In: Melanin: Biosynthesis, Functions and Health Effects

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Chapter VII

Melanic Pigmentation in Ectothermic

Vertebrates: Occurrence and Function
1

Classius de Oliveira1 and Lilian Franco-Belussi2

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

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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pigment cell differentiatioon and the traansfer of pigm

ments are deppendent on inntracellular leevels
of cyclic AM
MP [6].

Figure 1. Visceral pigmented cells of Euupemphix natttereri. A: Viscceral pigmenteed cells of paarietal
o melanocyte of testicle sho
owing
peritoneum shhowing dendrittic shape. B annd C: Electronn micrograph of
association w
with fibroblastt and presence of melanoossomes in the
t
citoplasm.. D-H: Stagees of
melanossome development. The granulees become ellectron dense by melanin accumulationn. M:
a
Nu: Nucleeolus.
Melanossomess. N: Nuleous. Na: Nuclear area.

Three main
m
hormonnes modulatte color chaange in verrtebrates. Thhe -Melano
ocyte
stimulating hhormone (-M
MSH) is amoong the bestt known of thhese. This hoormone reguulates
both the cutaaneous pigmeentation of ecctotherms andd extracutaneous pigmentaary system. In
n the
cutaneous pigmentation, -MSH reg
gulates the ddispersion off pigments iin melanophores,
xanthophoress, and erythhrophores [2]]. In the exttracutaneous pigmentary system, -M
MSH
increases thee expression of tyrosinasee gene, as deemonstrated by
b Guida et al. (2004) inn the
liver of Peloophylax lessoonae. The melanin-conce
m
entrating horrmone (MCH
H), which haas an
antagonistic effect to -M
MSH, is a cyyclic neuropepptide synthessized as a pre-hormone inn the
hypothalamuus of vertebbrates [7]. As
A a resultt, MCH inffluences the aggregationn of
melanosomes [3]. Melatoonin, a pineall hormone prooduced in daarkness, is ressponsible for skin
whitening inn amphibianss [8]. Melatoonin also indduced the agggregation of melanosomees in
cultures of melanophores
m
from fish andd amphibianss [9,10].

Melanosso
omes: An Organelle
O
T
That
Synthesizes and
d Stores Me
elanin
Melanossomes are org
ganelles that produce andd store melannin pigments.. These organnelle
posses distinnct stages of maturation,
m
w differentt shapes, sizes an electronn densities [111,23]
with
(Figure 1B-H
H). The initial, or pre-melaanosomes, haave no pigmeents but distinnctive features. At
the Stage I, melanosomes
m
s have irregullar fibrous sttructures and internal mem
mbranous vessicles
that resemble typical latee multivesicuular endosom
mes (also known as multivvesicular bod
dies).
Stage II mellanosomes are easily definned in transm
mission electrronic microsccopy due to their

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Classius de Oliveira and Lilian Franco-Belussi

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, LDOPA 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 melaninfilled 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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Figure 2. Orgaans and structuures of the abdoominal cavity of distinct anuurans species shhowing variatiion in
visceral pigmeentation in cattegories accorrding to Francco-Belussi et al.
a 2009. Categgory 0: absence of
melanic pigm
metnation. Cattegory 1: Prresence a few
w pigmented cells. Categoory 2: Modeerated
pigmentation. Category 3: Intense melaanic pigmentattion. O a: Od
dontophrynus americanus; E n:
H
magaalhaesi; H s: Hylodes
H
sazimaai, P cv: Physaalaemus cuvierri; I j:
Eupemphix naattereri; H m: Hylodes
Ischnocnema juipoca; P o: Physalaeemus olfersii;; PS f: Pseeudopaludicolaa falcipes; E b:
tylus binotatus; R o: Rhinellaa ornata; PR b:
b Proceratophhrys boiei; PR m: Proceratop
phrys
Eleutherodacty
melanopogon;; L fr: Leptoddactylus furnarrius; L b: Lepptodactylus bokermanni; D m
m: Dendropso
ophus
minutus; D n: Dendropsophus nanus.

Such stuudies found th

hat the pigmentation on thee surface of testes
t
of severral anurans varies
v
among speciies and generra. Members of genera Addelphobates, Colostethus,, Dendropsopphus,
and Leptodaactylus no prresent pigmeentation on tthe testes [24
4,25,26]. Onn the other hand,
h
members of the family Leiuperidae
L
a other Denndrobatidae have
and
h
melanicc pigments onn the
testes in various degrees of
o pigmentatiion [24,25].

218

Classius de Oliveira
O
and Lilian
L
Francoo-Belussi

Figure 3. Pressence of melan

nic pigmented cells in conjunntive membranne of nerves off the lumbar plexus
(A) and assocciated with reenal blood vesssels (B) of E
Eupemphix natttereri. C: Verrtebral column
n. K:
Kidney. N: Nerve
N
of the luumbar plexus. P: Lumbosacrral parietal peeritoneum. R:R
Renal blood veesels.
Arrow: pigmented cells.

Figure 4. Melanic pigmentaation in testiclees of Eupemphhix nattereri shhowed intense color black on
o the
w associated with blood veessels
surface (A, D)) and pigmenteed cells in inteerstitium of thiis organ (B) with
(C) and preseence of pigmeented cells in surface (D). L
L: Seminiferouus locule. T: Testicle. V: Blood
B
vessel. Arrow: pigmented ceells. B-D: Stainned with Hemaatoxilin-eosin.

So, thesee findings suuggest that thhe visceral ppigmentation shows a phyylogenetic sig
gnal.
Although, sttudies reporteed that the pigmentation
p
on testes vaaries. This vaariation is related
with breedinng season in the bufonidss Rhinella scchneideri [277] and Ateloppus spp. [288]. In
Physalaemuss cuvieri testiicular pigmenntation is present and not varies duringg breeding seeason
[27]. In the kidneys,
k
simiilar to testes, the pigmenttation on the dorsal surfacce increased from
the beginningg towards thee end of the breeding seaason in Dendrropsophus naanus [27]. On
n the

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219

heart, testess and kidneys pigmenntation in E

Eupemphix nattereri
n
inccrease follow
wing
administratioon with E. colllis lippopolyysacaride [266].
Pigmented cells in thhe epidermis and various organs are similar to melanocytes [299,21]
originated frrom the ectoddermal neuraal crest [30]. These cells are able to pproduce and store
melanin [31]]. However, additional stuudies about its
i occurrencce and anatom
mical distribu
ution
are needed inn order to determine their biological
b
funnctions.

T
The Melanin in
n Hematopoiettic Orga
ans
As menttioned previoously, a uniquue feature off ectothermic vertebrates iis the presencce of
an extracutanneous pigmenntary system in various tiissues and orrgans compossed of many cells
with melaniin-filled cytooplasm. In hematopoietic
h
c organs, a pigmentary cell types with
phagocytic aactivity are fo
ound. Few stuudies have innvestigated thhe functions oof the pigmen
ntary
system in animals. The majority
m
of stuudies have foocused on thee pigmented cells
c
of the sp
pleen
and liver.
These macrophage-li
m
ike cells [32]] are derivedd from hemaatopoietic stem cells [30] and
often aggreggate in pigmeented noduless called melaanomacrophagge centers [33]. These cen
nters
belong to thhe mononucclear phagocy
yte system and its mainn function iis related too the
phagocytosiss of cellular material
m
derived from cattabolism [34]]. This evideence suggestss that
melanomacroophage centeers are respponsible for the detoxifiication or reecycling of both
endogenous and exogenoous products [35]. There aare also evideences that melanomacrophage
centers are innvolved in thhe resistance against bacteerial pathogens, parasites,, and spores [36].
The storage of
o iron after erythrophago
e
cytosis is alsoo reported [31].

Figure 5. Liveer sections show

wing melanom
macrophages inn the tissue (A)) and presence of hemosiderin (B)
and lipofuscinn inside of pig
gmented cell. H:
H Hepatocytees. S: Sinusoidds. Coloration: Acid ferrocyanide
solution (B) annd Schmorls solution
s
(C).

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Classius de Oliveira and Lilian Franco-Belussi

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., FrancoBelussi 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

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Classius de Oliveira and Lilian Franco-Belussi

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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