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A simple and inexpensive chemical test for behavioral ecologists to determine the presence of carotenoid pigments in animal tissues

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Abstract

Animals use several different types of pigments to acquire their colorful ornaments. Knowing the types of pigments that generate animal colors often provides valuable information about the costs of developing bright coloration as well as the benefits of using these signals in social or sexual contexts. It is often assumed that red, orange, and yellow colors in animals are derived from carotenoid pigments, when in fact there are other pigments that confer similar colors on animals. These include the pteridine pigments in a wide range of organisms, hemoglobin in blood-filled sinuses, the psittacofulvins of parrot feathers, and the phaeomelanin pigments in rufous or yellow feathers and fur. In this paper, we describe a quick and easy, two-step chemical method for field biologists to determine if their study species uses carotenoid pigments as integumentary colorants. This laboratory procedure first employs a thermochemical extraction technique, in which acidified pyridine is used under high temperature to free carotenoid pigments from tissue to produce a colorful, pigmented solution. Red, orange, or yellow tissues containing pteridines, hemoglobin, or eumelanins do not release colored pigments into heated pyridine. However, psittacofulvins, and occasionally phaeomelanins, will also solubilize using this method. Thus, a follow-up test is needed, using solvent transfer, to confirm the presence of carotenoids in animal tissues. The use of absorbance spectrophotometry on the colorful solution may also provide information about the predominant carotenoids that bestow color on your study animal.

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Fig. 1
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Notes

  1. For certain carotenoid-enriched tissues (e.g., fruits, fish skin), other solvents, such as acetone (Grether et al. 2001) or tetrahydrofuran (McGraw et al. 2001), will also solubilize carotenoids. However, we recommend a standardized procedure that ensures that even the most tightly bound animal tissue matrices will release pigments into solution.

  2. This was the case for non-carotenoid pigments extracted from chicken, turaco, swan, and eider feathers and crow eggshells. However, macaw psittacofulvins did transfer to hexane:TBME, but have never been found in animals other than parrots, and carotenoids have never been found in parrot feathers.

  3. It is also worth noting here that lipophilic psittacofulvins and carotenoids have distinct λmax values (Hudon and Brush 1992), such that absorbance spectrophotometry can be used if necessary as a third diagnostic test to confirm the presence of carotenoids in animal tissues.

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Acknowledgements

This paper evolved from several inquiries and discussions at the 9th International Behavioral Ecology Congress in Montreal, Quebec, Canada. The Institutional Animal Care and Use Committee at Cornell University approved all procedures reported in our study (protocol no. 99–89). We thank T. Czeschlik and two anonymous referees for helpful comments on the manuscript. This research was funded by the Environmental Protection Agency (STAR fellowship to K.J.M.); during manuscript preparation, K.J.M. was supported by the United States Department of Agriculture (grant to K. Klasing) and by the College of Liberal Arts and Sciences and the School of Life Sciences at Arizona State University.

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  1. Kevin J. McGraw

    Present address: School of Life Sciences, Arizona State University, Tempe, AZ 85287–4501, USA

Authors and Affiliations

  1. Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA

    Kevin J. McGraw

  2. Provincial Museum of Alberta, 12845 102nd Avenue, Edmonton, AB, T5N 0M6, Canada

    Jocelyn Hudon

  3. Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA

    Geoffrey E. Hill

  4. Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA

    Robert S. Parker

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  1. Kevin J. McGraw
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Correspondence to Kevin J. McGraw.

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McGraw, K.J., Hudon, J., Hill, G.E. et al. A simple and inexpensive chemical test for behavioral ecologists to determine the presence of carotenoid pigments in animal tissues. Behav Ecol Sociobiol 57, 391–397 (2005). https://doi.org/10.1007/s00265-004-0853-y

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