In: Melanin: Biosynthesis, Functions and Health Effects ISBN: 978-1-62100-991-7 Editors: Xiao-Peng Ma and Xiao-Xiao Sun 2012 Nova Science Publishers, Inc.

Chapter VII

Melanic Pigmentation in Ectothermic Vertebrates: Occurrence and Function

Classius de Oliveira1 and Lilian Franco-Belussi2 1 So Paulo State University UNESP, IBILCE So Jos do Rio Preto,

Department of Biology. Rua Cristvo Colombo, 2265 So Jos do Rio Preto, SP, Zip code 15054-000, Brazil

2 Post-Graduate Program in Animal Biology (UNESP/IBILCE-SJRP), SP, Brazil

Abstract

Ectotherms have specialized chromatophores whose pigments are responsible for the different colors of the epidermis. Melanocytes are one type of chromatophore that produce and store melanin in organelles called melanosomes. In ectotherms, cells containing melanin pigments occur in several organs and tissues. These cells are found in the capsule and stroma of the organs, giving it a dark coloration. The function of visceral pigment cells is poorly known, but melanomacrophages are known to perform phagocytosis in hematopoietic organs and also act against bacteria, due to melanin. In addition, the distribution of visceral melanocytes varies with physiological factors, such as age, nutritional status; and also environmental one, such as temperature and photoperiod. On the other hand, the pigmentation in some organs seems to be conservative, and may help in phylogenetic reconstructions.

Keywords: Chromatophores; Melanin; Melanocytes; Melanomacrophages; Extracutaneous pigmentary system

Introduction Chromatophores are specialized cells found in invertebrates and ectothermic vertebrates

that store pigments. These cells have many cytoplasmic projections, giving it a dendritic aspect. Chromatophores originate in the neural tube during embryonary development and

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Classius de Oliveira and Lilian Franco-Belussi 214

later migrate to the skin. In the adult, chromatophores are found in the epidermis and dermis [1].

Chromatophores have many types of pigments. At least five types are described in vertebrates. The melanophores are black or brown colored cells with melanin granules. They are found in fish, amphibians, and reptiles. The erythrophores are reddish cells with pteridine pigments. The xanthophores also have pteridine, along with carotenoid pigments arranged in vesicles, which gives it a yellow color. The iridophores have a metallic color due to purine deposited in reflective crystals. As erythrophores and xanthophores, iridophores are also found in fish, amphibians, and reptiles. The leucophores are white colored cells with purine granules, and only occur in fish [1,2].

Chromatophores are found preferably in the dermis of animals. The xanthophores are the most superficial cells of this skin layer, located just below the basement membrane. Deep inside the skin there are iridophores, cells that have iridescent appearance. Even more deep, there are the melanophores. The arrangement of these pigment cells in the skin layers are closely related to their pigment type and which wavelengths they reflect or absorb [2].

In this chapter, we will discuss some hypotheses posed to explain the function of these cells in internal organs of ectothermic vertebrates.

Melanophores: Color Changes and Hormonal Control

Melanophores are found in the deepest layer of the dermis and in visceral organs of

ectotherms. These cells are dendritic in shape (Figure 1A), and in dermis are responsible for the dark color and the quick color change. The arrangement of these pigment cells in the skin layers are closely related to their pigment type and which wavelengths they reflect or absorb. This feature dictates its function [2].

Some ectotherms can quickly change the body color through the regulation of chromatophores. For example, the stimulus for aggregation or dispersion of pigments in fish may come directly from innervation or alternatively from hormonal control [3]. Contrarily, pigment migration in amphibians only occurs by hormonal control [2]. This quick color change is physiological and involves numerous types of chromatophores. It is also related to camouflage and social signaling [4]. Physiological color change occurs in animals that can quickly change their coloration through the bidirectional migration of pigment granules within pigment cells. Environmental stimuli that evoke these changes are mainly associated with light intensity, background color or social context [5,6].

Color change may also happens by means of morphological change in vertebrates. It is slower but lasting than the physiological one. A change in morphological coloration is defined as a gradual color change resulting from the increase or decrease in the number of pigment cells or the amount of pigment within cells. Such a change is usually associated with ontogenetic, sexual, feeding, or seasonal changes [5,6]. Melanosomes are dispersed and transferred to skin cells in mammals and amphibians. In these animals, the dispersion of pigments also stimulates the production of new pigment cells in the long term. The morphological change in color is also modulated by hormones, although the regulation of

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Classius de Oliveira and Lilian Franco-Belussi 216

regular, elongated and parallel fibers. These fibers serve as a mold for the deposition of eumelanin in mature melanosomes. As a result, melanossomes at Stage III are dark and thick. The accumulation of melanin causes a masking of the intraluminal structure of melanossomes at Stage IV. Thus, the formation of melanosomes with characteristic fibers precedes the eumelanin synthesis and is an essential step in eumelanogenesis [2,11].

Melanosomes are mobile structures and move inside cells by the action of motor proteins guided by microtubules: the cytoplasmic dynein, kinesin II, and myosin V. Each protein act differently in the movement of melanosomes, and also the aggregation and dispersion of pigments. Kinesin is responsible for the centrifugal transport, dispersion of melanosomes and consequent darkening of the animal. Dynein is the antagonist of kinesin and are responsible for the centripetal transport of melanosomes, aggregation of pigment cells, and consequent bleaching of the organism [2,12]. On the other hand, actin molecules are responsible for the short or cross transport. This transport is conducted by actin molecules because this route is deslocated from the axis of microtubules. It also allows a greater dispersion of pigments throughout the cytoplasm. In this type of transport, myosin V binds to melanosomes allowing the myosin-melanosome set to slide along the actin filament [2,12].

Melanin in Ectothermic Vertebrates Melanin is an endogenous complex polymer [13] that occurs in multifunctional forms

[14,15] in both vertebrates and invertebrates. The biosynthesis of this substance is initiated by hydroxylation of L-phenylalanine into L-tyrosine or directly from L-tyrosine, which is hydroxylated to L-dihydroxyphenylalanine (L-DOPA) by the tyrosinase enzyme. Lately, L-DOPA is oxidized to dopaquinone by the tyrosinase enzyme, which diverges into the synthesis of eu- or pheomelanin [14,15]. Melanin may absorb and neutralize free radicals, cations, and other potentially toxic agents derived from the degradation of phagocytosed cellular material [16]. Melanin is also a key agent against bacterial components in ectothermic vertebrates, due to the action of hydrogen-peroxidase and their quinone precursors, which act as a bactericide [17].

A unique characteristic of ectothermic vertebrates is the presence of an extracutaneous pigmentary system [18] in various tissues and organs composed of many cells with melanin-filled cytoplasm. The melanin often present in the liver, spleen, kidneys, peritoneum, lungs, heart, blood vessels, thymus, gonads, and meninges constitute the visceral pigmentation [17,19, 20, 21,22] (Figure 2). Visceral melanocytes are localized closed to vessels and conjunctive membranes (Figure 3) and these cells are distributed in both surface and interstitium of the organs stroma (Figure 4).

Visceral Pigmentation: Anatomical Patterns in Anurans In anurans, visceral pigmentation is present in several organs of the abdominal cavity, we

hypothesize that this pigmentation has a similar pattern of occurrence within taxonomic ranks (species, genera and families). In fishes, the presence of extracutaneous pigment is highly variable, even among closely related species [16].

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Classius de Oliveira and Lilian Franco-Belussi 220

The presence of melanomacrophages is reported in the spleen, liver, and kidneys. These cells are known as Kupffer cells in the liver and have phagocytic activity. They can be classified as small or large Kupffer cells, according to their melanin content [37,38]. These cells have peroxidase, lipase [18], and melanin granules, along with other substances, such as hemosiderin and lipofuscin, derived from the degradation of phagocytosed cellular material (Figure 5) [21, 39,40].

Haemosiderin is a granular substance composed of iron hydroxide and proteins. It is generated in tissues saturated with iron ions and have to accumulate in granules to remain stored inside the cell [41]. The hemosiderin have protein derived from the catabolism of hemoglobin, and therefore it is an intermediate metabolite in the recycling of components in the erythropoiesis [42]. The production of granules of denatured hemoglobin occurs during the catabolism of red cells, which takes place in digestive vacuoles. These vacuoles are yellow-brownish due to iron hydroxide and bile pigments. The color tends to fade out within three to four days, although some partially digested granules may remain in the tissue, producing a yellow color due to the absorption of bilirubin [41].

Lipofuscin, also known as the age pigment, is an intra-lysosomal pigment that are neither degraded by lysosomal hydrolases nor exocitated [43]. This pigment is produced by the oxidative polymerization of polyunsaturated fatty acids and accumulates in post-mitotic cells [44]. During the normal autophagic degradation of mitochondria and iron-containing proteins in lysosomes, iron is released in lysosomes, in which it may react with hydrogen peroxide to form hydroxyl radicals. Depending on the rate of formation of these highly reactive radicals, they can bind to lysosomal material to form lipofuscin or these reactive radicals can destabilize the lysosomal membrane, inducing apoptosis and necrosis [43,45].

Some studies have described drastic structural and functional alterations in the Kupffer cells during the hibernation cycle, a period characterized by low temperatures and reduced food supply. In an experiment with three amphibian species (Rana esculenta, Ichthyosaura alpestris, and Triturus carnifex), there were much more pigments in the hepatic parenchyma in the hibernation than in the active period [46,47]. In addition, an increase in liver pigmentation may be related to hemocatereses [48,49]. For example, the activation of hepatic melanogenesis in salamanders may be related to hypoxia [50]. Accordingly, the increase of melanin pigments in melanomacrophage centers in fish has been associated with diseases [36].

The Functions of Melanin in Visceral Pigmentation Moresco and Oliveira (2009) analyzed the extracutaneous pigmentation pattern of three

species of anuran amphibians (Dendropsophus nanus, Physalaemus cuvieri, and Rhinella schneideri) during the breeding season. In that study, the change in the pigmentation of structures during the reproductive period could not be associated with or compared among species, since the occurrence of pigmentation was different for each species. The authors reported that the pigmentation varied during the reproductive period in the toad R. schneideri. However, the same study showed that the testicular pigmentation was evenly distributed throughout the breeding season in P. cuvieri.

Melanic Pigmentation in Ectothermic Vertebrates 221

Accordingly, the gonads of D. nanus, D. minutus, D. elianeae, and D. sanborni had no pigmentation during the reproductive period [19]. These differences between species of distinct families can be related with similar phenotypic traits, in species that lives in similar environmental conditions [51]. Ours studies showed that is possible determine a pattern for each species, and identify a relationships among within of the taxon. These description of visceral pigmentation represent helpful information to evaluate biological relations in a phylogenetic and evolutionary perspective.

Pigment cells are not found in the gonads of the majority of anuran species (e.g., Franco-Belussi et al. 2011). When present, visceral melanocytes are closely related to the vascular system, as well as blood vessels of other organs and associated conjunctive membranes. Specifically, there is an intense pigmentation in the interstitium and the tunica albuginea of the gonads of Eupemphix nattereri, Physalaemus cuvieri, and P. marmoratus, giving the testicle a full or mixed black color [23,52,53,54].

These cells make up the connective tissue of the organ itself or of tissues associated with it, such as the tunica adventitia or serous membranes. Pigmented cells of most organs, such as gonads and rectum have typical dendritic melanocytes, which differentiate it from melanomacrophages of organs, which have a punctuated appearance. Melanocytes are distributed in both the surface and interstitium of the organs stroma. Its occurrence may vary from a few to a large concentration of cells, when an intense blackish color is observed on structures.

The visceral pigmentation on testes, heart, and kidneys of anurans increases after the administration of lipopolysaccharide (LPS) from Escherichia coli [26]. These cells responded to LPS intoxication promoting a rapid increase of pigmentation on the surface of the testes after 2 hours, followed by a decrease in the pigmentation after 24 hours of administration. These changes are probably related to the bactericide role of melanin, which neutralizes LPS effects [26].

Conclusion Finally, the pigmentation is an anatomical feature of an organism. However there are

very complex relationship between chromatophores and the organs in which they occur. Certainly melanocytes have multiple functions still waiting to be determined. Additional studies about its occurrence and anatomical distribution are needed in order to determine their biological functions.

Acknowledgments The authors are indebted to FAPESP (So Paulo Research Foundation grant #

02/08016-9, 05/02919-5, 09/13925-7, 06/57990-9, and 08/52389-0) and CNPq (Brazilian National Council for Scientific and Technological Development grant # 475248/2007-4 and 473499/2010-0) for financial support and fellowships. LFB received a doctoral fellowship from FAPESP (#2011/01840-7) during the final preparation of this chapter. We would like to thank Msc. Diogo Borges Provete for suggestions in the chapter and revision of the English

Classius de Oliveira and Lilian Franco-Belussi 222

language, and Dr. Lia Raquel de Souza Santos, Dr. Rodrigo Zieri, Msc. Rafaela Maria Moresco for contributing for the research project.

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