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Chromatophores are large, stellate and spectacular pigment bearing cells, typically located in the skin, that generate body colouration. In many animals, melanocytes/melanophores, the melanin containing chromatophores, are also present in various tissues inside the body, for instance in the ear, brain, abdominal cavity, around internal organs and skeleton. The presence of such internal pigmentation in puzzling as it is hidden from sight. While there is an enormous amount of studies and data on skin chromatophores, from the fine details of the motile machinery to animal behaviour (Aspengren et al., 2009), internal melanocytes have historically been largely ignored, until recently (Aoki et al., 2009; Brito and Kos, 2008; Randhawa et al., 2009; Yajima and Larue, 2008). Remarkably little is still known about their possible functions, and this uncertainty is problematic for the more general question of the role(s) of melanin in itself (Aspengren et al., 2009; Boissy and Hornyak, 2006; Braasch et al., 2009; Hill, 2000; Ito, 2009). While internal melanocytes are prevalent, internal erythrophores and xanthophores appear more uncommon. In fish, however, such cells can be found interspersed with melanocytes and reflective chromatophores in the highly pigmented peritoneum (the endothelium that covers the abdominal cavity) (Nilsson Sköld et al., 2008). In juveniles, as well as in adults of species with relatively transparent bodies, internal chromatophores may actually contribute to the overall body colouration, as shown in two-spotted goby females, where abdominal trunk biopsies were analysed (Nilsson Sköld et al., 2008).

In fishes, skin patterns can be rapidly modified by aggregation or dispersion of the pigment-containing organelles inside the chromatophores present in the skin (i.e. physiological colour change) for background adaptation or signalling displays (Aspengren et al., 2009; Fujii and Oshima, 1994). In comparison, colour change in internal chromatophores has been largely ignored as it has been generally considered that they are not responsive (Boissy and Hornyak, 2006). However, melanocytes in the peritoneum and around the skeleton of the ice goby, Leucopsarion petersii, do indeed adapt to the background by pigment translocations in vivo and in biopsies, thus providing the first evidence for internal colour change (Goda and Fujii, 1996). Recent work on biopsies from the two-spotted goby showed that also internal erythrophores and xanthophores can be responsive (Nilsson Sköld et al., 2008). As these examples come from relatively transparent species, it is possible that this phenomenon is more common than earlier believed, especially in species with some degree of body transparency. In order to test if a capacity for internal colour change was related to the degree of body transparency, and to reveal a possible function of these cells, we analysed the regulatory capability of peritoneal melanocytes in eight different teleost species, representing five different orders within the large super order Acanthopterygii and in one member of the super order Clupeomorpha, all with different degrees of body transparency. We used epinephrine and melatonin as potential pigment aggregating agents (see Appendix S1 for methodology). A positive relationship between body transparency and rate of internal colour change would suggest a special adaptive role for internal melanocytes in transparent fish species, and thus constitute a novel function for internal pigments.

Our results showed that peritoneal melanocytes were present in all investigated fish species. Especially high densities were found in the gobies and in pipefish, plaice and herring (Figure 1A, B). Internal erythrophores and xanthophores were also observed in the gobies, pipefish and in plaice, but not in the other species. A capacity to regulate peritoneal melanocytes by melatonin and epinephrine was detected in six out of the eight species (Figure 1A, B and Table S1). In all species tested, peritoneal melanocytes had dispersed pigment from the start of the experiment. The epidermal melanocytes of sand goby, black goby and wrasse responded within 30 min, whereas plaice and pipefish only responded appreciably after 2 h. In herring, both controls and treated peritoneum biopsies showed a response after 30 min with an only moderate further aggregation after 2 h. Sculpins and sticklebacks showed no response even after 3 h of observation. In the gobies, also peritoneal erythrophores and xanthophores responded to norepinephrine by pigment aggregation. In sand goby, pipefish and herring, the untreated control biopsy also responded slightly with time, although to considerable less degree than the melatonin and epinephrine treated biopsies (Table S1).

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Figure 1.  Biopsies of peritoneum from different species of fish were incubated in norepinephrine to trigger melanosome aggregation. The pair wise located pictures were taken on the same biopsies before and 1 h after addition of norepinephrine. The left panel show species with clear peritoneal melanosome aggregation (A), while the right panel show species with little or no peritoneal melanosome aggregation (B). Representative photographs of fishes placed on a light board and used for body transparency analysis (C). Scale bar = 100 μm.

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On the cutaneous side, epidermal melanocytes, or melanophores, in all species had fully dispersed pigment at the start of the experiment. The epidermal melanophores in all species but sculpin and stickleback responded by melanosome aggregation within 30 min. After 2 h, also stickleback and sculpin showed a response. The rate of response was similar for melatonin and epinephrine within most species, but stickleback epidermis did not respond to melatonin even after 3 h of observation. In pipefish and herring, the untreated control biopsies also responded, but the response was much less than for the hormone treated biopsies (Table S1).

The degree of body transparency differed between fish species, with highest levels in sand gobies followed in order by wrasse, black gobies, plaice, pipefish and sticklebacks. The lowest degree of transparency was found in herring and sculpins (Figure 1C, Table S2). The degree of response in peritoneal melanocytes (ΔMI) was significantly positively related to body transparency (r2 = 0.76, F1,6 = 19.5, P = 0.004 for melatonin, and r2 = 0.80, F1,6 = 23.8, P = 0.003 for epinephrine, Figure 2). The life style of plaice is different from the other species, in that it typically resides flat on the bottom and semi-covers its body with sand. It is therefore possible that the role of regulating body pigmentation is different in plaice compared to the other fishes. If excluding plaice from the analysis, the relationships were even stronger (r2 = 0.93, F1,6 = 61.7, P < 0.001 for melatonin, and r2 = 0.90, F1,6 = 46.22 and P = 0.001 for epinephrine). To test the robustness of these relationships, analyses were also performed using either MI after 30, 60 min or the slope value during the linear phase (results not shown). The outcomes were highly similar regardless of which parameter that was used. While there was a considerable difference in rate of colour change also in epidermal cells between species, there were no relationships with body transparency (r2 < 0.15, F1,6 < 1.1, P > 0.33 for melatonin or epinephrine). Furthermore, the degree of response in inner melanophores were not correlated to that of outer cells (Pearson correlations, t6 < 1.3, P > 0.26 for melatonin or and epinephrine). These results were repeatable also if using MI after 60 min or the slope value during the linear phase (not shown). Thus, only for the inner melanocytes was the capacity of colour change positively related to body transparency.

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Figure 2.  Significant positive relationship between body transparency and the capacity to aggregate peritoneal melanosomes in eight fish species after 30 min exposure to melatonin (A) and norepinephrine (B). The grade of melanosome aggregation was determined using the Melanophore Index, MI, where fully aggregated melanosomes give MI = 1 and fully dispersed melanosomes give MI = 5. The capacity of cells to respond (ΔMI) was measured as difference in MI before, and 30 min after, exposure.

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We show that the potential for internal colour change is common in fishes, but, importantly, that it is not ubiquitous. The degree of response was strongly related to the degree of body transparency. However, this was not the case for epidermal chromatophores. Thus, only for inner melanocytes was the capacity of colour change, which is a potentially costly process (Aspengren et al., 2009), connected to body transparency. The results were robust with respect to differences in methodology. For instance, the conclusion is the same even if changing time of measurement, if comparing to control biopsies, or if using the slopes taken from the individual aggregation-time reaction curves. When the data from plaice, a fish with a different life style than the other species, was excluded from the analysis, the relationship between the capacity of inner colour change and body transparency was even stronger. While pigment cells on the outside of animals enable colour change in many species, it has been widely believed that melanin containing cells in internal tissues does not contribute to this process, as they are not responsive (Boissy and Hornyak, 2006). Our results from eight species of fish, together with the previously two reported species, add credence to the idea that internal melanocytes are commonly responsive among teleost fishes.

Our results suggest a novel and potentially adaptive function of internal chromatophores, by showing that the capacity for internal colour change is greater in transparent than non-transparent species of fish. Inner chromatophores, such as those of the peritoneum, may therefore have a function in background adaptation and/or in social signalling in partially transparent fishes. Given that cutaneous chromatophores, retinal pigment cells, otic melanocytes as well as peritoneal melanocytes, can respond to external stimuli, these cells appear to share a relationship with environmental sensation. By providing communication and awareness of the surroundings, melanocytes in non-cutaneous tissues, such as in the peritoneum, may therefore be more connected to animal behaviour than previously considered. The presence of internal melanocytes in non-transparent species suggests that there are additional function(s) of these cells. For example, they may function as waste deposits for accumulated melanin, participate in the innate immune system, function as antioxidants and/or possibly help shield gonadal tissue from DNA damage, as speculated (Aspengren et al., 2009; Hill, 2000; Ito, 2009). We encourage further functional investigations and tests of these hypotheses on internal chromatophores and melanin.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. References
  4. Supporting Information

This study was conducted at Sven Lovén Centre for Marine Sciences at Kristineberg, Sweden, with help from Erik Selander, Tony Fagerberg, Emma Norén and Kristina Holm, and financially supported by the Swedish Research Council (VR-NT) and C.F. Lundström’s foundation to H.N.S.

References

  1. Top of page
  2. Acknowledgements
  3. References
  4. Supporting Information
  • Aoki, H., Yamada, Y., Hara, A., and Kunisada, T. (2009). Two distinct types of mouse melanocyte: differential signaling requirement for the maintenance of non-cutaneous and dermal versus epidermal melanocytes. Development 136, 25112521.
  • Aspengren, S., Hedberg, D., Nilsson Sköld, H., and Wallin, M. (2009). New insights into melanosome transport in vertebrate pigment cells. Int. Rev. Cell Mol. Biol. 272, 245302.
  • Boissy, R. E., and Hornyak, T. J. (2006). Extracutaneous melanocytes. In Pigmentary System, J. J. Nordlund, R. E. Boissy, V. J. Hearing, R. A. King, W.S. Oetting, and J-.P. Ortonne, eds. (Malden, MA: Blackwell Publishing), pp. 91107.
  • Braasch, I., Liedtke, D., Volff, J.-N., and Schartl, M. (2009). Pigmentary function and evolution of tyrp1 gene duplicates in fish. Pigment Cell Melanoma Res. 22, 839850.
  • Brito, F.C., and Kos, L. (2008). Timeline and distribution of melanocyte precursors in the mouse heart. Pigment Cell Melanoma Res. 21, 464470.
  • Fujii, R., and Oshima, N. (1994). Factors influencing motile activities of fish chromatophores. In Advances in Comparative and Environmental Physiology, R. Gilles, ed. (Berlin: Springer-Verlag), Vol. 2, pp. 154.
  • Goda, M., and Fujii, R. (1996). Biology of the chromatophores of the ice goby, Leucopsarion petersii. Zool. Sci. 13, 783793.
  • Hill, H. Z. (2000). The function of melanin or six blind people examining an elephant. Bioessays 14, 4956.
  • Ito, S. (2009). Melanins seem to be everywhere in the body, but for what? Pigment Cell Melanoma Res. 22, 1213.
  • Nilsson Sköld, H., Amundsen, T., Svensson, P.A., Mayer, I., Bjelvenmark, J., and Forsgren, E. (2008). Hormonal regulation of female nuptial coloration in a fish. Horm. Behav. 54, 549556.
  • Randhawa, M., Huff, T., Valencia, J.C., Younossi, Z., Chandhoke, V., Hearing, V.J., and Baranova, A. (2009). Evidence for the ectopic synthesis of melanin in human adipose tissue. FASEB J. 23, 835843.
  • Yajima, I., and Larue, L. (2008). The location of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color. Pigment Cell Melanoma Res. 21, 471476.

Supporting Information

  1. Top of page
  2. Acknowledgements
  3. References
  4. Supporting Information

Table S1. Capacity for outer (epidermis) and inner (peritoneum) colour change in fish.

Table S2. Body transparency in fish.

Appendix S1. Materials and methods.

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PCMR_674_sm_AppendicesS1-3_rev.doc716KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.