McClure & McCune, 2003). Our aim in this study is to assess the effect of some factors: 95 substratum albedo influence (white or black background), light period, temperature, and 96 behavioral selection of background. For this purpose, we used the primitive family Alytidae 97 (Pough et al., 2016) in order to get an example of implications of melanophore metachrosis in 98 basal anuran species. 99 Our first hypothesis is that albedo of substrate is influencing aggregation of 100 melanosomes of anuran larvae, and thus, influencing the colour of the Alytes tadpoles. As for 101 our next hypothesis, we aim to test two points: the role of environmental temperature in colour 102 change, and to evaluate if there is a possible effect of colour in body temperature (in case of 103 same environmental temperature) and thus, in larvae thermoregulation. We also test whether 104 photoperiod has a significant role in color change (a longer photoperiod means a longer 105 exposition to radiation). In addition, we tested whether different coloured tadpoles selected 106 different substrate background in relation with their colour. 107 107 108 The model species selected for this experiment was the Betic midwife toad (Alytes 109 dickhilleni). This is a basal anuran (Pough et al., 2016), with a tadpole that can reach relatively 110 large size (max. 6 cm) and with a prolonged larval period before reaching metamorphosis (from 111 3 to 16 months depending on temperature). Tadpoles (n=38) were obtained from an artificial 112 pool at Calar Alto (-2.52 W,37.24 N; 1833m; Almeria province, southeastern Spain). Larvae 113 (Stage 30; Gosner 1960) were maintained in two 10 L containers in the same room, with 114 dechlorinated water and the same background (grey). Changes of water was implemented every 115 two days, maintaining it well oxygenated with an air pump. Larvae were fed ad libitum once per 116 day with rabbit vegetable pellets (protein: 11.10%; fibre: 18.14%; fat: 7.05%; calcium: 0.25%, 117 Iron: 0.07%), and acclimatized to 18 ºC (Guerrero-Gómez et al., 2019) in order to slow down the 118 metamorphosis, with a 12-12 h photoperiod (elevation of the lab 7 m over sea level). 119 119 120 120 121 After a week of acclimatization, larvae were classified to one of two colour morphs: dark 122 or pale. We established a natural, biological marker as criteria for the assignation: Tadpoles with 123 the characteristic “V” of the species visible on the top of the body were classified as pale (see 124 Supplementary material 1), and tadpoles with a colouration that was not pale enough to see the 125 “V” were classified as dark (Supplementary material 1). This colouration classification can be 126 complemented with observation of melanophores with stereomicroscope, so colouration can 127 be easily assigned to one or other stage. 128 Then, tadpoles were randomly assigned to one of two treatments: One container (n= 129 19 tadpoles; 10 pale and 9 dark) was located over a black background (albedo=0.18) and the 130 other (n=19 tadpoles; 9 pale and 10 dark) over a white background (albedo=0.75). In the 131 laboratory, tadpole containers were kept 25 cm apart on the same shelf. For one week, the 132 number of pale or dark larvae (see supplementary material 1 for classification criteria) were 133 recorded in each treatment allowing us to obtain the temporal change of colouration each day. 134 At the end of this week pictures of pigment patches in the tail were taken using a Leica Stereo 135 Microscope MZ10 F. In order to confirm our results, as a control, these experiments were 136 repeated inverting the background of the containers after a week, repeating the same 137 procedure (hereinafter, we will call this experiment reversion experiment). 138 With the aim of understanding the role of tadpole colour on thermoregulation, first, we 139 measured superficial body temperature of each tadpole (using a no-contact Fluke® Precision 140 Infrared Thermometer 572). To avoid the bias of reflectance in infrared measurements we also 141 tested temperature with a cloacal mercury thermometer (Miller & Weber INC NY USA) placed 142 in the back of the body in a sub-sample of the animals, and reflectance was fitted for dark or 143 pale colourations. In addition, in order to ensure we were measuring animal temperature and 144 not the superficial water temperature, the temperature of water in the tanks was verified. Later, 145 we implemented a gradual 6 ºC room temperature reduction (2ºC per day to avoid possible 146 thermal shock) until reaching 12 ºC (final temperature). Temperature of the tadpoles 147 maintained in the same background treatments were recorded as described above. With this 148 final test, we aimed to observe if within the same substratum treatment there were differences 149 in the response under different temperature conditions. The temperature of this last test was 150 selected considering the average day temperature of a natural pond with presence of the 151 species (HOBO Pendant G temperature Data Logger-UA-004-64 placed in natural water bodies 152 of origin). 153 153 154 In order to investigate the role of light in the process of colour change, we implemented 155 a final test dividing each background treatment in two photoperiod treatments, one with 12D— 156 12L h lighting and one with 24 h lighting, resulting in four treatments (white background 12h of 157 light, n= 10; white background 24 h of light n= 9, black background 12 h of light, n= 10; and black 158 background 24 h of light, n= 9).The source of light was artificial (fluorescent tube). Room and 159 water temperature were maintained at 18ºC during all the experiments (water temperature was 160 reset to 18 ºC in the four days preceding this experiment in order to allow acclimatization), and 161 containers under 12 h lighting were covered by panels to avoid influence of any residual light of 162 the 24 h treatment lamp. 163 163 164 A behavioral test for substratum colour preference was conducted after the initial colour 165 change experiment: We placed the tadpoles in a 10L container with four patches (two black and 166 two white, see supplementary material 1), and we tested the colour selection of both groups 167 (dark tadpoles, n=19; pale tadpoles, n=19). We tested the response under two scenarios, 168 response of the whole group and individual response. For each group response, we 169 photographed and counted individually the larvae of each treatment (n=19+19= 38) located in 170 black or white patches during one-hour periods until completing 5 observations. In the case of 171 individual response, we selected individual tadpoles of both treatments and released them in 172 the container, recording the data after one-hour periods. 173 173 174 Statistical analyses were implemented using R Statistical software (R Core Team, 2013). 175 McNemar’s test (a test used on paired nominal data in order to detect changes in time) was used 176 to test for colouration change, reversion, behavioral group and single behavioral test. For this 177 we constructed the 2x2 contingency matrix with the dichotomic response of body colour and 178 time for each treatment. The Mann-Whitney U test (quantitative response in the case of body 179 temperature) were implemented in order to test this last hypothesis. Dependent variables were 180 Colour (proportion of dark tadpoles vs pale tadpoles) for colour change and photoperiod 181 experiments, and predictor variables being background colour treatment, and background 182 colour treatment + photoperiod respectively. In the case of body temperature experiments the 183 dependent variable was body temperature, and the predictor was tadpole colouration. In the 184 behavioral test the predictor was background colour treatment and the dependent variable was 185 colour selection. 186 186 187 In the case of colour change experiments, results show a significant increment of dark 188 tadpole proportion when they are placed on black background and an increment of pale 189 tadpoles on white background (Figure 1). Results of reversion experiments were also significant 190 under both treatments (change to pale colouration with white background and change to dark 191 colouration with black background). We also observed significant differences in body 192 temperature (0.49 ºC of mean temperature) between tadpoles of different body colour (p < 193 0.001), with a higher temperature in dark tadpoles (18.75 ºC ±0.15; pale tadpoles: 18.25±0.21) 194 with the same water temperature (18ºC). Differences for colour change in the decreasing 195 temperature scenario were also significant (Figure 1). 196 The pictures of tail patches show a clear response in the melanophore patterns (Figure 197 1). For pale tadpoles we can observe small isolated dots (melanosomes are aggregated at the 198 nucleus), and for dark tadpoles we can observe larger dots (melanosomes are dispersed 199 throughout the cell body and dendritic processes), creating a colour stain (supplementary 200 material 2) 201 In the case of the photoperiod influence, we found that the proportion of pale tadpoles 202 increased significantly at longer lighting periods (p-value < 0.005). This indicates that the longer 203 photoperiod may cause a shift towards pale colouration in tadpoles at the same temperature 204 and background treatments (Figure 2). 347 West-Eberhard, M.J. 2003. Developmental plasticity and evolution. Oxford University 348 Press. 349 Zamora-Camacho, F.J., Cortés-Manzaneque, S., Aragón, P. 2019. Simulated predation 350 pressure in Pelobates cultripes tadpoles modulates morphology at the metamorphic 351 stage. Current Zoology. 352 352 353 Figure captions 354 Figure 1. Percentage of dark coloured larvae in each treatment in initial (A), reversion (B) and 355 temperature experiments (C); including average colour change rate per day (CCRD). Initial colour 356 change experiment black background (χ²=10; p= 0.005), white background (χ²=10; p= 0.005); 357 Reversion experiment white background (χ²=15; p= 0.005), black background (χ²=15; p= 0.005); 358 temperature experiment (p= 0.024). We provide, in addition, section of larval tail crest at white 359 substratum treatment (D) and black substratum treatment (E), with details of black coloured 360 patches. We can observe the effect of melanosome aggregation (D) or dispersion (right) in the 361 melanophore cell. Black scale is 1mm. 362 Figure 2. Percentage of colouration response to background and photoperiod treatments at 363 18ºC. Black bars for dark tadpoles and grey bars for pale tadpoles. P-values < 0.05 for both, 364 white background treatment and lack background treatment. 365 Supplementary material captions 366 Supplementary material 1. Experiential design of water container for colour change and colour 367 selection experiments. Criteria to classify larvae under dark or pale coloration is also shown. 368 The characteristic “V” of the species was visible only in tadpoles classified as pale. 369 Supplementary material 2. Left: Schematized melanophore metachrosis under both treatments. 370 In light coloured tadpoles, melanosomes are aggregated in melanophore cell body. In dark 371 coloured tadpoles, melanosomes are dispersed radially by means of microtubules. Right: 372 Proposed environmental model explaining the mechanism implicated in melanophore 373 metachrosis observed in amphibian tadpoles 374 375 Figure 1 Click here to access/download;Figure;figure 1.jpg Figure 2 Click here to access/download;Figure;figure2.jpg Click here to access/download Supplementary File Sup matt.docx Supplementary File