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.
Melanic Pigmentation in Ectothermic Vertebrates: Occurrence and
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),
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 .
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 .
In this chapter, we will discuss some hypotheses posed to
explain the function of these cells in internal organs of
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 .
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 .
Contrarily, pigment migration in amphibians only occurs by hormonal
control . This quick color change is physiological and involves
numerous types of chromatophores. It is also related to camouflage
and social signaling . 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
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
pigment cell of cyclic AM
Figure 1. Visperitoneum shassociation wmelanossome
Three mstimulating hboth the cutacutaneous
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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
Melanin in Ectothermic Vertebrates Melanin is an endogenous
complex polymer  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 . 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 .
A unique characteristic of ectothermic vertebrates is the
presence of an extracutaneous pigmentary system  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
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 .
Figure 2. Orgavisceral pigmemelanic pigmpigmentation. Eupemphix
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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 , 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
. 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 . 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 .
Lipofuscin, also known as the age pigment, is an intra-lysosomal
pigment that are neither degraded by lysosomal hydrolases nor
exocitated . This pigment is produced by the oxidative
polymerization of polyunsaturated fatty acids and accumulates in
post-mitotic cells . 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 . Accordingly, the increase of melanin
pigments in melanomacrophage centers in fish has been associated
with diseases .
The Functions of Melanin in Visceral Pigmentation Moresco and
Oliveira (2009) analyzed the extracutaneous pigmentation pattern of
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
. These differences between species of distinct families can be
related with similar phenotypic traits, in species that lives in
similar environmental conditions . 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
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 . 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 .
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
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