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

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.